Limonene

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.

We will produce the flavoring substance limonene by expressing limonene synthase in S. cerevisiae, which naturally synthesizes the educt geranyl pyrophosphate.

Explanations on the figure

(+)-Limonene synthase 1 http://partsregistry.org/Part:BBa_K801061 BBa_K801061 and (+)-Limonene Synthase 1 with Strep-Tag and Yeast consensus sequence http://partsregistry.org/Part:BBa_K801060 BBa_K801060 could successfully cloned in a new yeast expression vector pTUM100. Western blot detection of both synthases after expression in Saccharomyces cerevisiae and proof of functionality of the enzymes by detecting limonene under GPP addition in vivo and in vitro via different GC-MS methods was effectually. Furthermore differences in protein expression in yeast depending on existence of consensus sequence could detected. For further genome integration experiments in yeasts we could established gene constructs of several promotors and terminators adjacent to the limonene synthase gene http://partsregistry.org/Part:BBa_K801062 BBa_K801062, http://partsregistry.org/Part:BBa_K801063 BBa_K801063, http://partsregistry.org/Part:BBa_K801064 BBa_K801064. Unfortunately we could not yet proof 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.

Thaumatin

Thaumatin is a natural α+β-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 slow and lasts long. It has been approved as a sweetener by the European Union (E957).

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.

The BioBrick for Prepro-Thaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as a expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expression in yeast, purified using a ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore the expession of Thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.

A proof of principle for the expression of Thaumatin was achieved. Further goals would include to increase the expression of the thaumatin and to investigate the secretion.

Caffeine

Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants (3). The molecule acts as a competitive antagonist on adenosine receptors and therefore increases indirectly neurotransmitter concentrations resulting in warding of drowsiness and restoring of alertness.

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.

• Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 N-methyl nucleosidase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each.
• 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.
• 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.
• Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in Saccharomyces cerevisiae INVSc1 in selective Sc minimal induction medium lacking uracil with 2% galactose.

The homologue expression of the three required enzymes for caffeine synthesis in Saccharomyces cerevisiae INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 succeeded. Further testing of caffeine production using crude extracts from lysed transformed yeast cells and working on genome integration of the expression cassette consisting of all three enzymes flanked by promoter and terminator for caffeine production during expression are in progress.

Xantohumol

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.

The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.

Explanations on the figure

The whole biosynthetic pathway for the production of Xantohumol 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: [http://partsregistry.org/Part:BBa_K801090 BBa_K801090] [http://partsregistry.org/Part:BBa_K801091 BBa_K801091] [http://partsregistry.org/Part:BBa_K801092 BBa_K801092] [http://partsregistry.org/Part:BBa_K801093 BBa_K801093] [http://partsregistry.org/Part:BBa_K801094 BBa_K801094] [http://partsregistry.org/Part:BBa_K801095 BBa_K801095] [http://partsregistry.org/Part:BBa_K801096 BBa_K801096] [http://partsregistry.org/Part:BBa_K801097 BBa_K801097] [http://partsregistry.org/Part:BBa_K801098 BBa_K801098]

The construction of the Xantohumol pathway was achieved whereas the expression and characterization has to be postponed after the european Jamboree or migth be an interesting task for next year's iGEM teams.

pTUM100

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 a vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.

Explanations on the figure

Using the pYES vector from Invitrogen we first deleted forbidden restriction sites in the vector back bone via side directed mutagenesis. Furthermore the original multiple cloning site was replaced for a multiple cloning site compatible to 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. 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 the yeast Saccharomyces cerevisiae. Moreover we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.

Outlook and conclusion

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. To cover even more demands we are planning to design a second vector template containing a His-tag.

Ethanol-inducible promoter

Explanations on the figure

50-100 words on results

Light-switchable promoter

The idea behind a lightswitchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelengths.

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 to the bavarian purity law.

50-100 words on results

As we can't obey the letter of the German Purity Law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is unacceptable for us to work with antibiotics to keep up the selective environment. Since we can't work with auxotrophies in beer either, we have to make sure the yeasts don't get rid of the biobricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve genome integration.

Plasmid backbone used for integration of our expression cassettes

50-100 words on results

Contrary to popular opinion, the chief ingredient of beer is not YPD but gyle, a carefully prepared mixture of malt, hop, barley and water. Although the name S. cerevisiae suggests that it is used in the beer brewing process, in fact the yeast employed in brewing have strongly adapted to gyle, as they are reutilized in every succeeding brewing cycle. Hence some investigation on how our yeast performs in gyle was necessary.

Explanations on the figure

Our experiments show that the growth of several different yeast strains is not impaired in gyle!

Expression assays proved the necessity of genome integration for a proper SynBio Beer.

As soon as cloning of integration vectors containing expression cassettes for our biosynthetic pathways are finished, we will repeat the brewing experiments.