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Xanthohumol

Background and principles

4-coumaroyl-CoA

Plant secondary metabolites have proven or assumed beneficial properties and health promoting effects. Stilbenoids, flavonoids or lignins can result from 4-coumaroyl-coenzyme A, which is a nodal compound of phenylproponaoid metabolism in plants.

Biosynthesis:

The biosynthetic pathway of 4-coumaroyl-coenzyme A starts with the conversion of L-Phenylalanine to cinnamate catalyzed by phenylalanin ammonia lyase (PAL). PAL also shows activity with converting tyrosine to p-coumarate, albeit to a lower efficiency. The cinnamate 4-hydroxylase (C4H) catalyzes the synthesis of p-hydroxycinnamate from cinnamate and 4-coumarate: CoA ligase (4CL) converts p-coumarate to its coenzyme-A ester, activating it for reaction with malonyl CoA Emmanouil Trantas et al., 2009.

metabolic pathway from phenylalanin to 4-coumarate

The flavonoid biosynthetic pathway starts with the condensation of one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA, yielding naringenin chalcone. This reaction is carried out by the enzyme chalcone synthase (CHS). Chalcone is isomerised to a flavanone by the enzyme chalcone flavanone isomerase (CHI). From these central intermediates, the pathway diverges into several side branches, each resulting in a different class of flavonoids, for example Xanthohumol. Media:TUM12_Biosynthesis_of_Xanthohumol_(2).jpg

Our project will focus on the production of Xanthohumol, due to its characteristic as a cancer chemopreventive agent (see below).

Xanthohumol and its cancer related bioactivities:

Xanthohumol

Inhibition of the metabolic activation of procarcinogens:

2-amino-3-methylimidazo[4,5-f]quinolone, found in cooked meat, verified as a procarcinogen in an ames salmonella mutagenicity test. The inhibition is probably a result of an inhibition of the cytochrome P 450 enzymes Cyp1A1, Cyp1B1 and Cyp1A2 (phase 1 enzymes). But in order to achieve a clear inhibition, plasma concentrations of 1 µM would be necessary. In a study with male rats oral administration of xanthohumol (50 mg/kg) led to concentration maximums of 65 -180 nM after 4 h. Improved resorption of Xanthohumol could be a possible target for innovation (Yilmazer et al., 2001, Miranda et al., 2000, Henderson et al., 2000, Gerhauser et al., 2002).

Induction of carcinogen-detoxifying enzymes (phase 2 enzymes):

P450-activated carcinogens get conjugated to endogenous ligands (gluthathione, glucoronic acid, acetate and sulfate) by phase 2 enzymes to facilitate excretion. Therefore the induction of phase 2 enzymes should enhance the protection against carcinogenesis. Xanthohumol cat concentrations of 2.1-10.1 µM could induce quinone reductase (detoxification of quinones by ceonversion to hydroquinones which can be conjugated) in hepatoma Hepa 1c1c7 cells. It was shown that xanthohumol could selectively induce quinone reductase without causing a transcriptional activation of Cyp1A1. (Miranda et al., 2000, Gerhauser et al., 2002)

Inhibition of tumor growth at an early stage:

Xantohumol showed an inhibition of the proliferation of breast cancer (MCF-7) and ovarian cancer (A-2780) in vitro at IC50 values of 13 and 0.52 µM (Miranda et al., 1999). Furthermore xanthohumol can inhibit the endogenous prostaglandin synthesis through inhibition of cyclooxygenase (COX-1 and COX-2) with IC50 values of 17 and 42 µM. An increased prostaglandin production has been associated with the uncontrolled proliferation of tumor cells (Gerhauser et al., 2002). Pharmacokinetic studies for xanthohumol based on beverages with an xanthohumol content of 50 mg/l in humans are part of actual research activities. According to a scientist at the TA-XAN AG the first results will be published in June at a conference in Florenz.

Antioxidant activities:

Xanthohumol at 5 µM decreased conjugated diene formation as a measure for lipid peroxidation by more than 70 % after 5 h of incubation in an in vitro assay (protection of LDL from Cu2+ induced oxidation). Furthermore Xanthohumol was shown to scavenge hydroxyl-, peroxyl- and superoxide anion radicals (Miranda et al., 2000).

Idea

The idea is to perform a heterologous gene expression of all enzymes required for Xanthohumol biosynthesis in Saccharomyces cerevisiae. First, each enzyme should be expressed individually and the activities should be tested individually to ensure the functionality. Each gene should be inserted in a yeast expression vector under the control of a GAL10 promotor.

The final goal is the expression of all required genes in a single modified yeast to produce Xanthohumol out of the substrate L-Tyrosin.

General remarks

Detail of Jiang et al., 2004

Proof of principle

Jiang et al succeed in the biosynthesis of several flavonoids in Saccharomyces cerevisiae by the assembly of a plasmid which contains three required enzymes (pKS2µHyg-PAL-4CL-CHS). The activity of each enzyme was demonstrated and the presence of naringenin, which forms the product of the three enzymes(PAL, 4CL, CHS; see also picture on the right) was shown. Hanxiao Jiang et al., 2004

Necessary enzymes for the biosynthesis of xanthohumol

Biosynthesis of Xanthohumol

ENZYME 1: PAL = phenylalanine ammonia lyase: L-phenylalanin --> trans-cinnamate

ENZYME 2: 4CL = 4-coumarate - coenzym A ligase: 4-coumarate --> 4-coumaroyl-CoA

ENZYME 3: CHS = naringenin - chalcone synthase: 4-coumaroyl-CoA --> naringeninchalcone

ENZYME 4: APT = aromatic prenyltransferase: naringeninchalcone --> desmethylxanthohumol

ENZYME 5: OMT1 = chalcone O-methyltransferase: desmethylxanthohumol --> xanthohumol

Source: http://biocyc.org/META/NEW-IMAGE?type=NIL&object=PWY-5135

Enzymes - Sequences, Translation and Assays

Sequences of PAL, 4Cl and CHS come from Paper Hanxiao Jiang et al., 2004

Enzyme 1: phenylalanine ammonia lyase (PAL)

we only use the sequence from start- to stopcodon (bold): 1-2151

Sequence

>gi|18698155|emb|AX366866.1| Sequence 18 from Patent WO0208402

       1 atggcaccct cgctcgactc gatctcgcac tcgttcgcaa acggcgtcgc atccgcaaag
      61 caggctgtca atggcgcctc gaccaacctc gcagtcgcag gctcgcacct gcccacaacc
     121 caggtcacgc aggtcgacat cgtcgagaag atgctcgccg cgccgaccga ctcgacgctc
     181 gaactcgacg gctactcgct caacctcgga gacgtcgtct cggccgcgag gaagggcagg
     241 cctgtccgcg tcaaggacag cgacgagatc cgctcaaaga ttgacaaatc ggtcgagttc
     301 ttgcgctcgc aactctccat gagcgtctac ggcgtcacga ctggatttgg cggatccgca
     361 gacacccgca ccgaggacgc catctcgctc cagaaggctc tcctcgagca ccagctctgc
     421 ggtgttctcc cttcgtcgtt cgactcgttc cgcctcggcc gcggtctcga gaactcgctt
     481 cccctcgagg ttgttcgcgg cgccatgaca atccgcgtca acagcttgac ccgcggccac
     541 tcggctgtcc gcctcgtcgt cctcgaggcg ctcaccaact tcctcaacca cggcatcacc
     601 cccatcgtcc ccctccgcgg caccatctct gcgtcgggcg acctctctcc tctctcctac
     661 attgcagcgg ccatcagcgg tcacccggac agcaaggtgc acgtcgtcca cgagggcaag
     721 gagaagatcc tgtacgcccg cgaggcgatg gcgctcttca acctcgagcc cgtcgtcctc
     781 ggcccgaagg aaggtctcgg tctcgtcaac ggcaccgccg tctcagcatc gatggccacc
     841 ctcgctctgc acgacgcaca catgctctcg ctcctctcgc agtcgctcac ggccatgacg
     901 gtcgaagcga tggtcggcca cgccggctcg ttccacccct tccttcacga cgtcacgcgc
     961 cctcacccga cgcagatcga agtcgcggga aacatccgca agctcctcga gggaagccgc
    1021 tttgctgtcc accatgagga ggaggtcaag gtcaaggacg acgagggcat tctccgccag
    1081 gaccgctacc ccttgcgcac gtctcctcag tggctcggcc cgctcgtcag cgacctcatt
    1141 cacgcccacg ccgtcctcac catcgaggcc ggccagtcga cgaccgacaa ccctctcatc
    1201 gacgtcgaga acaagacttc gcaccacggc ggcaatttcc aggctgccgc tgtggccaac
    1261 accatggaga agactcgcct cgggctcgcc cagatcggca agctcaactt cacgcagctc
    1321 accgagatgc tcaacgccgg catgaaccgc ggcctcccct cctgcctcgc ggccgaagac
    1381 ccctcgctct cctaccactg caagggcctc gacatcgccg ctgcggcgta cacctcggag
    1441 ttgggacacc tcgccaaccc tgtgacgacg catgtccagc cggctgagat ggcgaaccag
    1501 gcggtcaact cgcttgcgct catctcggct cgtcgcacga ccgagtccaa cgacgtcctt
    1561 tctctcctcc tcgccaccca cctctactgc gttctccaag ccatcgactt gcgcgcgatc
    1621 gagttcgagt tcaagaagca gttcggccca gccatcgtct cgctcatcga ccagcacttt
    1681 ggctccgcca tgaccggctc gaacctgcgc gacgagctcg tcgagaaggt gaacaagacg
    1741 ctcgccaagc gcctcgagca gaccaactcg tacgacctcg tcccgcgctg gcacgacgcc
    1801 ttctccttcg ccgccggcac cgtcgtcgag gtcctctcgt cgacgtcgct ctcgctcgcc
    1861 gccgtcaacg cctggaaggt cgccgccgcc gagtcggcca tctcgctcac ccgccaagtc
    1921 cgcgagacct tctggtccgc cgcgtcgacc tcgtcgcccg cgctctcgta cctctcgccg
    1981 cgcactcaga tcctctacgc cttcgtccgc gaggagcttg gcgtcaaggc ccgccgcgga
    2041 gacgtcttcc tcggcaagca agaggtgacg atcggctcga acgtctccaa gatctacgag
    2101 gccatcaagt cgggcaggat caacaacgtc ctcctcaaga tgctcgctta gacactcttc
    2161 ccactctcgc atcccttcca taccctatcc cgcctgcact cttaggactc gcttcttgtc
    2221 ggactcggat ctcgcatcgc ttctttcgtt cttggctgcc tctctagacc gtgtccgtat
    2281 tacctcgaga ttgtgaatac aagcagtacc catccacgca tccgataaat cagggagaga
    2341 atctacgctt gcgggagctt cttgcgcata aactgtcgag tgcgggcgtt agtgcgaagt
    2401 caacgaaggc gagtggcagc ggctcactac cgcctcgag

Translation

>gi|18698155|emb|AX366866.1| 1-2151

MAPSLDSISHSFANGVASAKQAVNGASTNLAVAGSHLPTTQVTQVDIVEKMLAAPTDSTLELDGYSLNLGDVVSAARKGRPVRVKDSDEIRSKIDKSVEFLRSQLSMSVYGVTTGFGGSADTRTEDAISLQKALLEHQLC
GVLPSSFDSFRLGRGLENSLPLEVVRGAMTIRVNSLTRGHSAVRLVVLEALTNFLNHGITPIVPLRGTISASGDLSPLSYIAAAISGHPDSKVHVVHEGKEKILYAREAMALFNLEPVVLGPKEGLGLVNGTAVSASMAT
LALHDAHMLSLLSQSLTAMTVEAMVGHAGSFHPFLHDVTRPHPTQIEVAGNIRKLLEGSRFAVHHEEEVKVKDDEGILRQDRYPLRTSPQWLGPLVSDLIHAHAVLTIEAGQSTTDNPLIDVENKTSHHGGNFQAAAVAN
TMEKTRLGLAQIGKLNFTQLTEMLNAGMNRGLPSCLAAEDPSLSYHCKGLDIAAAAYTSELGHLANPVTTHVQPAEMANQAVNSLALISARRTTESNDVLSLLLATHLYCVLQAIDLRAIEFEFKKQFGPAIVSLIDQHF
GSAMTGSNLRDELVEKVNKTLAKRLEQTNSYDLVPRWHDAFSFAAGTVVEVLSSTSLSLAAVNAWKVAAAESAISLTRQVRETFWSAASTSSPALSYLSPRTQILYAFVREELGVKARRGDVFLGKQEVTIGSNVSKIYE
AIKSGRINNVLLKMLA

Compatibility (iGEM and S. cerevisiae)

Name	Length	RFC10	RFC25	Codon Usage	NCBI
Phenylalanine ammonia lyase	2439bp bzw. 2151bp	ok	6xNgoMIV (1438,1684,1852,1995,2329)	0 AS<10%	[http://www.ncbi.nlm.nih.gov/nuccore/AX366866]

Purification and Assay

Name	used restriction sites	purification	assay
Phenylalanine ammonia lyase	XbaI, AgeI	Strep tag II	substrate: L-tyrosin, product: 4-coumarate

Enzyme 2: 4-coumarate - coenzym A ligase (4CL)

we only use the sequence from start- to stopcodon (bold): 6-1691

>gi|609339|gb|U18675.1|ATU18675 Arabidopsis thaliana 4-coumarate--coenzyme A ligase (At4CL1) mRNA, complete cds

       1 ttacaatggc gccacaagaa caagcagttt ctcaggtgat ggagaaacag agcaacaaca
      61 acaacagtga cgtcattttc cgatcaaagt taccggatat ttacatcccg aaccacctat
     121 ctctccacga ctacatcttc caaaacatct ccgaattcgc cactaagcct tgcctaatca
     181 acggaccaac cggccacgtg tacacttact ccgacgtcca cgtcatctcc cgccaaatcg
     241 ccgccaattt tcacaaactc ggcgttaacc aaaacgacgt cgtcatgctc ctcctcccaa
     301 actgtcccga attcgtcctc tctttcctcg ccgcctcctt ccgcggcgca accgccaccg
     361 ccgcaaaccc tttcttcact ccggcggaga tagctaaaca agccaaagcc tccaacacca
     421 aactcataat caccgaagct cgttacgtcg acaaaatcaa accacttcaa aacgacgacg
     481 gagtagtcat cgtctgcatc gacgacaacg aatccgtgcc aatccctgaa ggctgcctcc
     541 gcttcaccga gttgactcag tcgacaaccg aggcatcaga agtcatcgac tcggtggaga
     601 tttcaccgga cgacgtggtg gcactacctt actcctctgg cacgacggga ttaccaaaag
     661 gagtgatgct gactcacaag ggactagtca cgagcgttgc tcagcaagtc gacggcgaga
     721 acccgaatct ttatttccac agcgatgacg tcatactctg tgttttgccc atgtttcata
     781 tctacgcttt gaactcgatc atgttgtgtg gtcttagagt tggtgcggcg attctgataa
     841 tgccgaagtt tgagatcaat ctgctattgg agctgatcca gaggtgtaaa gtgacggtgg
     901 ctccgatggt tccgccgatt gtgttggcca ttgcgaagtc ttcggagacg gagaagtatg
     961 atttgagctc gataagagtg gtgaaatctg gtgctgctcc tcttggtaaa gaacttgaag
    1021 atgccgttaa tgccaagttt cctaatgcca aactcggtca gggatacgga atgacggaag
    1081 caggtccagt gctagcaatg tcgttaggtt ttgcaaagga accttttccg gttaagtcag
    1141 gagcttgtgg tactgttgta agaaatgctg agatgaaaat agttgatcca gacaccggag
    1201 attctctttc gaggaatcaa cccggtgaga tttgtattcg tggtcaccag atcatgaaag
    1261 gttacctcaa caatccggca gctacagcag agaccattga taaagacggt tggcttcata
    1321 ctggagatat tggattgatc gatgacgatg acgagctttt catcgttgat cgattgaaag
    1381 aacttatcaa gtataaaggt tttcaggtag ctccggctga gctagaggct ttgctcatcg
    1441 gtcatcctga cattactgat gttgctgttg tcgcaatgaa agaagaagca gctggtgaag
    1501 ttcctgttgc atttgtggtg aaatcgaagg attcggagtt atcagaagat gatgtgaagc
    1561 aattcgtgtc gaaacaggtt gtgttttaca agagaatcaa caaagtgttc ttcactgaat
    1621 ccattcctaa agctccatca gggaagatat tgaggaaaga tctgagggca aaactagcaa
    1681 atggattgtg atggatgatt tcaaccaaaa agcaaagatg atttcaatgt gtatatacat
    1741 acaactgttt gacccaacca aggaaacaaa ctcatacgaa ccattgtctt ttgttgttgt
    1801 tgttgttgtt gttgttgctg ttcttgcttg attcatgtaa tgagcctttg tgatgaaggt
    1861 ggtttcttt

Translation 6-1691

MAPQEQAVSQVMEKQSNNNNSDVIFRSKLPDIYIPNHLSLHDYIFQNISEFATKPCLINGPTGHVYTYSDVHVISRQIAANFHKLGVNQNDVVMLLLPNCPEFVLSFLAASFRGATATAANPFFTPAEIAKQAKASNTKLIITEARYVDKIKPLQNDDGV
VIVCIDDNESVPIPEGCLRFTELTQSTTEASEVIDSVEISPDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYFHSDDVILCVLPMFHIYALNSIMLCGLRVGAAILIMPKFEINLLLELIQRCKVTVAPMVPPIVLAIAKSSETEKYDL
SSIRVVKSGAAPLGKELEDAVNAKFPNAKLGQGYGMTEAGPVLAMSLGFAKEPFPVKSGACGTVVRNAEMKIVDPDTGDSLSRNQPGEICIRGHQIMKGYLNNPAATAETIDKDGWLHTGDIGLIDDDDELFIVDRLKELIKYKGFQVAPAELEALLIGH
PDITDVAVVAMKEEAAGEVPVAFVVKSKDSELSEDDVKQFVSKQVVFYKRINKVFFTESIPKAPSGKILRKDLRAKLANGL

Compatibility (iGEM and S. cerevisiae)

Name	Length	RFC10	RFC25	Codon Usage	NCBI
Arabidopsis thaliana 4-coumarate--coenzyme A ligase	1869 bp bzw. 1685 bp	2xEcoRI (149-154, 309-314), 1x Spe1 (679-684)	ok after RFC10	0 AS<10%	[http://www.ncbi.nlm.nih.gov/nucleotide/609339/]

Purification and Assay

Name	used restriction sites	purification	assay
Arabidopsis thaliana 4-coumarate--coenzyme A ligase	XbaI, PstI	crude protein extraction	substrate: 4-coumarate, product: 4-coumaryl-CoA

Enzyme 3: naringenin - chalcone synthase(CHS)

we only use the sequence from start- to stopcodon (bold): 76-1248

>gi|11096318|gb|AF315345.1| Hypericum androsaemum chalcone synthase mRNA, complete cds

       1 aaactctgtc accacattat tgtaccttgt aacagcaagg tgcttaactg gttgatttaa
      61 acataaacaa ggaagatggt gaccgtggaa gaagtcagga aggcgcagcg ggccgagggt
     121 ccggccaccg tgatggccat cggaacggcc gtcccgccga actgcgttga ccaagcgacg
     181 taccccgact attatttccg tatcaccaac agcgagcaca aggccgagct caaggagaag
     241 ttccaacgca tgtgtgataa gtctcaaatc aagaaacgtt acatgtacct gaacgaggag
     301 gtcctcaaag agaaccccaa tatgtgtgct tacatggcac cttctctgga tgctaggcaa
     361 gacattgtgg tggtggaagt gcccaaacta ggtaaagagg cagcagttaa ggccatcaag
     421 gaatggggcc agcctaagtc caagatcacc cacttggtct tttgcaccac tagtggagtg
     481 gacatgcccg gggccgacta ccagctcacc aagctattgg gcctccgccc gtcggtgaag
     541 cgcctcatga tgtaccagca gggctgcttt gccggtggca cggtcctccg tctcgccaag
     601 gatctcgccg agaacaacaa gggtgcacgc gtccttgtcg tctgctcgga gatcacggcc
     661 gttaccttcc gtgggcccac cgacactcac ctcgacagcc ttgtgggcca ggcattgttc
     721 ggtgacggcg ctgccgccat catcatcggc tcggacccga tccccgaagt cgagaagccc
     781 ttgttcgagc tggtctccgc agcccagacc attctaccgg acagtgaggg tgcgatagac
     841 ggacatctcc gcgaggttgg gcttacattc cacttgctca aggatgttcc cgggttgatc
     901 tctaagaacg ttgagaagag cctcactgag gccttcaaac cgttgggcat ttcagattgg
     961 aactccctgt tttggatcgc ccacccaggc ggcccagcaa tcttggacca ggtagaggcc
    1021 aagttgagcc tcaagcccga gaagctacgg gccacaaggc acgtactttc cgagtacgga
    1081 aacatgtcta gtgcctgtgt gcttttcatc ttagacgaga tgaggaggaa gtccaaggaa
    1141 gacgggctta agaccacagg ggaaggaatc gagtggggag tgctttttgg atttgggcct
    1201 gggcttaccg ttgagaccgt tgtccttcac agtgtcgcca ttaactaggt caaggtcgtt
    1261 gctttgcgtt ttttactttg ttgttgcctg taatattttc actacttggc gtcttttttt
    1321 cactttctaa cttctaatgt tttacctctg ggtcaaacat atgtggtgca gtgaaaaact
    1381 gaaaaaaaaa aaaaaaaaaa aa

Translation 76-1248

MVTVEEVRKAQRAEGPATVMAIGTAVPPNCVDQATYPDYYFRITNSEHKAELKEKFQRMCDKSQIKKRYMYLNEEVLKENPNMCAYMAPSLDARQDIVVVEVPKLGKEAAVKAIKEWGQPKSKITHLVFCTTSGVDMPGADYQLTKLLGLRPSVKRLMMY
QQGCFAGGTVLRLAKDLAENNKGARVLVVCSEITAVTFRGPTDTHLDSLVGQALFGDGAAAIIIGSDPIPEVEKPLFELVSAAQTILPDSEGAIDGHLREVGLTFHLLKDVPGLISKNVEKSLTEAFKPLGISDWNSLFWIAHPGGPAILDQVEAKLSLK
PEKLRATRHVLSEYGNMSSACVLFILDEMRRKSKEDGLKTTGEGIEWGVLFGFGPGLTVETVVLHSVAIN

Compatibility (iGEM and S. cerevisiae)

Name	Length	RFC10	RFC25	Codon Usage	NCBI
Hypericum androsaemum chalcone synthase	1402 bp	1xSpeI (469)	ok after RFC10	2 AS<10%	[http://www.ncbi.nlm.nih.gov/nucleotide/11096318/]

Purification and Assay

Name	used restriction sites	purification	assay
Hypericum androsaemum chalcone synthase	XbaI, AgeI	Strep tag II	subtrate:

Enzyme 4: aromatic prenyltransferase (APT)

we decided to have the gene synthesized

>gi|11096318|gb|(AB543053.1)| optimized sequence with restriction sites (XbaI and AgeI) (gene synthesis)

    1 tctagatggctttgtcatccgtttcttcattttctttgggtaccaacccattcatctcca 
   61 tcccacataacaacaacaacttgaaggtttcttcctactgctgcaaatctaagtccagag 
  121 ttatcaactccactaactctaaacattgctccccaaacaacaacaacaacacttctaaca 
  181 agaccacccatttgttgggtttatacggtcaatcaagatgcttgttgaagccattgtctt 
  241 tcatctcttgcaacgatcaaagaggtaactctattagagcttccgcccaaattgaagata 
  301 gaccaccagaatctggtaacttgtctgctttgactaacgttaaggatttcgtttctgttt 
  361 gctgggaatacgttagaccatatactgctaagggtgttatcatttgctcctcttgtttgt 
  421 tcggtagagaattattggaaaacccaaacttgttctccagaccattgattttcagagcct 
  481 tgttgggtatgttggctattttgggttcttgtttttacaccgccggtatcaatcaaatct 
  541 tcgatatggatatcgacagaatcaacaagccagatttgccattggtttccggtagaattt 
  601 ctgttgaatctgcttggttgttgactttgtccccagctattattggtttcatcttgatct 
  661 tgaagttgaactccggtcctttgttgacctcattatactgtttggcaatcttgtccggta 
  721 ctatctattctgttccaccttttagatggaagaagaatccaattaccgccttcttgtgca 
  781 ttttgatgattcatgctggtttgaacttctccgtttactatgcttcaagagctgctttgg 
  841 gtttggcttttgcttggtcaccatctttttctttcattaccgctttcatcaccttcatga 
  901 ctttgactttggcttcctctaaggatttgtccgatattaacggtgatagaaagttcggtg 
  961 ttgaaactttcgctacaaaattgggtgctaagaacatcaccttgttaggtactggtttgt 
 1021 tattattgaactacgttgctgctatttccaccgctattatttggcctaaagctttcaagt 
 1081 ccaacatcatgttgttgtcccatgctatcttggccttttcattgatctttcaagctagag 
 1141 aattggacagaactaactacactccagaagcttgtaagtccttctacgaatttatctgga 
 1201 ttttgttctccgccgaatacgttgtttacttgttcatcaccggt

Translation 1-1236

MELSSVSSFSLGTNPFISIPHNNNNLKVSSYCCKSKSRVINSTNSKHCSPNNNNNTSNKTTHLLGLYGQSRCLLKPLSFISCNDQRGNSIRASAQIEDRPPESGNLSALTNVKDFVSVCWEYVRPYTAKGVIICSSCLFGRELLENPNLFSRPLIFRALL
GMLAILGSCFYTAGINQIFDMDIDRINKPDLPLVSGRISVESAWLLTLSPAIIGFILILKLNSGPLLTSLYCLAILSGTIYSVPPFRWKKNPITAFLCILMIHAGLNFSVYYASRAALGLAFAWSPSFSFITAFITFMTLTLASSKDLSDINGDRKFGVE
TFATKLGAKNITLLGTGLLLLNYVAAISTAIIWPKAFKSNIMLLSHAILAFSLIFQARELDRTNYTPEACKSFYEFIWILFSAEYVVYLFI

Compatibility (iGEM and S. cerevisiae)

Name	Length	RFC10	RFC25	Codon Usage	NCBI
Humulus lupulus aromatic prenyltransferase	1236 bp	ok	ok	optimized by GeneArt	[http://www.ncbi.nlm.nih.gov/nuccore/AB543053]

Purification and Assay

Name	used restriction sites	purification	assay
Humulus lupulus aromatic prenyltransferase	XbaI, AgeI	Strep tag II	substrate:

Enzyme 5: chalcone O-methyltransferase(OMT1)

we only use the sequence from start- to stopcodon (50-1108bold):

Humulus lupulus O-methyltransferase 1 (OMT1) mRNA, complete cds, Genbank EU309725.1

              1 ggacacaatt caatctattt tacccaaaaa ataactaaga aagaccaata tggaatctct
      61 aagaggccaa gaacagatat ggcaactcat gttcagcttt gtcgactcca tggccttgaa
     121 atgcgccatc gagcttcgca ttgctgacat cattcactct catggcaaac ctataactct
     181 ctcccaaata gcttctggca ttcgatcaaa ctccaactcc tccatatctc cgaatattcc
     241 ttacctctct cgcatcatga gatttcttgt tcgaaagaat atcttcactg aacatcaaga
     301 agataatgat gaggtgatct cattgtacgg gctaagtgat agctcgagat ggctgttgcg
     361 ggattttaag tcaagcctgg ctcccatggt gctcatgcag actcatccat tgtcgatggc
     421 ggtgtggcat ttccttgagg attatgtgag aaacagcagc aacactttcg aaaaggctca
     481 cggttgtaac atttgggagt tttcctcagc caatccagat ttcaacaaga tcttcaacaa
     541 tgccatggcg agtattgtgc caatatacat gggggctgtg ctttcaagtt ataaggatgg
     601 tcttggttgt attaaaggaa cagtggtgga cgttgggggt ggtacgggcg gctccatatc
     661 agagcttatg aaatattatc caaacatcaa agggattaac tttgacctgc cacatgtgat
     721 tgccacagca ccggcattgg atggtgttac ccatattagt ggtgacatat tcgagtcaat
     781 tcctagtgct gatgcggttt taatgaaggg tgtactacat tgcttcagcg atgaaaaatg
     841 tgtaaaagta ttgagaaatt gtcgaaaagc aataacagac aaaaagaatg ggaagattat
     901 cattttggag attgtgttgg acccaaccag caatcaaata tttgacgaga ctcgaatggt
     961 gtacgattta ttgattccay tctttagtgg tggaaaagag agaactgagc ttgaatggaa
    1021 aaggctatta aacgaggctg gttttacttc tatcaaaatc accaaaattc caattatacc
    1081 tgctattatt gaggcctttc tagtgtgaca acrtcgatct atctatatat atataaacta
    1141 ggttatgttg ctttcaacaa taagttccct atgtactgtt acggttatgt atggtttgct
    1201 gtgattaata taatatgttg gc

Translation 50-1108

MESLRGQEQIWQLMFSFVDSMALKCAIELRIADIIHSHGKPITLSQIASGIRSNSNSSISPNIPYLSRIMRFLVRKNIFTEHQEDNDEVISLYGLSDSSRWLLRDFKSSLAPMVLMQTHPLSMAVWHFLEDYVRNSSNTFEKAHGCNIWEFSSANPDFNK
IFNNAMASIVPIYMGAVLSSYKDGLGCIKGTVVDVGGGTGGSISELMKYYPNIKGINFDLPHVIATAPALDGVTHISGDIFESIPSADAVLMKGVLHCFSDEKCVKVLRNCRKAITDKKNGKIIILEIVLDPTSNQIFDETRMVYDLLIPXFSGGKERTE
LEWKRLLNEAGFTSIKITKIPIIPAIIEAFLV

Compatibility (iGEM and S. cerevisiae)

Name	Length	RFC10	RFC25	Codon Usage	NCBI
Humulus lupulus O-methyltransferase 1	1058	ok	ok	2 AS<10%	[http://www.ncbi.nlm.nih.gov/nucleotide/167613934/]

Purification and Assay

Name	used restriction sites	purification	assay
Humulus lupulus O-methyltransferase 1	XbaI, AgeI	Strep tag II	subtrate:

References

C Gerhauser, A Alt, E Heiss, A Gamal-Eldeen, K Klimo, J Knauft, I Neumann, H.R Scherf, N Frank, H Bartsch, H Becker Cancer chemopreventive activity of xanthohumol, a natural product derived from hop Mol. Cancer Ther., 1 (2002), pp. 959–969

M.C Henderson, C.L Miranda, J.F Stevens, M.L Deinzer, D.R Buhler In vitro inhibition of human P450 enzymes by prenylated flavonoids from hops, Humulus lupulus Xenobiotica, 30 (2000), pp. 235–251

C.L Miranda, G.L Aponso, J.F Stevens, M.L Deinzer, D.R Buhler Prenylated chalcones and flavanones as inducers of quinone reductase in mouse Hepa 1c1c7 cells Cancer Lett., 149 (2000), pp. 21–29

C.L Miranda, J.F Stevens, A Helmrich, M.C Henderson, R.J Rodriguez, Y.H Yang, M.L Deinzer, D.W Barnes, D.R Buhler Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines Food Chem. Toxicol., 37 (1999), pp. 271–285

C.L Miranda, J.F Stevens, V Ivanov, M McCall, B Frei, M.L Deinzer, D.R Buhler Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro J. Agric. Food Chem., 48 (2000), pp. 3876–3884

C.L Miranda, Y.H Yang, M.C Henderson, J.F Stevens, G Santana-Rios, M.L Deinzer, D.R Buhler, Prenylflavonoids from hops inhibit the metabolic activation of the carcinogenic heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline, mediated by cDNA-expressed human CYP1A2 Drug Metab. Dispos., 28 (2000), pp. 1297–1302

M Yilmazer, J.F Stevens, M.L Deinzer, D.R Buhler In vitro biotransformation of xanthohumol, a flavonoid from hops (Humulus lupulus), by rat liver microsomes Drug Metab. Dispos., 29 (2001), pp. 223–231

M Yilmazer, J.F Stevens, D.R Buhler In vitro glucuronidation of xanthohumol, a flavonoid in hop and beer, by rat and human liver microsomes FEBS Lett., 491 (2001), pp. 252–256

Team:TU Munich/Project/Xanthohumol

From 2012.igem.org

Contents

Xanthohumol

Background and principles

Biosynthesis:

Xanthohumol and its cancer related bioactivities:

Inhibition of the metabolic activation of procarcinogens:

Induction of carcinogen-detoxifying enzymes (phase 2 enzymes):

Inhibition of tumor growth at an early stage:

Antioxidant activities:

Idea

General remarks

Proof of principle

Necessary enzymes for the biosynthesis of xanthohumol

Enzymes - Sequences, Translation and Assays

Enzyme 1: phenylalanine ammonia lyase (PAL)

Enzyme 2: 4-coumarate - coenzym A ligase (4CL)

Enzyme 3: naringenin - chalcone synthase(CHS)

Enzyme 4: aromatic prenyltransferase (APT)

Enzyme 5: chalcone O-methyltransferase(OMT1)

References