http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Larakuntz2012.igem.org - User contributions [en]2024-03-29T11:26:56ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-27T01:18:58Z<p>Larakuntz: /* Limonene */</p>
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<div>{{Team:TU_Munich/Header}}<br />
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{{Team:TU_Munich/ExCol}}<br />
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=Limonene=<br />
<hr/><br />
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[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
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'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
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Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
D-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We have achieved our goal of a beer with limonene. We have successfully cloned (+)-limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in yeast cell culture''' via headspace GC-MS. Furthermore, we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Last but not least, we have been able to proof the '''production of limonene in the beers''' we brewed. Hence, we have brewed''' iGEM's first SynBio beer''' containing limonene.<br />
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<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
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Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
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This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
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<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
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[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
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<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
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<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
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<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
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<div><br />
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</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. A drawback might be that the selection pressure might not be preserved in the gyle and hence the loss of the plasmid might be possible. Therefore, we also performed brewing experiments with yeast that carried genome integrated limonene synthase.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD.<br />
<br />
On the one hand, we have been able to brew a beer with a yeast strain that was transformed with a vector carrying a constitutive expression cassette for limonene. On the other hand, we brewed a beer with a yeast strain that carried limonene synthase after genome integration. We analyzed the beers for limonene content via headspace (SPME needle) GC-MS. We have been able to detect limonene to be produced in both beers.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-27T01:18:24Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
D-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We have achieved our goal of a beer with limonene. We have successfully cloned (+)-limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in yeast cell culture''' via headspace GC-MS. Furthermore, we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Last but not least, we have been able to proof the production of limonene in the beers we brewed. Hence, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. A drawback might be that the selection pressure might not be preserved in the gyle and hence the loss of the plasmid might be possible. Therefore, we also performed brewing experiments with yeast that carried genome integrated limonene synthase.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD.<br />
<br />
On the one hand, we have been able to brew a beer with a yeast strain that was transformed with a vector carrying a constitutive expression cassette for limonene. On the other hand, we brewed a beer with a yeast strain that carried limonene synthase after genome integration. We analyzed the beers for limonene content via headspace (SPME needle) GC-MS. We have been able to detect limonene to be produced in both beers.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-27T01:17:38Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
D-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We have achieved our goal of a beer with limonene. We have successfully cloned (+)-limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in yeast cell culture''' via headspace GC-MS. Furthermore, we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Furthermore, we have been able to proof the production of limonene in the beers we brewed. Hence, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. A drawback might be that the selection pressure might not be preserved in the gyle and hence the loss of the plasmid might be possible. Therefore, we also performed brewing experiments with yeast that carried genome integrated limonene synthase.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD.<br />
<br />
On the one hand, we have been able to brew a beer with a yeast strain that was transformed with a vector carrying a constitutive expression cassette for limonene. On the other hand, we brewed a beer with a yeast strain that carried limonene synthase after genome integration. We analyzed the beers for limonene content via headspace (SPME needle) GC-MS. We have been able to detect limonene to be produced in both beers.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-27T01:17:07Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
D-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We have achieved our goal of a beer with limonene. We have successfully cloned (+)-limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in yeast cell culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Furthermore, we have been able to proof the production of limonene in the beers we brewed. Hence, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. A drawback might be that the selection pressure might not be preserved in the gyle and hence the loss of the plasmid might be possible. Therefore, we also performed brewing experiments with yeast that carried genome integrated limonene synthase.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD.<br />
<br />
On the one hand, we have been able to brew a beer with a yeast strain that was transformed with a vector carrying a constitutive expression cassette for limonene. On the other hand, we brewed a beer with a yeast strain that carried limonene synthase after genome integration. We analyzed the beers for limonene content via headspace (SPME needle) GC-MS. We have been able to detect limonene to be produced in both beers.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-27T01:15:57Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
D-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We have achieved our goal of a beer with limonene. We have successfully cloned (+)-limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Furthermore, we have been able to proof the production of limonene in the beers we brewed. Hence, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. A drawback might be that the selection pressure might not be preserved in the gyle and hence the loss of the plasmid might be possible. Therefore, we also performed brewing experiments with yeast that carried genome integrated limonene synthase.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD.<br />
<br />
On the one hand, we have been able to brew a beer with a yeast strain that was transformed with a vector carrying a constitutive expression cassette for limonene. On the other hand, we brewed a beer with a yeast strain that carried limonene synthase after genome integration. We analyzed the beers for limonene content via headspace (SPME needle) GC-MS. We have been able to detect limonene to be produced in both beers.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-27T01:13:10Z<p>Larakuntz: /* Biosynthesis pathways */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-limonene synthase 1 (<partinfo>BBa_K801065</partinfo>) and (+)-limonene synthase 1 with yeast consensus sequence (<partinfo>BBa_K801060</partinfo>) were successfully cloned into our new yeast expression vector pTUM104. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators (<partinfo>BBa_K801062</partinfo>, <partinfo>BBa_K801063</partinfo> and <partinfo>BBa_K801064</partinfo>). <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. Furthermore, we have been able to proof the production of limonene in the beers we brewed. Further experiments regarding the concentrations of limonene produced will be carried out in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_Overviewcaffeine.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP (lane 1), [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] (lane 2) and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] (lane 3) in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]] (C) LC/MS spectra of ''in vitro'' caffeine synthesis enzyme assay, showing produced theobromine]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 theobromine synthase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed yeast cells which had previously been transformed with our caffeine synthesis expression cartridge has been done and we were successful in producing theobromine, the immediate precursor of caffein, which we detected by the use of LC/MS with multiple reaction monitoring (MRM). <br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of tyrosine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
PAL (<partinfo>BBa_K801090</partinfo>, <partinfo>BBa_K801091</partinfo>), 4CL (<partinfo>BBa_K801092</partinfo>, <partinfo>BBa_K801093</partinfo>), CHS (<partinfo>BBa_K801094</partinfo>, <partinfo>BBa_K801095</partinfo>), APT (<partinfo>BBa_K801096</partinfo> and OMT (<partinfo>BBa_K801097</partinfo>, <partinfo>BBa_K801098</partinfo>). <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization might be an interesting task for iGEM teams in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 the galactose inducible promoter pGAL1 is located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
pTUM100 (<partinfo>BBa_K801000</partinfo>), pTUM101 (<partinfo>BBa_K801001</partinfo>), pTUM102 (<partinfo>BBa_K801002</partinfo>), pTUM103 (<partinfo>BBa_K801003</partinfo>) and pTUM104 (<partinfo>BBa_K801004</partinfo>). <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol1.png|400px|thumb|right| '''A''': The KlADH4-promoter was cloned from genomic DNA of ''Kluyveromyces lactis''. The new BioBrick BBa_K801020 was inserted into our pTUM100 vector. eGFP served as a reporter gene for characterization in ''S. cerevisiae'' (plasmid name: pTUM100_KLADH4_eGFP). '''B''' Emission spectra of eGFP obtained during cultivation of ''S. cerevisiae'' transformed with pTUM100_KLADH4_eGFP using different carbon sources. Blue: Galactose was used as carbon source. The measured ethanol concentration was 1.7 % (v/v). The peak at 509 nm indicates that eGFP is expressed. Red: Glycerol was used as carbon source. The measured ethanol concentration was 0.2 % (v/v). No eGFP fluorescence could be detected.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_lightnew.png|thumb|right|450px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1<br />
<br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
Working with food, it is unacceptable to use antibiotics to keep up the selective pressure during the brewing process. Since we cannot work with auxotrophies in beer either, we have to make sure the yeast cells do not lose the plasmids harboring our BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process. The yeast strains generally employed in brewing have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle and experiments on the toxicity of the substances produced by our biosynthetic pathways were necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments on the different yeast strains show that the growth of several different yeast strains is '''not impaired in gyle'''! <br><br />
The toxicity test with the substances caffeine and limonene showed a toxicity for yeast cells at higher concentrations in the cultur media. <br><br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
At the day of the final Wiki-Freeze we could finish the brewing with three different ingredients (limonene, thaumatin and caffeine) whose biosynthesis we have engineered during this summer. This was done using yeast culture expressing BioBricks from a plasmid as well as using cultures in which the expression cassette was integrated into the genome.<br><br />
This gives us the great honor to present '''iGEM's first and finest SynBio Beer: TUM-Brew'''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-27T01:08:29Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We have achieved our goal of a beer with limonene. We have successfully cloned (+)-limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Furthermore, we have been able to proof the production of limonene in the beers we brewed. Hence, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
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'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
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<div class="mfull bezel"><br />
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==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
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This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
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RFC 25 compatible <br />
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'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
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<div class="mfull bezel"><br />
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==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
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This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
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'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
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<div style="clear:both"><br />
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===Characterization===<br />
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<hr><br />
==== Gel Picture of Finished Constructs ====<br />
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[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
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(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
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To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
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</div><br />
<hr><br />
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==== Investigation of Yeast Consensus Sequence ====<br />
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[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
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[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
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We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
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We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
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Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
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<hr><br />
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====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
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'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
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<hr><br />
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==== In Vitro Detection of Limonene====<br />
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[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
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To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
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The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
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All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
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<div><br />
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</div><br />
<hr><br />
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==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
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We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
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<div><br />
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</div><br />
<hr><br />
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==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. A drawback might be that the selection pressure might not be preserved in the gyle and hence the loss of the plasmid might be possible. Therefore, we also performed brewing experiments with yeast that carried genome integrated limonene synthase.<br><br />
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Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD.<br />
<br />
On the one hand, we have been able to brew a beer with a yeast strain that was transformed with a vector carrying a constitutive expression cassette for limonene. On the other hand, we brewed a beer with a yeast strain that carried limonene synthase after genome integration. We analyzed the beers for limonene content via headspace (SPME needle) GC-MS. We have been able to detect limonene to be produced in both beers.<br />
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<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
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<hr><br />
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==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
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At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
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==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-27T01:07:25Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
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{{Team:TU_Munich/ExCol}}<br />
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=Limonene=<br />
<hr/><br />
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[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
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'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
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The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
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Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
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Doesn't that make you thirsty? Then you're probably an Englishman - <br />
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Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
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d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
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Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
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We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
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Furthermore, we have been able to proof the production of limonene in the beers we brewed. Hence, we have brewed iGEM's first SynBio beer containing limonene.<br />
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== Background and Principles ==<br />
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<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
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=== Biosynthesis ===<br />
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Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
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''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
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[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
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=== Molecular and Physiological Effects of Limonene ===<br />
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====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
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====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
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====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
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==Results==<br />
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===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
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RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. A drawback might be that the selection pressure might not be preserved in the gyle and hence the loss of the plasmid might be possible. Therefore, we also performed brewing experiments with yeast that carried genome integrated limonene synthase.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD.<br />
<br />
On the one hand, we have been able to brew a beer with a yeast strain that was transformed with a vector carrying a constitutive expression cassette for limonene. On the other hand, we brewed a beer with a yeast strain that carried limonene synthase after genome integration. We analyzed the beers for limonene content via headspace (SPME needle) GC-MS. We have been able to detect limonene to be produced in both beers.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-27T01:04:47Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. A drawback might be that the selection pressure might not be preserved in the gyle and hence the loss of the plasmid might be possible. Therefore, we also performed brewing experiments with yeast that carried genome integrated limonene synthase.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD.<br />
<br />
On the one hand, we have been able to brew a beer with a yeast strain that was transformed with a vector carrying a constitutive expression cassette for limonene. On the other hand, we brewed a beer with a yeast strain that carried limonene synthase after genome integration. We analyzed the beers for limonene content via headspace (SPME needle) GC-MS. We have been able to detect limonene to be produced in both beers.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-27T01:03:11Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-limonene synthase 1 (<partinfo>BBa_K801065</partinfo>) and (+)-limonene synthase 1 with yeast consensus sequence (<partinfo>BBa_K801060</partinfo>) were successfully cloned into our new yeast expression vector pTUM104. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators (<partinfo>BBa_K801062</partinfo>, <partinfo>BBa_K801063</partinfo> and <partinfo>BBa_K801064</partinfo>). <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. Furthermore, we have been able to proof the production of limonene in the beers we brewed. Further experiments regarding the concentrations of limonene produced will be carried out in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_Overviewcaffeine.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP (lane 1), [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] (lane 2) and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] (lane 3) in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]] (C) LC/MS spectra of ''in vitro'' caffeine synthesis enzyme assay, showing produced theobromine]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 theobromine synthase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed yeast cells which had previously been transformed with our caffeine synthesis expression cartridge has been done and we were successful in producing theobromine, the immediate precursor of caffein, which we detected by the use of LC/MS with multiple reaction monitoring (MRM). <br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of tyrosine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
PAL (<partinfo>BBa_K801090</partinfo>, <partinfo>BBa_K801091</partinfo>), 4CL (<partinfo>BBa_K801092</partinfo>, <partinfo>BBa_K801093</partinfo>), CHS (<partinfo>BBa_K801094</partinfo>, <partinfo>BBa_K801095</partinfo>), APT (<partinfo>BBa_K801096</partinfo> and OMT (<partinfo>BBa_K801097</partinfo>, <partinfo>BBa_K801098</partinfo>). <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization might be an interesting task for iGEM teams in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 the galactose inducible promoter pGAL1 is located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
pTUM100 (<partinfo>BBa_K801000</partinfo>), pTUM101 (<partinfo>BBa_K801001</partinfo>), pTUM102 (<partinfo>BBa_K801002</partinfo>), pTUM103 (<partinfo>BBa_K801003</partinfo>) and pTUM104 (<partinfo>BBa_K801004</partinfo>). <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol1.png|400px|thumb|right| '''A''': The KlADH4-promoter was cloned from genomic DNA of ''Kluyveromyces lactis''. The new BioBrick BBa_K801020 was inserted into our pTUM100 vector. eGFP served as a reporter gene for characterization in ''S. cerevisiae'' (plasmid name: pTUM100_KLADH4_eGFP). '''B''' Emission spectra of eGFP obtained during cultivation of ''S. cerevisiae'' transformed with pTUM100_KLADH4_eGFP using different carbon sources. Blue: Galactose was used as carbon source. The measured ethanol concentration was 1.7 % (v/v). The peak at 509 nm indicates that eGFP is expressed. Red: Glycerol was used as carbon source. The measured ethanol concentration was 0.2 % (v/v). No eGFP fluorescence could be detected.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_lightnew.png|thumb|right|450px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1<br />
<br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
Working with food, it is unacceptable to use antibiotics to keep up the selective pressure during the brewing process. Since we cannot work with auxotrophies in beer either, we have to make sure the yeast cells do not lose the plasmids harboring our BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process. The yeast strains generally employed in brewing have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle and experiments on the toxicity of the substances produced by our biosynthetic pathways were necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments on the different yeast strains show that the growth of several different yeast strains is '''not impaired in gyle'''! <br><br />
The toxicity test with the substances caffeine and limonene showed a toxicity for yeast cells at higher concentrations in the cultur media. <br><br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
At the day of the final Wiki-Freeze we could finish the brewing with three different ingredients (limonene, thaumatin and caffeine) whose biosynthesis we have engineered during this summer. This was done using yeast culture expressing BioBricks from a plasmid as well as using cultures in which the expression cassette was integrated into the genome.<br><br />
This gives us the great honor to present '''iGEM's first and finest SynBio Beer: TUM-Brew'''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-27T01:02:05Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 (<partinfo>BBa_K801065</partinfo>) and (+)-limonene synthase 1 with yeast consensus sequence (<partinfo>BBa_K801060</partinfo>) were successfully cloned into our new yeast expression vector pTUM104. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators (<partinfo>BBa_K801062</partinfo>, <partinfo>BBa_K801063</partinfo> and <partinfo>BBa_K801064</partinfo>). <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. Furthermore, we have been able to proof the production of limonene in the beers we brewed. Further experiments regarding the concentrations of limonene produced will be carried out in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_Overviewcaffeine.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP (lane 1), [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] (lane 2) and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] (lane 3) in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]] (C) LC/MS spectra of ''in vitro'' caffeine synthesis enzyme assay, showing produced theobromine]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 theobromine synthase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed yeast cells which had previously been transformed with our caffeine synthesis expression cartridge has been done and we were successful in producing theobromine, the immediate precursor of caffein, which we detected by the use of LC/MS with multiple reaction monitoring (MRM). <br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of tyrosine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
PAL (<partinfo>BBa_K801090</partinfo>, <partinfo>BBa_K801091</partinfo>), 4CL (<partinfo>BBa_K801092</partinfo>, <partinfo>BBa_K801093</partinfo>), CHS (<partinfo>BBa_K801094</partinfo>, <partinfo>BBa_K801095</partinfo>), APT (<partinfo>BBa_K801096</partinfo> and OMT (<partinfo>BBa_K801097</partinfo>, <partinfo>BBa_K801098</partinfo>). <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization might be an interesting task for iGEM teams in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 the galactose inducible promoter pGAL1 is located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
pTUM100 (<partinfo>BBa_K801000</partinfo>), pTUM101 (<partinfo>BBa_K801001</partinfo>), pTUM102 (<partinfo>BBa_K801002</partinfo>), pTUM103 (<partinfo>BBa_K801003</partinfo>) and pTUM104 (<partinfo>BBa_K801004</partinfo>). <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol1.png|400px|thumb|right| '''A''': The KlADH4-promoter was cloned from genomic DNA of ''Kluyveromyces lactis''. The new BioBrick BBa_K801020 was inserted into our pTUM100 vector. eGFP served as a reporter gene for characterization in ''S. cerevisiae'' (plasmid name: pTUM100_KLADH4_eGFP). '''B''' Emission spectra of eGFP obtained during cultivation of ''S. cerevisiae'' transformed with pTUM100_KLADH4_eGFP using different carbon sources. Blue: Galactose was used as carbon source. The measured ethanol concentration was 1.7 % (v/v). The peak at 509 nm indicates that eGFP is expressed. Red: Glycerol was used as carbon source. The measured ethanol concentration was 0.2 % (v/v). No eGFP fluorescence could be detected.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_lightnew.png|thumb|right|450px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1<br />
<br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
Working with food, it is unacceptable to use antibiotics to keep up the selective pressure during the brewing process. Since we cannot work with auxotrophies in beer either, we have to make sure the yeast cells do not lose the plasmids harboring our BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process. The yeast strains generally employed in brewing have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle and experiments on the toxicity of the substances produced by our biosynthetic pathways were necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments on the different yeast strains show that the growth of several different yeast strains is '''not impaired in gyle'''! <br><br />
The toxicity test with the substances caffeine and limonene showed a toxicity for yeast cells at higher concentrations in the cultur media. <br><br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
At the day of the final Wiki-Freeze we could finish the brewing with three different ingredients (limonene, thaumatin and caffeine) whose biosynthesis we have engineered during this summer. This was done using yeast culture expressing BioBricks from a plasmid as well as using cultures in which the expression cassette was integrated into the genome.<br><br />
This gives us the great honor to present '''iGEM's first and finest SynBio Beer: TUM-Brew'''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-27T01:01:21Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 (<partinfo>BBa_K801065</partinfo>) and (+)-limonene synthase 1 with yeast consensus sequence (<partinfo>BBa_K801060</partinfo>) were successfully cloned into our new yeast expression vector pTUM104. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators (<partinfo>BBa_K801062</partinfo>, <partinfo>BBa_K801063</partinfo> and <partinfo>BBa_K801064</partinfo>). <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. Furthermore, we have been able to proof the production of limonene in the beers we brewed. Further experiments regarding the concentrations of limonene produced will be carried out in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_Overviewcaffeine.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP (lane 1), [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] (lane 2) and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] (lane 3) in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]] (C) LC/MS spectra of ''in vitro'' caffeine synthesis enzyme assay, showing produced theobromine]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 theobromine synthase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed yeast cells which had previously been transformed with our caffeine synthesis expression cartridge has been done and we were successful in producing theobromine, the immediate precursor of caffein, which we detected by the use of LC/MS with multiple reaction monitoring (MRM). <br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of tyrosine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
PAL (<partinfo>BBa_K801090</partinfo>, <partinfo>BBa_K801091</partinfo>), 4CL (<partinfo>BBa_K801092</partinfo>, <partinfo>BBa_K801093</partinfo>), CHS (<partinfo>BBa_K801094</partinfo>, <partinfo>BBa_K801095</partinfo>), APT (<partinfo>BBa_K801096</partinfo> and OMT (<partinfo>BBa_K801097</partinfo>, <partinfo>BBa_K801098</partinfo>). <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization might be an interesting task for iGEM teams in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 the galactose inducible promoter pGAL1 is located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
pTUM100 (<partinfo>BBa_K801000</partinfo>), pTUM101 (<partinfo>BBa_K801001</partinfo>), pTUM102 (<partinfo>BBa_K801002</partinfo>), pTUM103 (<partinfo>BBa_K801003</partinfo>) and pTUM104 (<partinfo>BBa_K801004</partinfo>). <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol1.png|400px|thumb|right| '''A''': The KlADH4-promoter was cloned from genomic DNA of ''Kluyveromyces lactis''. The new BioBrick BBa_K801020 was inserted into our pTUM100 vector. eGFP served as a reporter gene for characterization in ''S. cerevisiae'' (plasmid name: pTUM100_KLADH4_eGFP). '''B''' Emission spectra of eGFP obtained during cultivation of ''S. cerevisiae'' transformed with pTUM100_KLADH4_eGFP using different carbon sources. Blue: Galactose was used as carbon source. The measured ethanol concentration was 1.7 % (v/v). The peak at 509 nm indicates that eGFP is expressed. Red: Glycerol was used as carbon source. The measured ethanol concentration was 0.2 % (v/v). No eGFP fluorescence could be detected.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_lightnew.png|thumb|right|450px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1<br />
<br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
Working with food, it is unacceptable to use antibiotics to keep up the selective pressure during the brewing process. Since we cannot work with auxotrophies in beer either, we have to make sure the yeast cells do not lose the plasmids harboring our BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process. The yeast strains generally employed in brewing have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle and experiments on the toxicity of the substances produced by our biosynthetic pathways were necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments on the different yeast strains show that the growth of several different yeast strains is '''not impaired in gyle'''! <br><br />
The toxicity test with the substances caffeine and limonene showed a toxicity for yeast cells at higher concentrations in the cultur media. <br><br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
At the day of the final Wiki-Freeze we could finish the brewing with three different ingredients (limonene, thaumatin and caffeine) whose biosynthesis we have engineered during this summer. This was done using yeast culture expressing BioBricks from a plasmid as well as using cultures in which the expression cassette was integrated into the genome.<br><br />
This gives us the great honor to present '''iGEM's first and finest SynBio Beer: TUM-Brew'''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-27T00:58:14Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 (<partinfo>BBa_K801065</partinfo>) and (+)-limonene synthase 1 with yeast consensus sequence (<partinfo>BBa_K801060</partinfo>) were successfully cloned into our new yeast expression vector pTUM104. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators (<partinfo>BBa_K801062</partinfo>, <partinfo>BBa_K801063</partinfo> and <partinfo>BBa_K801064</partinfo>). <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. Furthermore, we have been able to detect limonene to be produced in the beers we brewed. Further experiments regarding the concentrations of limonene produced will be carried out in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_Overviewcaffeine.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP (lane 1), [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] (lane 2) and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] (lane 3) in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]] (C) LC/MS spectra of ''in vitro'' caffeine synthesis enzyme assay, showing produced theobromine]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 theobromine synthase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed yeast cells which had previously been transformed with our caffeine synthesis expression cartridge has been done and we were successful in producing theobromine, the immediate precursor of caffein, which we detected by the use of LC/MS with multiple reaction monitoring (MRM). <br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of tyrosine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
PAL (<partinfo>BBa_K801090</partinfo>, <partinfo>BBa_K801091</partinfo>), 4CL (<partinfo>BBa_K801092</partinfo>, <partinfo>BBa_K801093</partinfo>), CHS (<partinfo>BBa_K801094</partinfo>, <partinfo>BBa_K801095</partinfo>), APT (<partinfo>BBa_K801096</partinfo> and OMT (<partinfo>BBa_K801097</partinfo>, <partinfo>BBa_K801098</partinfo>). <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization might be an interesting task for iGEM teams in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 the galactose inducible promoter pGAL1 is located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
pTUM100 (<partinfo>BBa_K801000</partinfo>), pTUM101 (<partinfo>BBa_K801001</partinfo>), pTUM102 (<partinfo>BBa_K801002</partinfo>), pTUM103 (<partinfo>BBa_K801003</partinfo>) and pTUM104 (<partinfo>BBa_K801004</partinfo>). <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol1.png|400px|thumb|right| '''A''': The KlADH4-promoter was cloned from genomic DNA of ''Kluyveromyces lactis''. The new BioBrick BBa_K801020 was inserted into our pTUM100 vector. eGFP served as a reporter gene for characterization in ''S. cerevisiae'' (plasmid name: pTUM100_KLADH4_eGFP). '''B''' Emission spectra of eGFP obtained during cultivation of ''S. cerevisiae'' transformed with pTUM100_KLADH4_eGFP using different carbon sources. Blue: Galactose was used as carbon source. The measured ethanol concentration was 1.7 % (v/v). The peak at 509 nm indicates that eGFP is expressed. Red: Glycerol was used as carbon source. The measured ethanol concentration was 0.2 % (v/v). No eGFP fluorescence could be detected.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_lightnew.png|thumb|right|450px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1<br />
<br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
Working with food, it is unacceptable to use antibiotics to keep up the selective pressure during the brewing process. Since we cannot work with auxotrophies in beer either, we have to make sure the yeast cells do not lose the plasmids harboring our BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process. The yeast strains generally employed in brewing have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle and experiments on the toxicity of the substances produced by our biosynthetic pathways were necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments on the different yeast strains show that the growth of several different yeast strains is '''not impaired in gyle'''! <br><br />
The toxicity test with the substances caffeine and limonene showed a toxicity for yeast cells at higher concentrations in the cultur media. <br><br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
At the day of the final Wiki-Freeze we could finish the brewing with three different ingredients (limonene, thaumatin and caffeine) whose biosynthesis we have engineered during this summer. This was done using yeast culture expressing BioBricks from a plasmid as well as using cultures in which the expression cassette was integrated into the genome.<br><br />
This gives us the great honor to present '''iGEM's first and finest SynBio Beer: TUM-Brew'''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-27T00:54:34Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-Limonene synthase 1 (<partinfo>BBa_K801065</partinfo>) and (+)-limonene synthase 1 with yeast consensus sequence (<partinfo>BBa_K801060</partinfo>) were successfully cloned into our new yeast expression vector pTUM104. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators (<partinfo>BBa_K801062</partinfo>, <partinfo>BBa_K801063</partinfo> and <partinfo>BBa_K801064</partinfo>). <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. The last remaining step to a SynBio beer would be the integration of our gene constructs <partinfo>BBa_K801062</partinfo>, <partinfo>BBa_K801063</partinfo> and <partinfo>BBa_K801064</partinfo> into the yeast genome.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_Overviewcaffeine.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP (lane 1), [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] (lane 2) and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] (lane 3) in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]] (C) LC/MS spectra of ''in vitro'' caffeine synthesis enzyme assay, showing produced theobromine]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 theobromine synthase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed yeast cells which had previously been transformed with our caffeine synthesis expression cartridge has been done and we were successful in producing theobromine, the immediate precursor of caffein, which we detected by the use of LC/MS with multiple reaction monitoring (MRM). <br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of tyrosine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
PAL (<partinfo>BBa_K801090</partinfo>, <partinfo>BBa_K801091</partinfo>), 4CL (<partinfo>BBa_K801092</partinfo>, <partinfo>BBa_K801093</partinfo>), CHS (<partinfo>BBa_K801094</partinfo>, <partinfo>BBa_K801095</partinfo>), APT (<partinfo>BBa_K801096</partinfo> and OMT (<partinfo>BBa_K801097</partinfo>, <partinfo>BBa_K801098</partinfo>). <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization might be an interesting task for iGEM teams in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 the galactose inducible promoter pGAL1 is located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
pTUM100 (<partinfo>BBa_K801000</partinfo>), pTUM101 (<partinfo>BBa_K801001</partinfo>), pTUM102 (<partinfo>BBa_K801002</partinfo>), pTUM103 (<partinfo>BBa_K801003</partinfo>) and pTUM104 (<partinfo>BBa_K801004</partinfo>). <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol1.png|400px|thumb|right| '''A''': The KlADH4-promoter was cloned from genomic DNA of ''Kluyveromyces lactis''. The new BioBrick BBa_K801020 was inserted into our pTUM100 vector. eGFP served as a reporter gene for characterization in ''S. cerevisiae'' (plasmid name: pTUM100_KLADH4_eGFP). '''B''' Emission spectra of eGFP obtained during cultivation of ''S. cerevisiae'' transformed with pTUM100_KLADH4_eGFP using different carbon sources. Blue: Galactose was used as carbon source. The measured ethanol concentration was 1.7 % (v/v). The peak at 509 nm indicates that eGFP is expressed. Red: Glycerol was used as carbon source. The measured ethanol concentration was 0.2 % (v/v). No eGFP fluorescence could be detected.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_lightnew.png|thumb|right|450px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1<br />
<br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
Working with food, it is unacceptable to use antibiotics to keep up the selective pressure during the brewing process. Since we cannot work with auxotrophies in beer either, we have to make sure the yeast cells do not lose the plasmids harboring our BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process. The yeast strains generally employed in brewing have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle and experiments on the toxicity of the substances produced by our biosynthetic pathways were necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments on the different yeast strains show that the growth of several different yeast strains is '''not impaired in gyle'''! <br><br />
The toxicity test with the substances caffeine and limonene showed a toxicity for yeast cells at higher concentrations in the cultur media. <br><br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
At the day of the final Wiki-Freeze we could finish the brewing with three different ingredients (limonene, thaumatin and caffeine) whose biosynthesis we have engineered during this summer. This was done using yeast culture expressing BioBricks from a plasmid as well as using cultures in which the expression cassette was integrated into the genome.<br><br />
This gives us the great honor to present '''iGEM's first and finest SynBio Beer: TUM-Brew'''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-27T00:50:09Z<p>Larakuntz: /* Detection of Limonene in Beer */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. A drawback might be that the selection pressure might not be preserved in the gyle and hence the loss of the plasmid might be possible. Therefore, we also performed brewing experiments with yeast that carried genome integrated limonene synthase.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD.<br />
<br />
On the one hand, we have been able to brew a beer with a yeast strain that was transformed with a vector carrying a constitutive expression cassette for limonene. On the other hand, we brewed a beer with a yeast strain that carried limonene synthase after genome integration. We analyzed the beers for limonene content via headspace (SPME needle) GC-MS. We have been able to detect limonene to be produced in both beers.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-27T00:43:36Z<p>Larakuntz: /* Detection of Limonene in Beer */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We brewed a beer with a yeast strain that was transformed with a vector carrying a constitutive expression cassette on the one hand. On the other hand, we brewed with a yeast strain that carried limonene synthase after genome integration. We analyzed the beers for limonene content via headspace (SPME needle) GC-MS. We have been able to detect limonene to be produced in both beers.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-27T00:40:33Z<p>Larakuntz: /* Detection of Limonene in Beer */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We brewed a beer with a yeast strain that was transformed with a vector carrying a constitutive expression cassette on the one hand. On the other hand, we brewed with a yeast strain that had limonene synthase integrated into its genome. We analyzed the beers for limonene content via headspace (SPME needle) GC-MS. We have been able to detect limonene to be produced both beers.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Team/MembersTeam:TU Munich/Team/Members2012-10-26T16:39:03Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
__NOTOC__<br />
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<head><br />
<br />
<script style="text/js"><br />
$(document).ready(function(){<br />
$(".coumaryl").addClass("team");<br />
$(".limonene").addClass("team");<br />
$(".thaumatin").addClass("team");<br />
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<br />
=Team TU Munich=<br />
<hr/><br />
[[File:TUM12 Gruppenfoto 1.jpg|right|400px]]<br />
<br />
<br />
'''Griaß Eich!'''<br />
<br>'''Welcome to our team page!''' In the following we would like to present to you the mothers of brewing,<br />
the godfathers of drinking and the awkward uncles of inappropriate drunk jokes. We give you:<br />
<br />
The '''iGEM Team of the Technische Universität München 2012'''!<br />
<br />
<br />
== Students ==<br />
<hr/><br />
<br />
<div class="thaumatin"><br />
=== Thaumatin ===<br />
<div class="mleft"><br />
[[File:Alois_einzel_TUM12.jpg|left|150px]]<br />
====Alois Bräuer==== <br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;6th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 23<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Super Perforator<br />
<br>&nbsp;&nbsp;'''''BAC:''''' 12ng/µl<br />
</div><br />
<div class="mright"><br />
[[File:Martin_einzel_TUM12.jpg|right|150px]]<br />
====Martin Schappert==== <br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;6th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 22<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Hipster<br />
<br>&nbsp;&nbsp;'''''Achievement:''''' Not a single f*ck was<br />
<br>&nbsp;&nbsp;given that day<br />
</div></div><br />
<br />
<br />
<div class="limonene"><br />
<br />
=== Limonene ===<br />
<hr/><br />
<div class="mleft"><br />
[[File:Andrea_einzel_TUM12.jpg|left|150px]]<br />
====Andrea Richter====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 23<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Good Girl Gina<br />
<br>&nbsp;&nbsp;'''''Favourite SOP:''''' Mini prep<br />
</div><br />
<div class="mright"><br />
[[File:Lara_einzel_TUM12.jpg|right|150px]]<br />
<br />
====Lara Kuntz====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;4th Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 24<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Merry Margaret<br />
<br>&nbsp;&nbsp;'''''Hobbies:''''' Carpooling<br />
</div><br />
</div><br />
<br />
<div class="coumaryl"><br />
<br />
=== Xanthohumol ===<br />
<hr/><br />
<div class="mleft"><br />
[[File:Daniela_einzel_TUM12.jpg|left|150px]]<br />
====Daniela Dichtler====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 23<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The special One<br />
<br>&nbsp;&nbsp;'''''Motivation:''''' Profound<br />
</div><br />
<div class="mright"><br />
[[File:Ingmar_einzel_TUM12.jpg|right|150px]]<br />
<br />
====Ingmar Polte====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;4th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 21<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The go-to Guy <br />
<br>&nbsp;&nbsp;'''''Achievement:''''' Clultiple mosing nite<br />
</div><br />
<div class="mleft"><br />
[[File:Mary_einzel_TUM12.jpg|left|150px]]<br />
====Maria Trumpfheller==== <br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;4th Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 26<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The oldie woman <br />
<br>&nbsp;&nbsp;'''''Motivation Lvl:''''' over 9000 <br />
<br>&nbsp;&nbsp;"Why isn't anybody in the lab?"<br />
</div><br />
<br />
<div class="mright"><br />
[[File:Katrin_einzel_TUM12.jpg|right|150px]]<br />
<br />
====Katrin Fischer====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 20<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Pup<br />
<br>&nbsp;&nbsp;'''''BAC:''''' Minor<br />
</div><br />
</div><br />
<br />
<div class="caffeine"><br />
<br />
=== Caffeine ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Denis_einzel_TUM12.jpg|left|150px]]<br />
====Dennis Hell====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 25<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' Best rapper in town!<br />
</div><br />
<br />
<div class="mright"><br />
[[File:Roman_einzel_TUM12.jpg|right|150px]]<br />
<br />
====Roman Prechtl==== <br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;6th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 23<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Bavarian<br />
<br>&nbsp;&nbsp;'''''Quote:''''' "Woid i mai Bià weàma mi'm<br />
<br>&nbsp;&nbsp;Biàweàma, wa's Bià weàma wià da<br />
<br>&nbsp;&nbsp;Biàweàma"<br />
</div><br />
<br />
<div class="mleft"><br />
[[File:Saskia_einzel_TUM12.jpg|left|150px]]<br />
<br />
====Saskia König==== <br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 24<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Ginger<br />
<br>&nbsp;&nbsp;'''''Favourite garment:''''' Lab coat and a<br />
<br>&nbsp;&nbsp;hint of Chanel No.5<br />
</div><br />
</div><br />
<br />
<div class="light_switchable_promoter bezel mleft" style="margin:5px 0px;"><br />
<br />
=== Light switchable promoter ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Jeff_einzel_TUM12.jpg|left|150px]]<br />
====Jim Panse====<br />
&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;1st Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 7<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The strange guy from<br />
<br>&nbsp;&nbsp;Far&nbsp;East.<br />
<br>&nbsp;&nbsp;白人看不懂 =D...<br />
</div><br />
</div><br />
<br />
<div class="constitutive_promoter bezel mright" style="margin:5px 5px;"><br />
<br />
=== Constitutive promoters ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Georg_einzel_TUM12.jpg|left|150px]]<br />
====Georg "Schorsch" Schützinger====<br />
&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Biology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 27<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The lonesome Ranger<br />
<br>&nbsp;&nbsp;'''''Place to be:''''' Electrophoresis room<br />
</div><br />
</div><br />
<br />
<div class="ethanol_inducible_promoter bezel mleft" style="margin:5px 0px;"><br />
<br />
=== Alcohol-inducible promoter ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Simon_einzel_TUM12.jpg|left|150px]]<br />
====Simon Heinze====<br />
&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;6th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 22<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Bachelor<br />
<br>&nbsp;&nbsp;'''''Last request:''''' "May the person, who<br />
<br>&nbsp;&nbsp;has taken the Not1 return it by the <br />
<br>&nbsp;&nbsp;end of the week?"<br />
</div><br />
</div><br />
<br />
<div class="hrns"><br />
<br />
=== Human Practice and Sponsoring ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:David_einzel_TUM12.jpg|left|150px]]<br />
====David Wehner====<br />
<br>&nbsp;&nbsp;'''''Subjects of study:''''' <br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;4th Semester <br />
<br>&nbsp;&nbsp;TUM-BWL, Diploma,<br />
<br>&nbsp;&nbsp; 10th Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 25<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Godfather<br />
<br>&nbsp;&nbsp;'''''Budget:''''' Infinite<br />
</div><br />
<br />
<div class="mright"><br />
[[File:Jara_einzel_TUM12.jpg|right|150px]]<br />
====Jara Obermann====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 23<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The public relations Lady<br />
<br>&nbsp;&nbsp;'''''BAC:''''' Stone-cold sober<br />
</div><br />
<br />
<div class="mleft"><br />
[[File:Nadine_einzel_TUM12.jpg|left|150px]]<br />
<br />
====Nadine Gerstenberg====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Biology, B. Sc.<br />
<br>&nbsp;&nbsp;4th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 21<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Teacher<br />
<br>&nbsp;&nbsp;'''''Place to be:''''' At home<br />
</div><br />
</div><br />
<br />
<div class="theory bezel mright"><br />
<br />
=== Brewing and RFC ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Volker_einzel_TUM12.jpg|left|150px]]<br />
====Volker Morath====<br />
&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Diploma Thesis at Skerra lab<br />
<br>&nbsp;&nbsp;12th Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 25<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The King of Pop<br />
</div> <br />
</div><br />
<br />
<div class="modeling bezel mleft"><br />
<br />
=== Modeling ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Fabian_einzel_TUM12.jpg|left|150px]]<br />
====Fabian Froehlich==== <br />
&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Mathematics in Bioscience, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 22<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Konrad Zuse<br />
<br>&nbsp;&nbsp;'''''Rank:''''' Veteran<br />
</div> <br />
</div><br />
<br />
==Advisor==<br />
<hr/><br />
<div class="bezel mfull"><br />
[[File:TUM12_ASK.png|left|150px]]<br />
====[http://www.lrz.de/~Biologische-Chemie/ProfSkerra/Skerra.html Prof. Dr. Arne Skerra]==== <br />
Since 1998 Prof. Dr. Arne Skerra is Full Professor at the [[http://www.tum.de Technische Universität München]], where he heads the [[http://www.lrz.de/~Biologische-Chemie/index.html Institute of Biological Chemistry]]. He is internationally renowned for his comprehensive experience and his pioneering contributions in the fields of molecular biotechnology and protein engineering. In addition to his scientific excellence he strives to apply and economically utilize academic results, and to date he founded two start-ups. He advises our iGEM team scientifically and offers laboratory places at his chair.<br />
<br />
<br>&nbsp;&nbsp;<br />
</div><br />
<br />
==Instructors==<br />
<hr/><br />
<div class="bezel mfull"><br />
Doreen Schiller <br />
<br />
• Position: PhD Student<br />
<br />
Fong- Chin Huang <br />
<br />
• Position: Post Doc <br />
<br />
Katrin Franz <br />
<br />
• Position: PhD Student<br />
</div></div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Team/MembersTeam:TU Munich/Team/Members2012-10-26T16:33:18Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
__NOTOC__<br />
<html><br />
<head><br />
<br />
<script style="text/js"><br />
$(document).ready(function(){<br />
$(".coumaryl").addClass("team");<br />
$(".limonene").addClass("team");<br />
$(".thaumatin").addClass("team");<br />
$(".caffeine").addClass("team");<br />
$(".hrns").addClass("team");<br />
$(".team").addClass("ui-corner-all");<br />
});<br />
</script><br />
</head><br />
</html><br />
<br />
=Team TU Munich=<br />
<hr/><br />
[[File:TUM12 Gruppenfoto 1.jpg|right|400px]]<br />
<br />
<br />
'''Griaß Eich!'''<br />
<br>'''Welcome to our team page!''' In the following we would like to present to you the mothers of brewing,<br />
the godfathers of drinking and the awkward uncles of inappropriate drunk jokes. We give you:<br />
<br />
The '''iGEM Team of the Technische Universität München 2012'''!<br />
<br />
<br />
== Students ==<br />
<hr/><br />
<br />
<div class="thaumatin"><br />
=== Thaumatin ===<br />
<div class="mleft"><br />
[[File:Alois_einzel_TUM12.jpg|left|150px]]<br />
====Alois Bräuer==== <br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;6th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 23<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Super Perforator<br />
<br>&nbsp;&nbsp;'''''BAC:''''' 12ng/µl<br />
</div><br />
<div class="mright"><br />
[[File:Martin_einzel_TUM12.jpg|right|150px]]<br />
====Martin Schappert==== <br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;6th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 22<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Hipster<br />
<br>&nbsp;&nbsp;'''''Achievement:''''' Not a single f*ck was<br />
<br>&nbsp;&nbsp;given that day<br />
</div></div><br />
<br />
<br />
<div class="limonene"><br />
<br />
=== Limonene ===<br />
<hr/><br />
<div class="mleft"><br />
[[File:Andrea_einzel_TUM12.jpg|left|150px]]<br />
====Andrea Richter====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 23<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Good Girl Gina<br />
<br>&nbsp;&nbsp;'''''Favourite SOP:''''' Mini prep<br />
</div><br />
<div class="mright"><br />
[[File:Lara_einzel_TUM12.jpg|right|150px]]<br />
<br />
====Lara Kuntz====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;4th Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 24<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Margaret<br />
<br>&nbsp;&nbsp;'''''Hobbies:''''' Carpooling<br />
</div><br />
</div><br />
<br />
<div class="coumaryl"><br />
<br />
=== Xanthohumol ===<br />
<hr/><br />
<div class="mleft"><br />
[[File:Daniela_einzel_TUM12.jpg|left|150px]]<br />
====Daniela Dichtler====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 23<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The special One<br />
<br>&nbsp;&nbsp;'''''Motivation:''''' Profound<br />
</div><br />
<div class="mright"><br />
[[File:Ingmar_einzel_TUM12.jpg|right|150px]]<br />
<br />
====Ingmar Polte====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;4th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 21<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The go-to Guy <br />
<br>&nbsp;&nbsp;'''''Achievement:''''' Clultiple mosing nite<br />
</div><br />
<div class="mleft"><br />
[[File:Mary_einzel_TUM12.jpg|left|150px]]<br />
====Maria Trumpfheller==== <br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;4th Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 26<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The oldie woman <br />
<br>&nbsp;&nbsp;'''''Motivation Lvl:''''' over 9000 <br />
<br>&nbsp;&nbsp;"Why isn't anybody in the lab?"<br />
</div><br />
<br />
<div class="mright"><br />
[[File:Katrin_einzel_TUM12.jpg|right|150px]]<br />
<br />
====Katrin Fischer====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 20<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Pup<br />
<br>&nbsp;&nbsp;'''''BAC:''''' Minor<br />
</div><br />
</div><br />
<br />
<div class="caffeine"><br />
<br />
=== Caffeine ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Denis_einzel_TUM12.jpg|left|150px]]<br />
====Dennis Hell====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 25<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' Best rapper in town!<br />
</div><br />
<br />
<div class="mright"><br />
[[File:Roman_einzel_TUM12.jpg|right|150px]]<br />
<br />
====Roman Prechtl==== <br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;6th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 23<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Bavarian<br />
<br>&nbsp;&nbsp;'''''Quote:''''' "Woid i mai Bià weàma mi'm<br />
<br>&nbsp;&nbsp;Biàweàma, wa's Bià weàma wià da<br />
<br>&nbsp;&nbsp;Biàweàma"<br />
</div><br />
<br />
<div class="mleft"><br />
[[File:Saskia_einzel_TUM12.jpg|left|150px]]<br />
<br />
====Saskia König==== <br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 24<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Ginger<br />
<br>&nbsp;&nbsp;'''''Favourite garment:''''' Lab coat and a<br />
<br>&nbsp;&nbsp;hint of Chanel No.5<br />
</div><br />
</div><br />
<br />
<div class="light_switchable_promoter bezel mleft" style="margin:5px 0px;"><br />
<br />
=== Light switchable promoter ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Jeff_einzel_TUM12.jpg|left|150px]]<br />
====Jim Panse====<br />
&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;1st Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 7<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The strange guy from<br />
<br>&nbsp;&nbsp;Far&nbsp;East.<br />
<br>&nbsp;&nbsp;白人看不懂 =D...<br />
</div><br />
</div><br />
<br />
<div class="constitutive_promoter bezel mright" style="margin:5px 5px;"><br />
<br />
=== Constitutive promoters ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Georg_einzel_TUM12.jpg|left|150px]]<br />
====Georg "Schorsch" Schützinger====<br />
&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Biology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 27<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The lonesome Ranger<br />
<br>&nbsp;&nbsp;'''''Place to be:''''' Electrophoresis room<br />
</div><br />
</div><br />
<br />
<div class="ethanol_inducible_promoter bezel mleft" style="margin:5px 0px;"><br />
<br />
=== Alcohol-inducible promoter ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Simon_einzel_TUM12.jpg|left|150px]]<br />
====Simon Heinze====<br />
&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;6th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 22<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Bachelor<br />
<br>&nbsp;&nbsp;'''''Last request:''''' "May the person, who<br />
<br>&nbsp;&nbsp;has taken the Not1 return it by the <br />
<br>&nbsp;&nbsp;end of the week?"<br />
</div><br />
</div><br />
<br />
<div class="hrns"><br />
<br />
=== Human Practice and Sponsoring ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:David_einzel_TUM12.jpg|left|150px]]<br />
====David Wehner====<br />
<br>&nbsp;&nbsp;'''''Subjects of study:''''' <br />
<br>&nbsp;&nbsp;Molecular Biotechnology, B. Sc.<br />
<br>&nbsp;&nbsp;4th Semester <br />
<br>&nbsp;&nbsp;TUM-BWL, Diploma,<br />
<br>&nbsp;&nbsp; 10th Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 25<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Godfather<br />
<br>&nbsp;&nbsp;'''''Budget:''''' Infinite<br />
</div><br />
<br />
<div class="mright"><br />
[[File:Jara_einzel_TUM12.jpg|right|150px]]<br />
====Jara Obermann====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Molecular Biotechnology, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 23<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The public relations Lady<br />
<br>&nbsp;&nbsp;'''''BAC:''''' Stone-cold sober<br />
</div><br />
<br />
<div class="mleft"><br />
[[File:Nadine_einzel_TUM12.jpg|left|150px]]<br />
<br />
====Nadine Gerstenberg====<br />
<br>&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Biology, B. Sc.<br />
<br>&nbsp;&nbsp;4th Semester<br />
<br>&nbsp;&nbsp;'''''Age:''''' 21<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Teacher<br />
<br>&nbsp;&nbsp;'''''Place to be:''''' At home<br />
</div><br />
</div><br />
<br />
<div class="theory bezel mright"><br />
<br />
=== Brewing and RFC ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Volker_einzel_TUM12.jpg|left|150px]]<br />
====Volker Morath====<br />
&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Diploma Thesis at Skerra lab<br />
<br>&nbsp;&nbsp;12th Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 25<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The King of Pop<br />
</div> <br />
</div><br />
<br />
<div class="modeling bezel mleft"><br />
<br />
=== Modeling ===<br />
<hr/><br />
<br />
<div class="mleft"><br />
[[File:Fabian_einzel_TUM12.jpg|left|150px]]<br />
====Fabian Froehlich==== <br />
&nbsp;&nbsp;'''''Subject of study:'''''<br />
<br>&nbsp;&nbsp;Mathematics in Bioscience, M. Sc.<br />
<br>&nbsp;&nbsp;2nd Semester <br />
<br>&nbsp;&nbsp;'''''Age:''''' 22<br />
<br>&nbsp;&nbsp;'''''Team Role:''''' The Konrad Zuse<br />
<br>&nbsp;&nbsp;'''''Rank:''''' Veteran<br />
</div> <br />
</div><br />
<br />
==Advisor==<br />
<hr/><br />
<div class="bezel mfull"><br />
[[File:TUM12_ASK.png|left|150px]]<br />
====[http://www.lrz.de/~Biologische-Chemie/ProfSkerra/Skerra.html Prof. Dr. Arne Skerra]==== <br />
Since 1998 Prof. Dr. Arne Skerra is Full Professor at the [[http://www.tum.de Technische Universität München]], where he heads the [[http://www.lrz.de/~Biologische-Chemie/index.html Institute of Biological Chemistry]]. He is internationally renowned for his comprehensive experience and his pioneering contributions in the fields of molecular biotechnology and protein engineering. In addition to his scientific excellence he strives to apply and economically utilize academic results, and to date he founded two start-ups. He advises our iGEM team scientifically and offers laboratory places at his chair.<br />
<br />
<br>&nbsp;&nbsp;<br />
</div><br />
<br />
==Instructors==<br />
<hr/><br />
<div class="bezel mfull"><br />
Doreen Schiller <br />
<br />
• Position: PhD Student<br />
<br />
Fong- Chin Huang <br />
<br />
• Position: Post Doc <br />
<br />
Katrin Franz <br />
<br />
• Position: PhD Student<br />
</div></div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-26T16:23:06Z<p>Larakuntz: /* Outlook: Characterization of Enzymatic Activity */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We analyzed this first beer for limonene content via headspace (SPME needle) GC-MS. Unfortunately we have not yet been able to proof a significant difference between the beer supposed to contain enhanced amounts of limonene and the negative control beer. This is probably due to a loss of the plasmid which encodes limonene synthase. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will repeat the experiment.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==== Outlook: Characterization of Enzymatic Activity====<br />
[[file:TUM12_pyrophosphate.png|thumb|center|600px| Fig. 13: Enzymatic assay that will be used to characterize limonene synthase. The principle is well known from pyrosequencing and couples the production of pyrophosphate to the luminescence produced by the firefly luciferase being dependent on ATP.]]<br />
<div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-26T16:22:06Z<p>Larakuntz: /* Detection of Limonene in Beer */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We analyzed this first beer for limonene content via headspace (SPME needle) GC-MS. Unfortunately we have not yet been able to proof a significant difference between the beer supposed to contain enhanced amounts of limonene and the negative control beer. This is probably due to a loss of the plasmid which encodes limonene synthase. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will repeat the experiment.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM101 with limonene synthase. Green: Beer brewed with yeast that supposedly carries limonene synthase in genome (after genome integration).]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==== Outlook: Characterization of Enzymatic Activity====<br />
[[file:TUM12_pyrophosphate.png|thumb|center|600px| Figure 13: Enzymatic assay that will be used to characterize limonene synthase. The principle is well known from pyrosequencing and couples the production of pyrophosphate to the luminescence produced by the firefly luciferase being dependent on ATP.]]<br />
<div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-26T16:16:54Z<p>Larakuntz: /* Detection of Limonene in Beer */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We analyzed this first beer for limonene content via headspace (SPME needle) GC-MS. Unfortunately we have not yet been able to proof a significant difference between the beer supposed to contain enhanced amounts of limonene and the negative control beer. This is probably due to a loss of the plasmid which encodes limonene synthase. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will repeat the experiment.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectra obtained from GC-MS analysis of brewed beer. Brown: Beer brewed with yeast that carries pTUM ]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==== Outlook: Characterization of Enzymatic Activity====<br />
[[file:TUM12_pyrophosphate.png|thumb|center|600px| Figure 13: Enzymatic assay that will be used to characterize limonene synthase. The principle is well known from pyrosequencing and couples the production of pyrophosphate to the luminescence produced by the firefly luciferase being dependent on ATP.]]<br />
<div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-26T16:13:57Z<p>Larakuntz: /* Detection of Limonene in Beer */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We analyzed this first beer for limonene content via headspace (SPME needle) GC-MS. Unfortunately we have not yet been able to proof a significant difference between the beer supposed to contain enhanced amounts of limonene and the negative control beer. This is probably due to a loss of the plasmid which encodes limonene synthase. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will repeat the experiment.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Fig. 11: GC spectrum of GC-MS analysis of brewed beer. ]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==== Outlook: Characterization of Enzymatic Activity====<br />
[[file:TUM12_pyrophosphate.png|thumb|center|600px| Figure 13: Enzymatic assay that will be used to characterize limonene synthase. The principle is well known from pyrosequencing and couples the production of pyrophosphate to the luminescence produced by the firefly luciferase being dependent on ATP.]]<br />
<div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-26T16:13:06Z<p>Larakuntz: /* Outlook: Characterization of Enzymatic Activity */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We analyzed this first beer for limonene content via headspace (SPME needle) GC-MS. Unfortunately we have not yet been able to proof a significant difference between the beer supposed to contain enhanced amounts of limonene and the negative control beer. This is probably due to a loss of the plasmid which encodes limonene synthase. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will repeat the experiment.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Bildbeschriftung]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==== Outlook: Characterization of Enzymatic Activity====<br />
[[file:TUM12_pyrophosphate.png|thumb|center|600px| Figure 13: Enzymatic assay that will be used to characterize limonene synthase. The principle is well known from pyrosequencing and couples the production of pyrophosphate to the luminescence produced by the firefly luciferase being dependent on ATP.]]<br />
<div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-26T16:12:50Z<p>Larakuntz: /* Toxicity Assay */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We analyzed this first beer for limonene content via headspace (SPME needle) GC-MS. Unfortunately we have not yet been able to proof a significant difference between the beer supposed to contain enhanced amounts of limonene and the negative control beer. This is probably due to a loss of the plasmid which encodes limonene synthase. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will repeat the experiment.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Bildbeschriftung]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 12: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==== Outlook: Characterization of Enzymatic Activity====<br />
[[file:TUM12_pyrophosphate.png|thumb|center|600px| Figure 12: Enzymatic assay that will be used to characterize limonene synthase. The principle is well known from pyrosequencing and couples the production of pyrophosphate to the luminescence produced by the firefly luciferase being dependent on ATP.]]<br />
<div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-26T16:12:24Z<p>Larakuntz: /* Outlook: Characterizing Enzymatic Activity */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We analyzed this first beer for limonene content via headspace (SPME needle) GC-MS. Unfortunately we have not yet been able to proof a significant difference between the beer supposed to contain enhanced amounts of limonene and the negative control beer. This is probably due to a loss of the plasmid which encodes limonene synthase. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will repeat the experiment.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Bildbeschriftung]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 11: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==== Outlook: Characterization of Enzymatic Activity====<br />
[[file:TUM12_pyrophosphate.png|thumb|center|600px| Figure 12: Enzymatic assay that will be used to characterize limonene synthase. The principle is well known from pyrosequencing and couples the production of pyrophosphate to the luminescence produced by the firefly luciferase being dependent on ATP.]]<br />
<div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-26T16:06:30Z<p>Larakuntz: /* Limonene */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A Common Saying Is "If Life Gives You Lemons - Make Lemonade". For Us It Is Rather "If Life Gives You Limonene - Make Beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of Yeast Consensus Sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of Recombinant Limonene Synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel Filtration of Purified Protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In Vitro Detection of Limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In Vivo Detection of Limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of Limonene in Beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We analyzed this first beer for limonene content via headspace (SPME needle) GC-MS. Unfortunately we have not yet been able to proof a significant difference between the beer supposed to contain enhanced amounts of limonene and the negative control beer. This is probably due to a loss of the plasmid which encodes limonene synthase. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will repeat the experiment.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Bildbeschriftung]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 11: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==== Outlook: Characterizing Enzymatic Activity====<br />
[[file:TUM12_pyrophosphate.png|thumb|center|600px| Figure 12: Enzymatic assay that will be used to characterize limonene synthase. The principle is well known from pyrosequencing and couples the production of pyrophosphate to the luminescence produced by the firefly luciferase being dependent on ATP]]<br />
<div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-26T16:03:51Z<p>Larakuntz: /* Gel picture of finished constructs */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A common saying is "If life gives you lemons - make lemonade". For us it is rather "If life gives you limonene - make beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel Picture of Finished Constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of the yeast consensus sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of recombinant limonene synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel filtration of purified protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In vitro detection of limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In vivo detection of limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of limonene in beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We analyzed this first beer for limonene content via headspace (SPME needle) GC-MS. Unfortunately we have not yet been able to proof a significant difference between the beer supposed to contain enhanced amounts of limonene and the negative control beer. This is probably due to a loss of the plasmid which encodes limonene synthase. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will repeat the experiment.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Bildbeschriftung]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 11: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==== Outlook: Characterizing the enzymatic activity====<br />
[[file:TUM12_pyrophosphate.png|thumb|center|600px| Figure 12: Enzymatic assay that will be used to characterize limonene synthase. The principle is well known from pyrosequencing and couples the production of pyrophosphate to the luminescence produced by the firefly luciferase being dependent on ATP]]<br />
<div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/LimoneneTeam:TU Munich/Project/Limonene2012-10-26T16:03:31Z<p>Larakuntz: /* Background and principles */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Limonene=<br />
<hr/><br />
<br />
[[File:Gruppe_limonen_TUM12.jpg|200px|thumb||Responsible: Lara Kuntz and Andrea Richter]]<br />
<br />
<br />
'''A common saying is "If life gives you lemons - make lemonade". For us it is rather "If life gives you limonene - make beer"'''.<br />
<br />
<br />
The obvious thing to do, isn't it? Beer with lemonade is a very popular beverage throughout Germany, e.g. "Radler", "Alsterwasser", "Russ'n". So why not take the shortcut and skip the intermediary? <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a chilled brew. <br />
<br />
Doesn't that make you thirsty? Then you're probably an Englishman - <br />
<br />
Just think of the refreshing sensation of lemons paired with the complex richness of a <s>chilled</s> ''tepid'' brew.<br />
<br />
d-limonene is used as a component of flavorings and fragrances since it has an orange/lemon-like odor. '''Limonene''' has been shown to inhibit rat mammary and other tumor development [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]]. Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
Producing the flavoring substance limonene in our beer might result in a fresh, '''lemon-like taste''' on the one hand. On the other hand, we might have '''beneficial effects on health''' such as preventive activity against cancer, dissolution of gallstones and relief of heartburn.<br />
<br />
<br />
We are close to achieving our goal of a limonene tasting beer. We have successfully cloned (+)-Limonene synthase 1 into our newly established yeast expression vector pTUM100. We transformed ''Saccharomyces cerevisiae'' with the plasmid carrying limonene synthase. We '''proved expression''' of limonene synthase via western blot. We verified the '''functionality of (+)-limonene synthase 1''' by in vitro assay with purified limonene synthase on the one hand. On the other hand, we proved functional '''limonene production in the yeast culture''' via headspace GC-MS. Furthermore we analyzed differences in protein expression in yeast depending on existence of the yeast '''consensus sequence'''. To survey the toxic concentration of limonene for yeast cells, a '''toxicity assay''' was performed. We showed an inhibition of growth at 1 mM and a lethal effect at 100 mM. These concentrations should not be reached by expression of limonene synthase in yeast. <br />
<br />
<br />
Unfortunately we could not yet prove a significant content of limonene in self brewed beer. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will '''repeat the brewing experiment'''.<br />
<br />
== Background and Principles ==<br />
----<br />
<div><br />
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral liquid with the molecular mass of 136.24 g/mol. The (R)-enantiomer smells like oranges and is content of many fruits, while the (S)-enantionmer has a piney odor [[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]]. Therefore, d-limonene ((+)-limonene, (R)-enantiomer) is used as a component of flavorings and fragrances. <br />
<br />
=== Biosynthesis ===<br />
<br />
Limonene is produced by '''limonene synthase'''. Limonene synthase uses geranyl pyrophosphate (GPP), which is the universal precursor of monoterpenoids, as educt. (+)-limonene synthase from ''Citrus limon'' consists of 606 aminoacids (EC=4.2.3.20) and catalyzes the following reaction: '''Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate''' (see Fig. 1).<br />
<br />
[[File:TUM12_ReactionLimoneneSynthase.jpg|thumb|900px|Fig. 1: Reaction catalyzed by limonene synthase.]]<br />
<br />
''Saccharomyces cerevisiae'' produces geranyl pyrophosphate via the mevalonate pathway (see Fig. 2). GPP occurs as an intermediate of farnesyl pyrophosphate (FPP) synthesis [[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]]. It has been established that ''S. cerevisiae'' has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions [[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008], [http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]].<br />
<br />
[[File:TUM12_TerpeneSynthesis.png|thumb|900px|Fig. 2: Simplified isoprenoid pathway in ''S. cerevisiae'', including the branch point to linalool. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase. [<html><a href="http://www.ncbi.nlm.nih.gov/pubmed/20675444">Rico et al., 2010</a></html>]]]<br />
<br />
=== Molecular and Physiological Effects of Limonene ===<br />
<br />
====Flavor and Aliment====<br />
Because of its pleasant citrus flavour and very low toxicity (oral LD50 for mice = 5.6 and 6.6 g/kg body weight), d-limonene is widely used as a flavor and fragrance additive. It is listed in the Code of Federal Regulations as a generally recognized as safe ('''GRAS''') flavoring agent [[http://www.fda.gov/downloads/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/UCM264589.pdf FDA]]. It can be found in common food items such as fruit juices, soft drinks, baked goods, ice cream, and pudding in typical concentrations of 50 ppm to 2,500 ppm [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. The normal daily consume of d-limonene is 0.27 mg/kg body weight per day [[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. As '''natural compound''' of plants, limonene has practical advantages with regard to availability, suitability for oral application, regulatory approval and mechanisms of action. It does not pose a mutagenic, carcinogenic or nephrotoxic risk to humans [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Cancer Inhibition====<br />
Monoterpenes show anticarcinogenic effects in animal experiments. They have been shown to inhibit rat mammary, gastric, lung and skin tumor development by several discussed mechanisms such as apoptosis induction and modulation of oncogene signal transduction [[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004], [http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]]. d-limonene induces phase I and phase II carcinogen-metabolizing enzymes such as cytochrome p450. These metabolize carcinogens into less toxic forms on the one hand. On the other hand, they prevent the interaction of chemical carcinogens with DNA. Limonene has also been shown to inhibit tumor cell proliferation, to accelerate the rate of tumor cell death and induce tumor cell differentiation. Furthermore, d-limonene regulates cell growth and/or transformation by inhibiting protein isoprenylation [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
<br />
====Solvation of Gallstones====<br />
d-limonene is used as excellent '''solvent of cholesterol''', therefore it has been used clinically to dissolve cholesterol-containing gallstones [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]]. A study with 200 patients showed that direct infusion of 20-30 ml d-limonene (97% solution) completely or partially dissolved gallstones in 141 patients. Gallstones completely dissolved in 96 cases (48%); partial dissolution was observed in 29 cases (14.5%); and in 16 cases (8%) complete dissolution was achieved with the inclusion of hexamethaphosphate (HMP), a chelating agent that can dissolve bilirubin calcium stones [[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]]. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn [[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]].<br />
</div><br />
<br />
==Results==<br />
----<br />
<br />
===BioBricks===<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801060 BBa_K801060] '''(+)-Limonene synthase 1''' with '''''Strep''-tag''' and yeast '''consensus sequence''' ====<br />
<br />
<br />
<br />
This part contains (+)-limonene synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal ''Strep''-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801061 BBa_K801061] '''(+)-Limonene synthase 1''' coding region from ''Citrus limon'' ====<br />
<br />
Improved version of BBa_I742111.<br />
<br />
Does not contain a stop codon. Needs to be used with RFC 25.<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801062 BBa_K801062] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by TEF1 promoter and CYC1 terminator.<br />
<br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801063 BBa_K801063] '''(+)-Limonene synthase 1 expression cassette for yeast''' ====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF1 promoter and yeast TEF1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801064 BBa_K801064] '''(+)-Limonene synthase 1 expression cassette for yeast'''====<br />
<br />
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controlled by yeast TEF2 promoter and yeast CYC1 terminator. <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801065 BBa_K801065] '''(+)-Limonene synthase 1''' with '''''Strep''-Tag''' ====<br />
<br />
This part contains the coding region of (+)-limonene synthase from Citrus limon with a C-terminal ''Strep''-tag. This part is based on BBa_K801061.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801066 BBa_K801066] '''(+)-Limonene synthase 1''' with yeast '''consensus sequence''' for improved expression====<br />
<br />
This part is second version of BBa_K801060. It contains (+)-limonene synthase and the consensus sequence for enhanced expression in yeast. It does not contain a C-terminal ''Strep''-tag as BBa_K801060. The sequence does not contain a stop codon so that RFC 25 has to be used.<br />
<br />
RFC 25 compatible <br />
<br />
'''Further information:'''<br />
* [http://www.ncbi.nlm.nih.gov/nucleotide/21435702 NCBI]<br />
* UniProt entry: [http://www.uniprot.org/uniprot/Q8L5K3 Q8L5K3]<br />
* E.C. Number: [http://enzyme.expasy.org/EC/4.2.3.20 4.2.3.20]<br />
* Origin of the enzyme: ''Citrus limon''<br />
</div><br />
<div style="clear:both"><br />
<br />
===Characterization===<br />
</div><br />
<hr><br />
==== Gel picture of finished constructs ====<br />
<div><br />
[[file:TUM12_limonenegel.png|thumb|300px|right|Fig. 3: Gel electrophoresis of [http://partsregistry.org/Part:BBa_K801060 K801060] and [http://partsregistry.org/Part:BBa_K801061 K801061] after analytical restrigtion digest with EcoR1 and Pst1.]]<br />
<br />
<br />
(+)-Limonene synthase 1 coding region without yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] in pSB1C3 is shown next to (+)-limonene synthase 1 with ''Strep''-tag and yeast consensus sequence [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] after restriction digest with EcoR1 and Pst1 restriction enzymes. To check success of ligation, DNA fragments were separated by agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain. As expected, the 1665 bp fragment of [[http://partsregistry.org/Part:BBa_K801061 BBa_K801061]] and the 1708 bp fragment of [[http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] were detected additionally to the pSB1C3 vector that has 2070 bp (see Fig. 3).<br />
<br />
To compare limonene synthase expression in yeast depending on yeast consensus sequence, we produced duplicates of biobricks; one of each has the consensus sequence, the other one does not. For yeast expression experiments, the biobricks were cloned into the yeast expression vector (pTUM100) recently designed by us. <br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Investigation of the yeast consensus sequence ====<br />
<br />
[[file:TUM12_consensus.png|thumb|450px|right|Fig. 4: Comparison of limonene synthase biobricks ([http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]) with and without yeast consensus sequence.]]<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] reported of a consensus sequence upstream of the AUG start codon in yeast.<br />
Although not as strong as the mammalian Kozak translation initiation sequence, the yeast consensus sequence is thought to have a 2–3-fold effect on the efficiency of translation initiation [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
We designed duplicates of limonene synthase encoding biobricks; one having the yeast consensus sequence, the other one not having the consensus sequence.<br />
<br />
We have only been able to show slight differences in expression between the two biobricks [[http://partsregistry.org/Part:BBa_K801065 BBa_K801065] and [http://partsregistry.org/Part:BBa_K801060 BBa_K801060]] via coomassie staining and western blot. The difficulties of showing the difference via SDS-page may result from variations in the amount of protein applied.<br />
<br />
Our in vivo analysis strongly indicates that the consensus sequence does lead to a 2-3-fold enhanced expression in yeast, though (see Fig. 8B). This is consistent with findings of others [[http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf pYES2 manual]].<br />
<br />
<br />
<br />
<hr><br />
<br />
====Purification of recombinant limonene synthase====<br />
[[File:TUM12_SApurification.png|thumb|200px|left|Fig. 5: Streptavidin Affinity (SA) chromatography.]]<br />
[[File:TUM12_gelfiltration.png|thumb|450px|right|Fig. 6: Analytical gelfiltration of limonene synthase.]]<br />
'''Streptavidin affinity chromatography'''<br><br />
The yeast cell extract that was obtained by cell lysis using glass beads of 0.5 mm was centrifuged at 11000 RPM in an SLA-3000 rotor for 60 minutes and subsequently dialysed against 5 liters of 1x Streptavidin Affinity buffer (SA-buffer) over night. The cell extract was then filtrated using a syringe filter with a pore size of 0.45μm and susequently applied on an SA-column. After sample application, the column was washed with 1x SA buffer until a base line was reached. Subsequently, bound protein was eluted using 5 mM Biotin in 1x SA buffer. The chromatogram of the purification is shown on the left side.<br><br><br />
<br />
'''Gel filtration of purified protein'''<br />
The protein sample obtained was concentrated using a centrifugation concentrator with a molecular size limit of 30 kDa subsequent filtration. 250μl of the solution were applied to an analytical gel filtration column Superdex 200 10/30 with 1x PBS as running buffer at a flow rate of 0.5 ml/min. In the chromatogram (shown in the figure on the right in section B), there is an aggregate peak at the exclusion limit that may be caused by the preceding concentration and a major peak at an elution volume of 13.580 ml. The calibration line that was obtained from the calibration proteins b-amylase, alcohol dehydrogenase, BSA, ovalbumine, carboanhydrase, cytochrome C and aprotinin filtrated with the same experimental setup resulted in a regression line with the formula y = -39206 x + 3.3463. Using this formula and the elution volume of the limonene synthase, an apparent molecular mass of 70.1 kDa could be determined for the produced limonene synthase. This fits quite well the theoretical molecular mass that was calculated using [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy ProtParam] to be 65977.1 Da. The four kilo daltons difference could be caused by posttranslational modifications. This hypothesis should be tested using mass spectrometry.<br />
<br />
<br />
<br />
<br />
<hr><br />
<br />
==== In vitro detection of limonene====<br />
<br />
[[file:TUM12_limonene_invivo.png|450px|thumb|right|Fig. 7: Spectrum of in vitro detection of limonene (enzyme assay with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]) and reference spectrum.]]<br />
<br />
To test the functionality of purified limonene synthase in vitro, we used an optimized protocol of an enzyme assay with extraction of limonene [[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007]]. The limonene synthase was purified via ''Strep''-tag. The enzyme assay was carried out in 25 mM Tris-HCl buffer with 5% Glycerol, 1 mM DTT and cofactors (10 mM MgCl2, 1 mg/ml BSA). 50 µM substrate (geranyl pyrophosphate) and 10 ng purified recombinant limonene synthase were added to the reaction batch. Negative controls were reaction batches without enzyme. The reaction was incubated for 15 min at room temperature. Afterwards, limonene was extracted with pentane, dried with sodiumsulfate and reduced under a stream of nitrogen. Three replicates were done of both sample and negative control.<br />
<br />
The pentane extracts were analyzed with gas chromatography-mass spectrometry ("5890 Series II GC" coupled to a "Finnigan Mat 55 S MS") to identify the enzymatically synthesized products.<br />
<br />
<br />
All enzyme reactions (three replicates) led to the production of limonene while the negative controls did not show limonene. Therefore, we showed that our purified '''limonene synthase is functional and leads to the production of limonene'''. <br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== In vivo detection of limonene====<br />
[[file:TUM12_limoneneinvivo.png|450px|thumb|right|Fig. 8: Detection of limonene in headspace above cell culture supernatant. [A] Spectrum of limonene obtained when analyzing cell culture that was transformed with pTUM104 containing construct of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]. [B] Overview about different measurements.]]<br />
Because limonene is a VOC (volatile organic compound) [[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]], we expected limonene to be present in the gaseous phase above the cell culture as well as in the cell culture. Therefore, we measured limonene via headspace (SPME needle) GC-MS.<br />
<br />
We showed limonene to be produced by yeast that was transformed with pTUM104 carrying limonene synthase coding regions (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801061 BBa_K801061] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801060 BBa_K801060]).<br />
<br />
We detected a greater amount of limonene in the sample that contained limonene synthase with consensus sequence. Hence, we showed that the yeast consensus sequence might increase the expression of limonene synthase and therefore might lead to enhanced limonene production.<br />
<br />
Furthermore, we were not able to detect a significant difference between samples that had additional GPP (educt) versus the ones that did not. This might be due to the inability of GPP to diffuse into the cells (hydrophilic character). Since we were able to detect limonene in both samples, it implies that the GPP present in the cells is sufficient for limonene production. This is consistent with the findings of [http://www.ncbi.nlm.nih.gov/pubmed/18155949| [Herrero et al., 2008]] that showed that ''S. cerevisiae'' cells (from laboratory and wine strains) contain enough free GPP to be catalytically transformed by monoterpene synthases into monoterpenes.<br />
<br />
<br />
<div><br />
<br />
<br />
<br />
</div><br />
<hr><br />
<br />
==== Detection of limonene in beer====<br />
[[File:TUM12_SPME.jpg|200px|thumb|left|Fig. 9: Preparation of sample for GC-MS with SPME.]]<br />
[[File:TUM12_LSbeer.jpg|200px|thumb|right||Fig. 10: iGEM's first and finest SynBio Beer with limonene.]]<br />
A first attempt to use our genetically engineered yeasts to brew a SynBio Beer were conducted using a transient transfection with a constitutive promoter. The drawback is that in the gyle the selection pressure is not preserved and the loss of the plasmid is possible.<br><br />
<br />
Three liters of gyle were inoculated with 100ml of a stationary yeast culture grown in YPD that was transiently transfected with a plasmid harboring a constitutive expression cassette for the limonene synthase.<br />
<br />
We analyzed this first beer for limonene content via headspace (SPME needle) GC-MS. Unfortunately we have not yet been able to proof a significant difference between the beer supposed to contain enhanced amounts of limonene and the negative control beer. This is probably due to a loss of the plasmid which encodes limonene synthase. We will try to integrate the limonene synthase expression cassette into the genome of yeast and afterwards we will repeat the experiment.<br />
<br />
<br><br><br />
[[file:TUM12_limonene_in_beer.png|thumb|center|600px| Bildbeschriftung]]<br />
<br />
<hr><br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
To establish whether limonene has an effect on yeast cells, we inoculated three different yeast strains with different concentrations of limonene. Limonene was added to the medium. We examined the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in grocery stores. <br />
<br />
At high concentrations, limonene affects the growth of yeast cells. We could show an inhibition of growth at 1 mM and even a lethal effect at 100 mM. At lower concentrations (1 µM, 10 µM, 100 µM) no inhibition could be observed. The growth rates of yeast cells which were incubated with low concentrations of limonene do not show a difference compared to the negative control (incubation of analogous yeast strains with YPD without limonene).<br />
<br />
<center><br />
[[File:TUM12_Toxicity_Limonene.png|800px|thumb|center|Fig. 11: Limonene toxicity assay evaluation.]]<br />
</center><br />
</div><br />
<br />
==== Outlook: Characterizing the enzymatic activity====<br />
[[file:TUM12_pyrophosphate.png|thumb|center|600px| Figure 12: Enzymatic assay that will be used to characterize limonene synthase. The principle is well known from pyrosequencing and couples the production of pyrophosphate to the luminescence produced by the firefly luciferase being dependent on ATP]]<br />
<div><br />
<br />
==References==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11605760 Fietzek et al., 2001]] Fietzek, C., Hermle, T., Rosenstiel, W., Schurig, V. (2001) Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks. ''Fresenius J Anal Chem.'', 371(1):58-63.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC340751/ Hamilton et al., 1987]] Hamilton, R., Watanabe, C. K., De Boer, A. H. (1987) Compilation and comparison of the sequence context around the AUG startcodons in ''Saccharomyces cerevisiae'' mRNAs. ''Nucleic Acids Res.'', 15(8):3581–3593.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18155949 Herrero et al., 2008]] Herrero, O., Ram ́on, D., and Orejas, M. (2008). Engineering the ''Saccharomyces cerevisiae'' isoprenoid pathway for de novo production of aromatic monoterpenes in wine. ''Metab Eng'', 10(2):78–86.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/1988264 Igimi et al., 1991]] Igimi, H., Tamura, R., Toraishi, K., Yamamoto, F., Kataoka, A., Ikejiri, Y., Hisatsugu, T., Shimura, H. (1991). Medical dissolution of gallstones. Clinical experience of d-limonene as a simple, safe, and effective solvent. '''Dig Dis Sci.''', 36(2):200-8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al., 2007]] Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., and Schwab, W. (2007). Cloning and functional characterization of three terpene synthases from lavender (''Lavandula angustifolia''). ''Arch Biochem Biophys'', 465(2):417–29.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12084056 Lücker et al., 2002]] Lücker, J., El Tamer, M. K., Schwab, W., Verstappen, F. W. A., van der Plas, L. H. W., Bouwmeester, H. J., and Verhoeven, H. A. (2002). Monoterpene biosynthesis in lemon (''Citrus limon''). cDNA isolation and functional analysis of four monoterpene synthases. ''Eur J Biochem'', 269(13):3160–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/17096665 Oswald et al., 2007]] Oswald, M., Fischer, M., Dirninger, N., and Karst, F. (2007). Monoterpenoid biosynthesis in ''Saccharomyces cerevisiae''. ''FEMS Yeast Res'', 7(3):413–21.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15763095 Pierucci et al., 2005]] Pierucc, P., Porazzi, E., Martinez, MP., Adani, F., Carati, C., Rubino, FM., Colombi, A., Calcaterra, E., Benfenati, E. (2005). Volatile organic compounds produced during the aerobic biological processing of municipal solid waste in a pilot plant.''' Chemosphere''', 59(3):423-30.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/20675444 Rico et al., 2010]] Rico, J., Pardo, E., and Orejas, M. (2010). Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme a reductase catalytic domain in ''Saccharomyces cerevisiae''. ''Appl Environ Microbiol'', 76(19):6449–54.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18072821 Sun, 2007]] Sun, J. (2007). D-limonene: safety and clinical applications. ''Altern Med Rev'', 12(3):259–64.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed?term=Tsuda%20limonene%202004 Tsuda et al., 2004]] Tsuda, H., Ohshima, Y., Nomoto, H., Fujita, K., Matsuda, E., Iigo, M., Takasuka, N., Moore, MA. (2004). Cancer prevention by natural compounds. ''Drug Metab Pharmacokinet.'' 19(4):245-63.<br />
*[[http://www.mri.bund.de/fileadmin/Institute/PBE/Sekundaere_Pflanzenstoffe/Monoterpene.pdf Watzl, 2002]] Watzl, B. (2002). Monoterpene. ''Ernährungs-Umschau'' 49 Heft 8. 322-324.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/9724535 Williams et al., 1998]] Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (19</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:29:15Z<p>Larakuntz: /* Caffeine Synthesis Pathway Composite Part BBa_K801077 */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Biobricks created for caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===Characterization===<br />
<br />
==== Western Blot Detection of Expressed Enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by using yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II. The Strep-tag coding sequence has been fused to each of the enzyme coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
==== Composition of Expression Cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been checked by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
<br />
==== Caffeine Synthesis Pathway Composite Part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constitutive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the left.<br />
<br />
We have been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway. Therefore, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at a pH of 8.0.<br />
<br />
Detection of single metabolites of the caffeine biosynthesis pathway was '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, prior to LC/MS analysis.<br />
<br />
The picture below shows that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which is the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not yet say whether or not the ''in vitro'' synthesis of caffeine has worked. Further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:27:46Z<p>Larakuntz: /* Caffeine Synthesis Pathway Composite Part BBa_K801077 */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Biobricks created for caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===Characterization===<br />
<br />
==== Western Blot Detection of Expressed Enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by using yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II. The Strep-tag coding sequence has been fused to each of the enzyme coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
==== Composition of Expression Cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been checked by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
<br />
==== Caffeine Synthesis Pathway Composite Part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constitutive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the left.<br />
<br />
We have been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway. Therefore, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at a pH of 8.0.<br />
<br />
Detection of single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, prior to the LC/MS analysis.<br />
<br />
The picture below shows that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which is the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not yet say whether or not the ''in vitro'' synthesis of caffeine has worked. Further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:23:54Z<p>Larakuntz: /* Caffeine Synthesis Pathway Composite Part BBa_K801077 */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Biobricks created for caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===Characterization===<br />
<br />
==== Western Blot Detection of Expressed Enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by using yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II. The Strep-tag coding sequence has been fused to each of the enzyme coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
==== Composition of Expression Cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been checked by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
<br />
==== Caffeine Synthesis Pathway Composite Part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constitutive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
We have been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway. Therefore, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at a pH of 8.0.<br />
<br />
Detection of single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, prior to the LC/MS analysis.<br />
<br />
The picture below shows that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which is the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not yet say whether or not the ''in vitro'' synthesis of caffeine has worked. Further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:20:00Z<p>Larakuntz: /* Caffeine Synthesis Pathway composite part BBa_K801077 */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Biobricks created for caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===Characterization===<br />
<br />
==== Western Blot Detection of Expressed Enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by using yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II. The Strep-tag coding sequence has been fused to each of the enzyme coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
==== Composition of Expression Cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been checked by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
<br />
==== Caffeine Synthesis Pathway Composite Part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:17:08Z<p>Larakuntz: /* Composition of Expression Cassettes */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Biobricks created for caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===Characterization===<br />
<br />
==== Western Blot Detection of Expressed Enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by using yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II. The Strep-tag coding sequence has been fused to each of the enzyme coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
==== Composition of Expression Cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been checked by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
<br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:16:01Z<p>Larakuntz: /* Composition of expression cassettes */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Biobricks created for caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===Characterization===<br />
<br />
==== Western Blot Detection of Expressed Enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by using yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II. The Strep-tag coding sequence has been fused to each of the enzyme coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
==== Composition of Expression Cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
<br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:15:18Z<p>Larakuntz: /* Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Biobricks created for caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===Characterization===<br />
<br />
==== Western Blot Detection of Expressed Enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by using yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II. The Strep-tag coding sequence has been fused to each of the enzyme coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:12:10Z<p>Larakuntz: /* Results */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Biobricks created for caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
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<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
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<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:10:02Z<p>Larakuntz: /* Results */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
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<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
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<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
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'''NCBI access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
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<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
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'''NCBI access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
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'''NCBI access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:09:40Z<p>Larakuntz: /* BBa_K801071 RFC10 compatible BioBrick encoding the enzyme CaMXMT1 */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:09:24Z<p>Larakuntz: /* BBa_K801070 RFC10 compatible BioBrick encoding the enzyme CaXMT1 */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:07:50Z<p>Larakuntz: /* BioBricks */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''Coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to high similarity of the sequences of the three enzymes, we also changed this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene-synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slightly acidic environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:04:22Z<p>Larakuntz: /* The Molecular and Physiological Effects of Caffeine */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
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<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence, it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work...<br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves). Therefore, we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
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===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to the high similarity of the sequences of the three enzymes, we also exchanged this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene- synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl-donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slighty acid environment during beer brewing.<br />
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<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
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'''NCBI- Access number of original gene sequence:''' AB048793<br />
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This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
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'''NCBI- Access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
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'''NCBI- Access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
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This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
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This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
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</div><br />
<div class="mfull bezel"><br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
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This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
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This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
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===Characterization===<br />
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==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
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<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
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==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
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[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
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==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
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</div><br />
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==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T15:01:27Z<p>Larakuntz: /* The molecular and physiological effects of caffeine */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
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=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The Molecular and Physiological Effects of Caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine-alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter-release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work... <br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves), so we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to the high similarity of the sequences of the three enzymes, we also exchanged this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene- synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl-donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slighty acid environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T14:55:37Z<p>Larakuntz: /* Biosynthesis and Metabolic Engineering */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture (Fig. 1). Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The molecular and physiological effects of caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine-alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter-release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work... <br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves), so we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to the high similarity of the sequences of the three enzymes, we also exchanged this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene- synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl-donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slighty acid environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T14:52:56Z<p>Larakuntz: /* Biosynthesis and Metabolic Engineering */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture. Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. The presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The molecular and physiological effects of caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine-alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter-release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work... <br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves), so we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to the high similarity of the sequences of the three enzymes, we also exchanged this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene- synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl-donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slighty acid environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T14:51:12Z<p>Larakuntz: /* Biosynthesis and Metabolic Engineering */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture. Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was shown to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. The ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants. Therefore, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]]. Because of both, the presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by ''McCarthy and McCarthy, 2007''. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The molecular and physiological effects of caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine-alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter-release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work... <br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves), so we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to the high similarity of the sequences of the three enzymes, we also exchanged this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene- synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl-donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slighty acid environment during beer brewing.<br />
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<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
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<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
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'''NCBI- Access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
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<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
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==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
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Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
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The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
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[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
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==== Toxicity Assay ====<br />
<div><br />
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Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
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During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
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[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
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</div><br />
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==References==<br />
<hr/><br />
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*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T14:28:52Z<p>Larakuntz: /* Biosynthesis and Metabolic Engineering */</p>
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<div>{{Team:TU_Munich/Header}}<br />
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{{Team:TU_Munich/ExCol}}<br />
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=Caffeine=<br />
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[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
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<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
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=== Biosynthesis and Metabolic Engineering===<br />
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The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture. Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was declared to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. Because the ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants, the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]], because of both, the presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by ''McCarthy and McCarthy, 2007''. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
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The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
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<br />
===The molecular and physiological effects of caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine-alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter-release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work... <br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves), so we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
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== Results ==<br />
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===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to the high similarity of the sequences of the three enzymes, we also exchanged this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene- synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
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All mentioned methyltransferases use SAM as methyl-donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slighty acid environment during beer brewing.<br />
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<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
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'''NCBI- Access number of original gene sequence:''' AB048793<br />
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This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
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'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
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<div class="mfull bezel"><br />
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==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
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'''NCBI- Access number of original gene sequence:''' AB048794<br />
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This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
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'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
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'''NCBI- Access number of original gene sequence:''' AB084125<br />
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This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
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'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
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This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
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This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
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</div><br />
<div class="mfull bezel"><br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
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This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
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This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
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===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T14:26:34Z<p>Larakuntz: /* Biosynthesis and Metabolic Engineering */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture. Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was declared to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. Because the ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants (and without any further nucleosidase enzyme), the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]], because of both, the presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by ''McCarthy and McCarthy, 2007''. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The molecular and physiological effects of caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine-alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter-release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work... <br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves), so we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to the high similarity of the sequences of the three enzymes, we also exchanged this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene- synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl-donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slighty acid environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T14:25:53Z<p>Larakuntz: /* Biosynthesis and Metabolic Engineering */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture. Being involved in the catabolism of all purine nucleosides, PNP is an essential enzyme for organisms and was declared to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis, having been accomplished by [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. Because the ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants (and without any further nucleosidase enzyme), the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]], because of both, the presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by ''McCarthy and McCarthy, 2007''. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The molecular and physiological effects of caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine-alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter-release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work... <br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves), so we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to the high similarity of the sequences of the three enzymes, we also exchanged this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene- synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl-donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slighty acid environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T14:24:33Z<p>Larakuntz: /* Biosynthesis and Metabolic Engineering */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
<br />
The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine-metabolism of all organisms.''' Necessary for its production are '''three distinct N-methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine-nucleoside phosphorylase (PNP) or by the first N-methyl transferase of the reaction cascade shown in the picture. Being involved in the catabolism of all purin-nucleosides, PNP is an essential enzyme for organisms and was declared to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis, having been accomplished by [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. Because the ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants (and without any further nucleosidase enzyme), the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]], because of both, the presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by ''McCarthy and McCarthy, 2007''. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
<br />
The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
<br />
<br />
The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
===The molecular and physiological effects of caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine-alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter-release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work... <br />
<br />
Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves), so we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
<br />
</div><br />
<br />
== Results ==<br />
<hr/><br />
===BioBricks===<br />
<br />
[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
<br />
'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to the high similarity of the sequences of the three enzymes, we also exchanged this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene- synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
<br />
All mentioned methyltransferases use SAM as methyl-donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slighty acid environment during beer brewing.<br />
<br/><br/><br />
<br />
<div class="mfull bezel"><br />
==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048793<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
</div><br />
<br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB048794<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
<br />
'''NCBI- Access number of original gene sequence:''' AB084125<br />
<br />
This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
<br />
'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
<br />
This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
<br />
This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
<br />
This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
<br />
This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
</div><br />
<div class="mfull bezel"><br />
<br />
==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
<br />
This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
<br />
The accuracy of this BioBrick has not yet been proven by sequencing.<br />
</div><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<br />
<br />
===Characterization===<br />
<br />
==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
<br />
We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
<br />
* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
<br />
The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
<br />
* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
<br />
<br />
==== Composition of expression cassettes ====<br />
<br />
In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
<br />
The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
<br/><br/><br />
==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
<br />
This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
<br />
Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
<br />
Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
<br />
The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
<br />
<br/><br/><br/><br/><br />
<br />
[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
<br />
==== Toxicity Assay ====<br />
<div><br />
<br />
Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
<br />
We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
<br />
During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
<br />
<br />
[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
<br />
</div><br />
<br />
==References==<br />
<hr/><br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntzhttp://2012.igem.org/Team:TU_Munich/Project/CaffeineTeam:TU Munich/Project/Caffeine2012-10-26T14:22:27Z<p>Larakuntz: /* Background and principles */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
{{Team:TU_Munich/ExCol}}<br />
<br />
=Caffeine=<br />
<hr/><br />
<br />
[[File:Gruppe_Koffein_TUM12.jpg|350px|thumb||Responsible: Saskia König, Roman Prechtl and Dennis Hell]]<br />
<br />
<div style="text-align:justify;">Hops is not only one of the main ingredients of beer, it is also responsible for the '''sedative effect of beer''' [[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al. 2012]].<br />
In contrast, '''caffeine wards off drowsiness''' and already enjoys great popularity as additive in a multitude of beverages.<br />
As hops is essential to the brewing process, omitting hops is not an option. This makes caffeine a '''desirable agent to counteract the soporific effect of beer.'''<br />
<br />
<br />
We were successful in expressing ('''SDS-PAGE''' and '''western blot analysis''') all three genes which are necessary for caffeine biosynthesis (in plants) in yeast strain INVSc1 after having cloned the genes in the new yeast expression vector pTUM104. The proof of '''enzyme functionality''' by detection of synthesized caffeine via LC-MS is already in progress. The three single genes were '''submitted as BioBricks''' [http://partsregistry.org/Part:BBa_K801070 BBa_K801870], [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] and [http://partsregistry.org/Part:BBa_K801072 BBa_K801072]. <br />
<br />
Furthermore, we generated the '''caffeine synthesis BioBrick''' [http://partsregistry.org/Part:BBa_K801077 BBa_K801077], which contains all the genes necessary for caffeine synthesis - each gene with a promoter and terminator. The strength of the promoters has been chosen individually.<br />
<br />
In order to investigate the growth of our yeast cells under the influence of caffeine at different concentrations, we also performed a '''toxicity assay'''.<br />
<br />
Caffeine production in tobacco plants [[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005],[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] and in an ''in vitro'' assay has already been shown, but has '''never been performed in yeast'''.<br />
<br />
==Background and Principles==<br />
<hr/><br />
<br />
=== Biosynthesis and Metabolic Engineering===<br />
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The biosynthetic pathway of caffeine (1,3,7 Trimethylxanthine) starts with xanthosine, which is a '''natural component of the purine-metabolism of all organisms.''' Necessary for its production are '''three distinct N- methyl transferases and one nucleosidase'''. However, it remains to be elucidated whether the nucleosidase reaction is catalyzed by an unspecific purine-nucleosid phosphorylase (PNP) or by the first N- methyl transferase of the reaction cascade shown in the picture. Being envolved in the catabolism of all purin-nucleosides, PNP is an essential enzyme of organisms and was declared to catalyze the removal of the ribose moiety of 7-methylxanthine in ''in vitro'' caffeine biosynthesis, having been accomplished by [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. Because the ''in vivo'' synthesis of caffeine has also been shown by expression of the three N-methyl transferases in tobacco plants (and without any further nucleosidase enzyme), the assumption that the first methyl transferase is '''bifunctional and catalyzes the nucleosidase reaction''' is favored [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]], [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]], because of both, the presence of PNP or equal enzymes in plants has not yet been proven and a catalytic mechanism of the nucleosidase reaction is rather thinkable and has been explained by ''McCarthy and McCarthy, 2007''. As a matter of fact, a single N-methyl nucleosidase, as it is shown on the depicted pathway below, has indeed been partially purified out of tea leaves [[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]], but neither the native enzyme nor its DNA have ever been isolated [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]].<br />
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The enzyme caffeine synthase (last reaction step) can catalyze both the conversion of 7-methyl xanthine to theobromine and the methylation of theobromine to caffeine. Unfortunately, the affinity of this enzyme to 7-methyl xanthosine is less than one sixth of that of the other isoform CaMXMT1 [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. To obtain the best results, we decided to express both enzymes. One can also see the K<sub>m</sub> values for the required enzymes in this paper - it shows that the substrate affinity decreases continuously towards the endpoint (caffeine), "making the reaction proceed irreversibly and stepwise" [[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]]. [[File:TUM12_Xanthosine_Picture.PNG|right|thumb|480px| '''Fig. 1: Xanthosine providing metabolic pathways''']]<br />
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The chemical compound xanthosine is produced via '''at least four different routes''', shown in the picture "xanthosine routes". To improve caffeine production, these pathways could be a '''possible target for metabolic engineering in the future'''.<br />
[[File:TUM12_Caffeinebiosys.png|thumb|900px|'''Fig. 2: Pathway for ''in vivo'' caffeine biosynthesis]]<br />
During the degradation of caffeine, it is demethylated to theophylline by 7N-demethylase (main pathway). The decreased rate of this reaction is the '''reason for the accumulation of caffeine in the plant.''' Afterwards, theophylline is degraded to xanthine via 3-methylxanthine and xanthine enters the conventional purine catabolism pathway (degradation to CO<sub>2</sub> and NH<sub>3</sub>) [[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]]. This catabolistic pathway is '''another possible target''' for metabolic engineering to increase the amount of caffeine.<br />
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===The molecular and physiological effects of caffeine===<br />
[[File:TUM12_CaffeineAdenosine.png|right|thumb|250px| '''Fig. 3: Similar structures of caffeine and adenosine''']]<br />
Caffeine is a '''purine-alkaloid''' and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence it can '''block specific receptors in the hypothalamus'''. Adenosine binding leads to decreased neurotransmitter-release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine '''antagonizes adenosine and increases neuronal activity''', it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea about 25 - 90 mg. At higher doses (1 g), caffeine leads to higher pulse rates and hyperactivity, but until that, the alcohol will already have done its work... <br />
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Moreover, caffeine was shown to decrease the growth of ''E. Coli'' and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves), so we do not expect to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.<br />
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== Results ==<br />
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===BioBricks===<br />
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[[File:TUM12_new_Coffein_Biobricks.jpg|right|thumb|300px|'''Fig. 4: Created biobricks in the caffeine synthesis''']]<br />
All the generated BioBricks are based on mRNA sequences that have been isolated from ''coffea arabica'' by Hiroshi Sano et al., 2003, and registered at [http://www.ncbi.nlm.nih.gov/pubmed/ NCBI] (see numbers below). However, these '''sequences were modified''' in several ways, to make them '''iGEM compatible''' and '''improve the usage''' in general.<br />
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'''Modifications:'''<br />
* the 5' UTR and 3' UTR of the original sequences were removed<br />
* the '''yeast consensus sequence''' for improved ribosome binding (TACACA) was added 5' of the start codon ATG <br />
* according to the '''N- end rule''' and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine) to optimize protein stability and mRNA translation. This decision was made after analysis of the 3D- structure of the enzyme CaDXMT1. Because the first two residues of the amino acid sequence are not shown in the crystallized structure (probably because of high flexibility) we chose to exchange this amino acid. Further study (uniprot entry) showed that the first two residues of the sequence are not immediately involved in ligand binding in one of the three enzymes. Due to the high similarity of the sequences of the three enzymes, we also exchanged this amino acid in the enzymes CaXMT1 and CaMXMT1.<br />
* we added a '''C- terminal ''Strep''-tag''' for purification and detection <br />
* the remaining coding sequence was extended with the standard '''RFC10 prefix and suffix'''<br />
* we made an '''optimization of the coding sequences''' with respect to the '''codon usage in yeast and mRNA structures''' (online tool of gene- synthesis company)<br />
* we exchanged all critical restriction sites (RFC10 and RFC25)<br />
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All mentioned methyltransferases use SAM as methyl-donor and are located in the cytoplasm of the plants. Furthermore, they exist as homodimers, being also able to form heterodimers with each other (see [http://www.brenda-enzymes.info Brenda], also for further characteristics). The temperature- and pH-optima of all three enzymes are quite similar (between 20°C - 37°C and 7,5 - 8,5). The pH-optimum is compatible with the slighty acid environment during beer brewing.<br />
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==== [http://partsregistry.org/Part:BBa_K801070 BBa_K801070] RFC10 compatible BioBrick encoding the enzyme CaXMT1 ====<br />
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'''NCBI- Access number of original gene sequence:''' AB048793<br />
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This RFC10 compatible BioBrick encodes the enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica''). It catalyzes the first reaction step of the caffeine biosynthesis pathway. <br />
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'''Further information:'''<br />
* UniProt entry: Q9AVK0<br />
* E.C. Number: 2.1.1.158 <br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2eg5 ''C. canephora'' CaXMT1]<br />
* Part length: 1158 bp<br />
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==== [http://partsregistry.org/Part:BBa_K801071 BBa_K801071] RFC10 compatible BioBrick encoding the enzyme CaMXMT1 ====<br />
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'''NCBI- Access number of original gene sequence:''' AB048794<br />
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This RFC10 compatible BioBrick encodes the enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the third reaction step of the caffeine biosynthesis pathway. <br />
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'''Further information:'''<br />
* UniProt entry: Q9AVJ9<br />
* E.C. Number: 2.1.1.159<br />
* Part length: 1176 bp<br />
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==== [http://partsregistry.org/Part:BBa_K801072 BBa_K801072] RFC10 compatible BioBrick encoding the enzyme CaDXMT1 ====<br />
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'''NCBI- Access number of original gene sequence:''' AB084125<br />
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This RFC10 compatible BioBrick encodes the enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica''). It catalyzes the fourth reaction step of the caffeine biosynthesis, leading to caffeine. <br />
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'''Further information:'''<br />
* UniProt entry: Q8H0D2<br />
* 2.1.1.160<br />
* PDB: 3D- Structure: [http://www.pdb.org/pdb/explore/explore.do?structureId=2efj ''C. canephora'' CaDXMT1]<br />
* Part length: 1194 bp<br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801073 BBa_K801073] Generator BioBrick for CaXMT1 ====<br />
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This BioBrick generates the enzyme '''xanthosine N-methyltransferase 1 (CaXMT1)'''. It is regulated by the constitutive promoter ''Tef2'', which is a strong yeast promoter. The used terminator is ''Adh1'', a widely used yeast terminator. <br />
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This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801074] Generator BioBrick for CaMXMT1====<br />
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This BioBrick generates the enzyme '''7-methylxanthine N-methyltransferase 1(CaMXMT1)''' (= theobromine synthase). It is regulated by the constitutive promoter ''Tef1'', which is one of the strongest yeast promoters. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
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This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801075 BBa_K801075] Generator BioBrick for CaDXMT1 ====<br />
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This BioBrick generates the enzyme '''3,7-dimethylxanthine N-methyltransferase 1 (CaDXMT1)''' (= caffeine synthase), i.e. the last enzyme involved in caffeine biosynthesis. It is regulated by the constitutive promoter ''Tef2'', which is also used at the expression cassette of CaXMT1. The used terminator is ''Adh1'', as it is among the other expression cassettes. <br />
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This BioBrick is also a part of the '''"caffeine synthesis device"''' (see below) and the accuracy of the sequence has been proven by sequencing.<br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801076 BBa_K801076] Generator BioBrick for CaXMT1 and CaMXMT1 ====<br />
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This BioBrick generates the first two enzymes envolved in caffeine biosynthesis: '''methylxanthosine N-methyltransferase 1''' and '''7- methylxanthine N-methyltransferase 1'''. It is made up of the two single generators (see above), which means that CaXMT1 is regulated by the ''Tef2'' promoter and CaMXMT1 is regulated by the ''Tef1'' promoter. In both cases, the ''Adh1'' terminator was used.<br />
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==== [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801077 BBa_K801077] Generator BioBrick for CaXMT1, CaMXMT1 and CaDXMT1 ====<br />
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This BioBrick is the final '''"caffeine synthesis device"'''. It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be transformed directly into competent yeast cells or cloned into an adequate yeast genome integration vector.<br />
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The accuracy of this BioBrick has not yet been proven by sequencing.<br />
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===Characterization===<br />
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==== Western Blot detection of expressed enzymes CaXMT1, CaMXMT1 and CaDXMT1 (BBa_K801070, BBa_K801071 and BBa_K801072) ====<br />
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We were successful in expressing all three enzymes which are necessary for the caffeine biosynthesis pathway in yeast by use of the yeast expression vector pTUM104. The results can be seen on the right picture. Detection was performed by using a specific antibody against ''Strep''-tag II, which has been fused on each of the coding sequences.<br />
[[File:TUM12_WBs.png|500px|thumb|right| '''Fig. 5: Western Blot of protein crude extracts:''' Left: (1) CaXMT1 (uninduced);(2) CaMXMT1 (uninduced);(3) eGFP (20h);(4) CaMXMT1 (20h); Right: (1) CaDXMT1 (uninduced); (2) CaDXMT1 (20h)]]<br />
The theoretical weights of the enzymes (predicted with the online tool "ProtParam") are as follows:<br />
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* CaXMT1: 42998,4 Da<br />
* CaMXMT1: 43903,3 Da<br />
* CaDXMT1: 44473,7 Da<br />
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The real weights of the expressed enzymes are likely to be influenced by several posttranslational modifications after expression in yeast, and that is probably the reason why the detected proteins showed a higher apparent mass on the western blot. Amongst others, [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp '''ExPASy Proteomics tools'''] predicts the following modifications:<br />
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* acetylation at serine (second amino acid)<br />
* O-GlcNAc modifikation at several positions<br />
* phosphorylation at several positions<br />
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==== Composition of expression cassettes ====<br />
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In order to be able to perform a continuous expression of the three enzymes, we created several expression cassettes with individual promoters and the widely used ''Adh1'' terminator. The BioBricks BBa_K801073, BBa_K801074 and BBa_K801075 are enzyme generators, which provide continuous expression of the particular enzymes and are all part of the final '''caffeine synthesis pathway''' composite part. The choice for the respective promoters was made to support the irreversible production of caffeine, as it is ''in vivo'' in coffee plants, being assured by continuously increasing K<sub>m</sub> values of the three enzymes (H. Sano et al., 2003). Besides, we focused on prohibiting metabolic stress reactions. Thus, we used the ''Tef2'' promoter for the first enzyme CaXMT1 and the stronger ''Tef1'' promoter for the next enzyme, which is CaMXMT1, to establish high concentrations of the caffeine precursor theobromine. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. The last enzyme CaDXMT1 is then again regulated by the ''Tef2'' promoter.<br />
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The sequences of all three expression cassettes have been proved by sequencing, but expression has not yet been investigated.<br />
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==== Caffeine Synthesis Pathway composite part BBa_K801077====<br />
[[file:TUM12_BBa_K801077.jpg|left|thumb|200px|'''Fig. 6: Gel electrophoresis of BBa_K801077 digested with Xba1 and Pst1''']]<br />
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This part is our '''final product''', made up of all '''three expression cassettes''' mentioned above. As previously said, it provides '''constituvive expression''' of all three neccessary enzymes for caffein biosynthesis. It can be cloned in a '''yeast expression vector''' or be used for '''genome integration''' by the use of appropriate vectors. An analytical restriction digest can be seen on the right.<br />
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Since we had been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway, we used this composite part BBa_K801077 for expression of the enzymes, followed by '''enzymatic analyses''', in order to prove the functionality of our enzymes. After having cloned it into our vector '''pTUM100''', we performed an enzyme assay as described in ''H. Sano et al., 2003'', with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at an pH of 8.0.<br />
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Detection of the single metabolites of the caffeine biosynthesis pathway was to be '''performed by LC/MS analysis''', using multiple reaction monitoring (MRM). The solutions of several reaction batches were subjected to chloroform extraction, dried at 60°C and resuspended in 70% methanol, previously to the LC/MS analysis.<br />
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The picture below shows, that we '''were in fact able''' to perform an ''in vitro'' synthesis of theobromine, which the '''immediate precursor of caffeine'''. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time. Thus, we can not truly say wether or not the ''in vitro'' synthesis of caffeine has worked, but further experiments are running.<br />
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[[File:TUM12_CaffeineChromatograms.jpg|thumb|center|900px|'''Fig. 7: LC/MS chromatograms of the enzyme assay''' A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay]]<br />
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==== Toxicity Assay ====<br />
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Since high doses of caffeine (> 10 mM) have mutagenic effects on yeast ([http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]) we investigated the effect of different caffeine concentrations on different yeast strains. <br />
The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. Caffeine was added to the YPD medium in concentrations from 1 µM up to 100 mM and the growth rate was measured after a defined period of time. <br />
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We confirmed the toxic effect of caffeine on yeast at the concentration of 100 mM and the growth inhibition at the concentration of 10 mM. Furthermore we showed the correlation between decreasing caffeine concentration and growth rate. Cells incubated with caffeine concentrations in the range of micro molar showed a similar growth rate than the negative control (incubation without caffeine). <br />
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During our brewing experiments, we won’t exceed the level of toxicity (>10 mM) of caffeine.<br />
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[[File:TUM12_Toxicitiy_Caffeine.png|900px|thumb|center|'''Fig. 7: Evaluation of the Toxicity Assay for Caffeine''']]<br />
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==References==<br />
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*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.<br />
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.<br />
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.</div>Larakuntz