Conversion of stilbenoids

The present invention relates to the production and recovery of stilbenoids of the general formula 1:

Formula 1

Stilbenoids having the general formula 1 wherein Rl, R2, R3, R4, and R5 independently are either -H or -OH are phytoalexins produced in certain plants examples of which have been found to have antioxidant and anti-tumour properties of interest.

In pinosylvin, all the variable R groups are hydrogen. para-Resveratrol, where R^ is -OH, and piceatannol where R^ and R3 are -OH are of particular interest. The production of resveratrol and of pinosylvin by microbial expression has been described (WO06/089898 and WO2008/009728) . South African Patent 2004/8194 (University of Stellenbosch) and Becker et al disclosed a Saccharomyces cerevisiae for fermenting wine must having introduced therein a coumarate- coenzyme-A ligase encoding gene (4CL216) and a grapevine resveratrol synthase gene (vstl) . WO2006/125000 discloses oleaginous cells having resveratrol production capacity. WO2006/124999 discloses bacteria producing resveratrol.

Resveratrol can be produced (in micro-organisms) via two pathways, starting from phenylalanine or tyrosine. The pathway from phenylalanine requires 4 enzymes, phenylalanine ammonia lyase (PAL) , cinnamate-4-hydroxylase (C4H) and

preferably its electron supporting donor cytochrome p450 reductase (CPR) , 4-coumaroyl-CoA ligase (4CL) and resveratrol synthase (VST) (Figure I)) . The pathway from tyrosine requires only three enzymes tyrosine ammonia lyase (TAL) , 4- coumaroyl-CoA ligase (4CL) and resveratrol synthase (VST) .

Due to the complex nature of the 5 enzyme phenylalanine pathway (Fig 1) a second hydroxystilbene compound, called pinosylvin (see above) , can be formed as by-product if the phenylalanine ammonia lyase activity is higher than the subsequent hydroxylating activity of the C4H/CPR complex when converting cinnamate to coumarate (Fig 1) . The pinosylvin is formed because the 4CL and VST genes are rather promiscuous enzymes and can accept both cinnamic acid and coumaric acid precursors . Pinosylvin is not converted to resveratrol by the enzymes in this pathway. In particular, the normal cinnamate hydroxylase from the plant Arabidopsis thaliana does not carry out the conversion, which involves the addition of one -OH group (Chen and Morgan, 2007) . A minor amount of resveratrol has been identified as a product when pinosylvin is exposed to rat liver microsomes (Roupe et al, 2005) but the mechanism involved was not disclosed. Both human and rat liver microsomes have been found to have a similar metabolite profile when fed with non- hydroxylated stilbene. The trans-stilbene was hydroxylated in the para-position by certain liver cytochrome P450 enzymes (CYPs) in both rat and human (Sanoh et al,2002) . Resveratrol has been found to be converted to piceatannol by CYPlBl in human liver microsomes (Potter et al) . The present invention provides a method for producing a cis or trans (preferably trans-) target stilbenoid having the general formula 1

Formula 1

wherein Rl, R2, R3, R4, and R5 independently are either -H or -OH with the proviso that at least one of Rl, R2, R3, R4, and R5 is -OH comprising:

supplying to a micro-organism capable of reproduction, or producing in a micro-organism capable of reproduction, a substrate stilbenoid of the formula 2

Formula 2 wherein Rl, R2, R3, R4, and R5 independently are either -H or -OH with the proviso that at least one of Rl, R2, R3, R4, and

R5 is -H, and producing in said micro-organism a hydroxylase capable of hydroxylating said substrate stilbenoid to produce therefrom a said target stilbenoid having one or more additional ones of the R groups as an -OH group compared to the substrate stilbenoid.

The target stilbenoid can in particular be resveratrol or piceatannol.

This enables one to obtain resveratrol or other hydroxylated stilbenoids by conversion of pinosylvin. This may be carried out in a micro-organism having a metabolic pathway producing pinosylvin. Alternatively, pinosylvin may be supplied to a micro-organism producing said hydroxylase for conversion by said hydroxylase. In principle, a first micro-organism producing pinosylvin and a second microorganism producing said hydroxylase may be cultured together if compatible. Alternatively, pinosylvin may be recovered from a culture of a said first micro-organism (which may also produce hydroxylated stilbenoid, e.g. resveratrol) and may be supplied to a separate culture of said second micro-organism.

Said hydroxylating enzyme may be a cytochrome enzyme, especially a cytochrome p-450 (CYP) . The enzyme may be a mammalian enzyme, for instance derived from the rat or human, and may be codon optimised for expression in said micro- organism. It may be CYP2C19.

P450 monooxygenases such as CYP2C19 function as an enzyme complex with P450 reductase. To improve enzymatic activity and turnover rate of CYP2C19 in heterologous and homologous expression systems, co-expression of P450 reductase or construction of a chimeric protein containing monooxygenase and reductase activity would give this effect (Dodhia et al . , 2006, Parikh et al . , 1997, Hayashi et al . , 2000) . This strategy may be used to increase activity of all P450 monooxygenases including those involved in the phenylpropanoid pathway.

The stilbenoid producing micro-organism may be of a genus or species as described in WO06/089898 and WO2008/009728 or genetically engineered according to the

principles or practice there described. In particular, it may be a resveratrol producing micro-organism as described generally or by way of example in WO2006/089898 or a pinosylvin producing micro-organism as described generally or by way of example in WO2008/009728 or in WO2006/124999 or

WO2006/125000. The stilbenoid may be secreted by the microorganism or retained in the cells thereof.

Preferably, the micro-organism may be one having an operative metabolic pathway comprising at least one enzyme activity, said pathway producing 4-coumaric acid and producing resveratrol therefrom, or an oligomeric or glycosidically-bound derivative thereof preferably by a reaction catalysed by an enzyme in which endogenous malonyl- CoA is a substrate. Preferably the resveratrol is produced from 4-coumaroyl-CoA by a resveratrol synthase expressed in said micro-organism from nucleic acid coding for said enzyme which is not native to the micro-organism.

The 4-coumaric acid may be produced from trans-cinnamic acid by a cinnamate 4-hydroxylase not native to the micro- organism.

4-coumaric acid may be produced from tyrosine in a reaction catalysed by a L-phenylalanine ammonia lyase or a tyrosine ammonia lyase not native to the micro-organism. Trans-cinnamic acid may be produced from L-phenylalanine in a reaction catalysed by a L-phenylalanine ammonia lyase not native to the micro-organism. 4-coumaroyl-CoA may be formed in a reaction catalysed by a 4-coumarate-CoA ligase introduced into the micro-organism.

A native NADPH: cytochrome P450 reductase (CPR) may be expressed in the micro-organism or may recombinantly introduced.

Thus, the micro-organism may be one containing one or more copies of an heterologous DNA sequence encoding

phenylalanine ammonia lyase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding cinnamate-4-hydroxylase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding 4-coumarate CoA-ligase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding resveratrol synthase operatively associated with an expression signal, or may be one lacking cinnamate-4-hydroxylase activity, and containing one or more copies of a heterologous DNA sequence encoding tyrosine ammonia lyase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding 4-coumarate CoA-ligase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding resveratrol synthase operatively associated with an expression signal.

For the production of pinosylvin, the micro-organism may have an operative metabolic pathway comprising at least one enzyme activity, said pathway producing pinosylvin from cinnamic acid and preferably producing cinnamic acid and produces pinosylvin therefrom. Said pinosylvin may be produced in a reaction catalysed by an enzyme in which endogenous malonyl-CoA is a substrate, suitably from cinnamoyl-CoA by a stilbene synthase, suitably expressed in the micro-organism from nucleic acid coding for said enzyme which is not native to the micro-organism.

Cinnamic acid is preferably produced in said pathway from L-phenylalanine in a reaction catalysed by a L- phenylalanine ammonia lyase (PAL) which may be not native to the micro-organism.

Said PAL is preferably one accepting phenylalanine as a substrate and producing cinnamic acid therefrom, such that if the PAL also accepts tyrosine as a substrate and forms coumaric acid therefrom, the ratio Km (phenylalanine) /Km (tyrosine) for said PAL is less than 1:1 and preferably such that the ratio K _cat (PAL) /K _cat (C4H) is at least 2:1.

Cinnamoyl-CoA may be formed in a reaction catalysed by a 4-coumarate-CoA ligase or a cinnamoyl-CoA ligase which may be not native to the micro-organism.

Any or all of at least one copy of a genetic sequence encoding a phenylalanine ammonia lyase, at least one copy of a genetic sequence encoding a 4-coumarate-CoA ligase or cinnamate-CoA ligase, at least one copy of a genetic sequence encoding a resveratrol synthase or a pinosylvin synthase may be present operatively linked to an expression signal not natively associated with said genetic sequence.

Thus the micro-organism may be one containing one or more copies of an heterologous DNA sequence encoding phenylalanine ammonia lyase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding 4-coumarate CoA-ligase or cinnamate-CoA ligase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding resveratrol synthase operatively associated with an expression signal or may be one containing one or more copies of an heterologous DNA sequence encoding phenylalanine ammonia lyase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding 4- coumarate CoA-ligase or cinnamate-CoA ligase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding

pinosylvin synthase operatively associated with an expression signal .

In all cases, expression of the gene ACCl may be boosted to increase the pool of malonyl-CoA available in the metabolic pathway.

The micro-organism may be a fungus, whether filamentous or unicellular, or may be a bacterium.

In the accompanying drawings:

Figure 1 shows two metabolic pathways to resveratrol and pinosylvin respectively;

Figure 2 shows an HPLC chromatogram of the extraction according to example 8 of shake flask cultivation according to example 7 from strain FSSC-PAL2-CYP2C19. 1: Peak with the same retention time as the cinnamic acid standard;

Figure 3 shows an HPLC chromatogram of the extraction according to example 8 of shake flask cultivation according to example 7 from strain FSSC-PAL2-CYP2C19. 1: Peak with the same retention time as the cinnamic acid standard;

Figure 4 shows a UV absorption spectrum from peak 1 in figure 2. The spectrum matches the cinnamic acid standard;

Figure 5 shows an HPLC chromatogram of the extraction according to example 8 of shake flask cultivation according to example 7 of strain FSSC-PAL2-CYP2C19-4CL2-VST1. 1: Peak with the same retention time as the trans-resveratrol standard. 2: Peak with the same retention time as the trans- pinosylvin standard;

Figure 6 shows an HPLC chromatogram of the extraction according to example 8 of shake flask cultivation according to example 7 of strain FSSC-PAL2-CYP2C19-4CL2-VST1. 1: Peak with the same retention time as the trans-resveratrol standard. 2: Peak with the same retention time as the trans- pinosylvin standard;

Figure 7 shows a UV absorption spectrum from peak 1 in figure

6. The spectrum matches the trans-resveratrol standard;

Figure 8 shows a UV absorption spectrum from peak 2 in figure

6. The spectrum matches the trans-pinosylvin standard;

Figure 9 shows an HPLC chromatogram of a mix of 1: coumaric acid, 2 : trans-resveratrol, 3: cinnamic acid and 4 :pinosylvin .

Figure 10 shows UV absorption spectrum from coumaric acid (peak 1) in figure 9;

Figure 11 shows a UV absorption spectrum from trans- resveratrol (peak 2) in figure 9;

Figure 12 shows a UV absorption spectrum from cinnamic acid (peak 3) in figure 9;

Figure 13 shows a UV absorption spectrum from trans- pinosylvin (peak 4) in figure 9.

When resveratrol is produced via the pathway shown in Figure 1, via cinnamic acid and coumaric acid, it is trans- para-resveratrol that is produced. However, we have found that by feeding the micro-organism with appropriate coumaric acid isomers, other regioisomers of resveratrol can be

obtained. Thus, feeding ortho-coumaric acid produces ortho- resveratrol (R^ = -OH) and feeding meta-coumaric acid produces meta-resveratrol (R^ = -OH) . These compounds are distinguishable by HPLC and UV-spectroscopy . Feeding caffeic acid produces piceatannol. This is true for the resveratrol producing S. cerevisiae of WO06/089898 and has been demonstrated also in E. coli (Katsuyama et al, 2007) .

Recovery of stilbenoids may be effected by solvent extraction of micro-organism cultures, including both extraction from the supernatant and extraction from sedimented or centrifugible solids, especially micro-organism cells. Suitable solvents include ethyl acetate and other esters, especially those as described in GB 0714671.5, i.e. an ester which preferably is of the general formula R ⁶ -COO- R ⁷ , and R ⁶ is H or an aliphatic straight or branched chain hydrocarbon moiety of from 1-6 carbon atoms and R ⁷ is an aliphatic straight or branched chain hydrocarbon moiety of from 2-16 carbon atoms, or a heteroatom containing hydrocarbon moiety of from 2 to 16 carbon atoms or an aromatic or heteroaromatic moiety of from 5 to 16 carbon atoms . R ⁷ may have from 3 to 9 carbon atoms . R ⁶ may have from 1 to 4 carbon atoms .

Preferably, said ester is an octyl acetate, especially n-octyl acetate. Optionally, said liquid comprises or further comprises an alkane. It may consist of a said alkane and a said ester. Said alkane may be a Ce to Ciε straight or branched chain alkane, e.g. a C9-14 alkane, e.g. a C12 alkane. Preferably, said alkane is n-dodecane. Optionally, the stilbenoid is secreted into the cultivation medium in an amount substantially exceeding the solubility limit of the target stilbenoid such that it precipitates, usually as crystals having a size significantly

in excess of the size of the micro-organism cells. The stilbenoid may then be partially recovered by mechanical separation of the solid stilbenoid from the medium and from the micro-organism cells, e.g. by filtration using a medium having a pore size such that the stilbenoid is retained and micro-organism cells are allowed to pass. To encourage the formation of larger stilbenoid crystals, the medium may be rested for a period sufficient to allow crystal growth and/or may be cooled to reduce the solubility of the stilbenoid in the medium, e.g. cooled to below 10 ⁰ C, e.g. below 5 ⁰ C.

The micro-organism is required to be capable of reproduction, i.e. it should be present as viable cells which can reproduce, particularly under the conditions of the method. In an alternative aspect, the invention includes exposing a substrate stilbenoid, preferably pinosylvin, optionally produced in situ by a stilbenoid producing microorganism, to the action of a purified enzyme capable of hydroxylating said substrate stilbenoid to form a target stilbenoid having one or more -OH groups more than the substrate stilbenoid. The stilbenoids and the enzymes for use in this aspect of the invention may be as described above .

The invention will be further described and illustrated by the following description of non-limiting examples.

Example 1 Screening of human synthetic liver CYPs

Six individual human CYPs as purified enzymes (Sigma- Aldrich and Codexis) were screened by supplying each purifed CYP with NADPH and pure pinosylvin standard (from

Gentaur (France) and ArboNova (Finland)) . One CYP, CYP2C19, was found to convert pinosylvin to a compound (peak in HPLC

chromatogram) with a UV-absorbance spectrum and retention time and molecular mass similar to pure resveratrol standard.

CYP2C19 was found to be capable of biotransforming Pinosylvin to Resveratrol with a turnover number of approx 10 min ^x , with minor amounts of the meta-hydroxy regioisomer (1- 2%) as a by-product. The affinity of CYP2C19 for pinosylvin was determined to be high: no significant differences in specific activity (all activities were within 10%) were found when initial concentrations of pinosylvin of 250, 100, and 20 μM were used. On the basis of these results, the Km value of CYP2C19 for Pinosylvin is estimated to be well below 10 μM. No significant concentrations of piceatannol were formed during the incubations of pinosylvin with CYP2C19. However, when incubated with 100 uM resveratrol for 120 minutes, Cyp2C19 gave rise to very low - but significant - amounts of piceatannol formation (2-3 μM) . This would indicate a turnover rate of about 1-2 hr ^x . Although this activity is at least 100 times lower than the activity of pinosylvin to resveratrol, in the case of the expression of CYP2C19 in a micro-organism producing pinosylvin, some through conversion of piceatannol may be expected to occur, the extent of this being very much dependent on the expression levels of CYP2C19 and the concentration of resveratrol accumulating inside the cell. CYP1A2 was the only P450 that gave rise to a well- measurable formation rate of piceatannol from resveratrol (18 μM in 120 minutes, so a conversion rate of approx 0.2 min ¹ ) .

Example 2. A) Construction of a yeast vector for expression of CYP2C19

To verify the activity of CYP2C19 as a pinosylvin hydroxylase with in vivo activity an expression vector containing CYP2C19 is constructed. A codon optimized version

targeted for 5. cerevisiae is created and inserted into a high copy number expression vector, such as the 2 micron yeast expression vector. To drive transcription, a strong constitutive promoter, such as TEFl or TDH3 is sufficient. To verify the in vivo activity of CYP2C19, appropriate yeast cells containing the constructed high copy number expression vector are grown in shake flasks and fed with pinosylvin in the exponential growth phase. An HPLC with a UV detector attached is used to detect the formation of resveratrol from pinosylvin.

B) Construction of a yeast vectors for the expression of

CYP2C19, PAL2, 4CL2 and VSTl

To verify the resveratrol yielding activity of CYP2C19 expressed together with the PAL2, 4CL2 and VSTl, two construct are constructed.

A codon optimized version of 4CL2 and VSTl targeted for

5. cerevisiae is created and inserted into a high copy number expression vector, such as the 2 micron yeast expression vector. To drive transcription a strong constitutive promoters, such as TEFl or/and TDH3 is sufficient.

A codon optimized version of CYP2C19 and PAL2 targeted for 5. cerevisiae is created and inserted into a high copy number expression vector such as the 2 micron yeast expression vector. To drive transcription a strong constitutive promoters, such as TEFl or/and TDH3 is sufficient .

To verify the in vivo activity of CYP2C19, appropriate yeast cells containing the two constructed high copy number expression vectors are grown in shake flasks and samples are taken throughout the course of the fermentation. An HPLC with a UV detector attached, is used to detect the formation of resveratrol and intermediates in illustrated in figure 1.

Example 3 Production of piceatannol in yeast

The CYP2C19 converted pinosylvin mainly to trans- resveratrol. It also converted hundred times less efficiently resveratrol further into piceatannol. Thus in principle the combination of CYP2C19 in an organism with the trans- resveratrol pathway enzymes would enable the production of piceatannol from phenylalanine and/or tyrosine. However, better CYPs for conversion of resveratrol to piceatannol are known, such as CYPlBl (Potter et al, 2002) . Thus in principle the combination of CYPs CYP2C19 and CYPlBl in an organism with the trans-resveratrol pathway enzymes would better enable the production of piceatannol from phenylalanine and/or tyrosine.

Example 4

Isolation of genes encoding PAL, CYP2C19, 4CL2 , and VSTl

The PAL2 gene encoding Arabidopsis thaliana phenylalanine ammonia lyase (Cochrane et al . , 2004) was synthesized by GenScript Corporation (Piscataway, NJ) . The amino acid sequence was used as template to generate a synthetic gene codon optimized for expression in 5. cerevisiae (SEQ ID NO: 1) . The synthetic PAL2 gene was delivered inserted in E. coli pUC57 vector. The synthetic gene was purified from the pUC57 vector by amplifying it by forward primer 5-GGC GGC CGC ACT AGT ATG GAC CAA ATT GAA GCA- 3 SEQ ID NO 10 and reverse primer 5-AGA ATT GTT AAT TAA TTA GCA GAT TGG AAT AGG TG-3 SEQ ID NO 11 and purified from agarose gel using the QiaQuick Gel Extraction Kit (Qiagen) .

The CYP2C19 gene encoding Homo sapiens putative pinosylvin hydroxylase was synthesized by GenScript Corporation

(Piscataway, NJ) . The amino acid sequence was used as template to generate a synthetic gene codon optimized for expression in 5. cerevisiae (SEQ ID NO: 2) . The synthetic CYP2C19 gene was delivered inserted in E. coli pUC57 vector. The synthetic gene was purified from the pUC57 vector by amplifying it by forward primer 5-G GCC CGG GCG TCG ACA TGG ACC CAT TTG TTG TTT-3 SEQ ID NO 12 and reverse primer 5-CC AAG CTT ACT CGA GTT AGA CAG GGA TAA AAC ATA ATT-3 SEQ ID NO 13 and purified from agarose gel using the QiaQuick Gel Extraction Kit (Qiagen) .

4-coumarate : CoenzymeA ligase (4CL2) (Hamberger and Hahlbrock 2004; Ehlting et al . , 1999; SEQ ID NO: 3) was isolated via PCR from A. thaliana cDNA (BioCat, Heidelberg, Germany) using forward primer 5-GCG AAT TCT TAT GAC GAC ACA AGA TGT GAT AGT CAA TGA T-3 SEQ ID NO 14 and reverse primer 5-GCA CTA GTA TCC TAG TTC ATT AAT CCA TTT GCT AGT CTT GC-3 SEQ ID NO 15.

The VSTl gene encoding Vitis vinifera (grapevine) resveratrol synthase (Hain et al . , 1993) was synthesized by GenScript Corporation (Piscataway, NJ) . The amino acid sequence was used as template to generate a synthetic gene codon optimized for expression in S. cerevisiae (SEQ ID NO: 4) . The synthetic VSTl gene was delivered inserted in E. coli pUC57 vector flanked by BamHl and Xhol restriction sites. The synthetic gene was amplified using forward primer 5-CCG GAT CCT CAT GGC ATC CGT CGA AGA GTT CAG G-3 SEQ ID NO 16 and reverse primer 5-CGC TCG AGT TTT AGT TAG TAA CTG TGG GAA CGC TAT GC-3 SEQ ID NO 17 and purified from agarose gel using the QiaQuick Gel Extraction Kit (Qiagen) .

Example 5

A) Construction of a yeast vector for constitutive expression using the divergent Tefl and Tdh3 promoter

The 600 base pair TDH3 (GPD) promoter was amplified from 5. cerevisiae genomic DNA using the forward primer 5-GCG AGC TC AGT TTA TCA TTA TCA ATA CTC GCC ATT TCA AAG-3 SEQ ID NO 18 containing a Sacl restriction site and the reverse primer 5- CGT CTA GAA TC CGT CGA AAC TAA GTT CTG GTG TTT TAA AAC TAA AA-3 SEQ ID NO 19 containing a Xbal restriction site. The amplified TDH3 fragment was digested with Sacl/Xbal and ligated into Sacl/Xbal digested plasmid pRS416 (Sikorski and Hieter, 1989) as described previously (Mumberg et al, 1995) resulting in plasmid pRS416-TDH3.

The 400 base pair TEF2 promoter was amplified from 5. cerevisiae genomic DNA using the forward primer 5-GCG AGC TC ATA GCT TCA AAA TGT TTC TAC TCC TTT TTT ACT CTT-3 SEQ ID NO 20 containing a Sacl restriction site and the reverse primer 5-CGT CTA GA AAA CTT AGA TTA GAT TGC TAT GCT TTC TTT CTA ATG A-3 SEQ ID NO 21 containing a Xbal restriction site. The amplified TEF2 fragment was digested with Sacl/Xbal and ligated into Sacl/Xbal digested plasmid pRS416 (Sikorski and Hieter, 1989) as described previously (Mumberg et al, 1995) resulting in plasmid pRS416-TEF2.

A divergent fusion fragment between TEF2 promoter and TDH3 promoter was constructed starting from PRS416-TEF and PRS416-TDH3.

The 600 base pair TDH3 fragment was reamplified from PRS416-TDH3 using the forward primer 5- TTG CGT ATT GGG CGC TCT TCC GAG CTC AGT TTA TCA TTA TCA ATA CTC GC-3 SEQ ID NO 22 containing the underlined overhang for fusion PCR to TEF fragment and the reverse primer 5-ATG GAT CC TCT AGA ATC CGT CGA AAC TAA GTT CTG-3 SEQ ID NO 23 containing the underlined

BamHl restriction site. This resulted in a fragment ready for fusion to the below TEF2 fragment.

The 400 base pair TEF2 fragment including a 277 base pair spacer upstream of the Sacl restriction site was reamplified from PRS416-TEF2 using the forward primer 5-ATG AAT TC TCT AGA AAA CTT AGA TTA GAT TGC TAT GCT TTC-3 SEQ ID NO 24 containing the underlined EcoRl restriction site and the reverse primer 5-TGA TAA TGA TAA ACT GAG CTC GGA AGA GCG CCC AAT ACG CAA AC-3 SEQ ID NO 25 containing the underlined overhang for fusion to the TDH3 fragment. This resulted in a 680 base pair fragment ready for fusion to the TDH3 fragment.

The 600 base pair TEF2 fragment and the 600 base pair TDH3 fragments were joined together (fused) using fusion PCR with the forward primer 5-TAG TGC GGC CGC CCT TTA GTT CTA GAA AAC TTA GAT TAG ATT GCT A-3 SEQ ID NO 26 and the reverse primer 5-CGC CCG GGC CCT ATA GTG AGT CTA GAA TCC GTC GAA ACT AAG-3 SEQ ID NO 27 resulting in the divergent fragment <=TEF2/TDH3=> (Sequence ID NO:5) .

The vector pESC-URA with divergent galactose inducible promoters GAL1/GAL10 was amplified with forward primer 5-CTC ACT ATA GGG CCC GGG-3 SEQ ID NO 28 and reverse primer 5-ACT AAA GGG CGG CCG CA-3 SEQ ID NO 29 to remove the GALl /GALlO promoters .

The divergent constitutive <=TEF2/TDH3=> promoter fragment was amplified as in example 5a and fused using

Infusion™ (Clontech, Delaware, USA) into the above vector without the GALl/GallO fragment. This resulted in a vector pTDHTEF-URA with replaced promoters, from GALl/GallO to TEF2/TDH3 (Sequence NO: 6) .

B) Construction of a yeast vector for constitutive expression of CYP2C19

The gene encoding CYP2C19 was isolated as described in example 4. The amplified CYP2C19 PCR-product was inserted into an expression vector described in example 5a digested with Sail and Xhol by Infusion™ technology (Clontech, Delaware, USA), resulting in vector pTDHTEF-URA-CYP2C19.

Two different clones of pESC-HIS-4CL were sequenced to verify the sequence of the cloned gene (Sequence NO: 7) .

C) Construction of a yeast vector for constitutive expression of CYP2C19 and PAL2

The gene encoding PAL2 was isolated as described in example 4. The amplified PAL2 PCR-product was inserted into Pad and Spel digested expression vector created in example 5b by Infusion™ technology (Clontech, Delaware, USA) vector Stratagene) , resulting in vector pTDHTEF-URA-PAL2-CYP2C19. Two different clones of pTDHTEF-URA-PAL2-CYP2C19 were sequenced to verify the sequence of the cloned gene (Sequence NO: 8) . D) Construction of a yeast vector for constitutive expression induced of 4CL and VSTl

The gene encoding 4CL was isolated as described in example 4. The amplified 4CL PCR-product was digested with EcoRl/Spel and ligated into EcoRl/Spel digested pESC-HIS vector (Stratagene), resulting in vector pESC-HIS-4CL . Two different clones of pESC-HIS-4CL were sequenced to verify the sequence of the cloned gene.

The gene encoding VSTl was isolated as described in example 4. The amplified synthetic VSTl gene was digested with BamHl/Xhol and ligated into BamHl/Xhol digested pESC- HIS-4CL. The resulting plasmid, pESC-HIS-4CL-VSTl, contained

the genes encoding 4CL and VSTl under the control of the divergent galactose induced <=GAL1/GAL1O=> promoters. The sequence of the gene encoding VSTl was verified by sequencing of two different clones of pESC-HIS-4CL-VSTl . The vector pESC-HIS-4CL-VSTl with divergent galactose inducible promoters GAL1/GAL10 was sequentially digested with EcoRl and BamHl to remove the GAL1/GAL10 promoters.

The divergent constitutive <=TEF2/TDH3=> promoter fragment was sequentially digested with EcoRl and BamHl and ligated into the above vector without the GALl/GallO fragment. This resulted in a vector pesc-HIS-TEF2-4CL-TDH3- VSTl with replaced promoters, from GALl/GallO to TEF2/TDH3 (Sequence NO: 9) .

Example 6

Expression of the pathway to resveratrol in the yeast S. cerevisiae using PAL2, CYP2C19, 4CL2 and VSTl

Yeast strains containing the appropriate genetic markers were transformed with the vectors described in example 5. The transformation of the yeast cell was conducted in accordance with methods known in the art by using competent cells, an alternative being for instance, electroporation (see, e.g., Sambrook et al . , 1989) . Transformants were selected on medium lacking uracil and/or histidine and streak purified on the same medium.

5. cerevisiae strain FS01529 (MATa HISl ura3) was co- transformed with pTDHTEF-URA-PAL2-CYP2C19 (example 5c) and pTDHTEF-HIS-4CL2-VSTl (example 5d) , and the transformed strain was named FSSC-PAL2-CYP2C19-4CL2-VST1. As reference 5. cerevisiae strain FS01227 (MATa ura3) was transformed with

pTDHTEF-URA-PAL2-CYP2C19 (example 5c) and the transformed strain was named FSSC-PAL2-CYP2C19

Example 7

Fermentation with recombinant yeast strains in shake flasks

The recombinant yeast strains were inoculated from agar plates with a sterile inoculation loop and grown in 100 ml defined mineral medium (Verduyn et al . , 1992) that contained vitamins, trace elements and 40 g/1 glucose.

The 500 ml shake flasks with two baffles were incubated for three days at 30 °C and 150 rpm.

Example 8

a) Extraction of stilbenoids

Cells were harvested and an aliquot of 0.5 ml of cell broth was extracted once with 0.5 ml ethanol by vigorously mixing. The sample was centrifugation at 5000 g for 5 minutes. The supernatant was put into into HPLC vials.

b) Analysis of stilbenoids HPLC

For quantitative analysis of cinnamic acid, coumaric acid, resveratrol and pinosylvin, samples were subjected to separation by high-performance liquid chromatography (HPLC) Agilent Series 1100 system (Hewlett Packard) prior to uv- diode-array detection at λ = 306 nm. A Phenomenex (Torrance, CA, USA) 15 Luna 3 micrometer C18 (100 X 2.00 mm) column was used at 40 ⁰ C. As mobile phase a gradient of acetonitrile and milliq water (both containing 50 ppm trifluoroacetic acid) was used at a flow of 0.8 ml/min. The gradient profile was S-shaped from 15 % acetonitrile to 100 % acetonitrile over 7.5 min. The elution time was approximately 1.5-2.1

minutes for coumaric acid. The elution time was approximately 4.2-4.8 minutes for trans-resveratrol . The elution time was approximately 5.5-6.1 minutes for cinnamic acid. The elution time was approximately 6.4-7.1 minutes for trans-pinosylvin . (Figures 9-13)

Results

FSSC-PAL2-CYP2C19 and FSSC-PAL2-CYP2C19-4CL2-VST1 were cultivated as described in example 7, and analyzed for their content of stilbenoids. The HPLC chromatogram from a sample taken from cultivation with strain FSSC-PAL2-CYP2C19 produced one peak with a retention time corresponding to cinnamic acid (Figure 2, Figure 9) . The UV absorption spectrum of the sample (Figure 3) and the standard (Figure 12) was similar supporting the retention time identification of the peak as being cinnamic acid. The HPLC-analysis showed that strain FSSC-PAL2-CYP2C19 only had the capacity to produce cinnamic acid in low amounts. Strain FSSC-PAL2-CYP2C19-4CL2-VST1 had the capacity to produce cinnamic acid, the precursor of cinnamoyl-CoA which can be transformed into trans-pinosylvin by VSTl and trans-resveratrol. (figure 2 and 3) .

The HPLC chromatogram from a sample taken from a cultivation with strain FSSC-PAL2-CYP2C19-4CL2-VST1 produced two peak with a retention time corresponding to trans- resveratrol and trans-pinosylvin (Figure 5, Figure 9) . The UV absorption spectrum of peak one (Figure 6) and the standard of trans-resveratrol (Figure 10) was similar supporting the retention time identification of the peak as being trans-resveratrol. The UV absorption spectrum of peak two (Figure 7) and the standard of trans-pinosylvin (Figure 13) was similar supporting the retention time identification of the peak as being trans-pinosylvin.

The intermediates in the phenylpropanoid leading from phenylalanine to pinosylvin and resveratrol were confirmed by UV absorption spectra which was similar to the corresponding standards. Thus it can be concluded that CYP2C19 did not have the ability to convert cinnamic acid into coumaric acid and have the ability to convert Trans-pinosylvin into trans- resveratrol. The resveratrol was produced in an amount of 1 mg/L . In this specification, unless expressly otherwise indicated, the word 'or' is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator 'exclusive or' which requires that only one of the conditions is met. The word 'comprising' is used in the sense of 'including' rather than in to mean 'consisting of . All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.

REFERENCES

Becker et al, Ferns Yeast Research, 4, 2003, 79-85

Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine related antioxidant resveratrol

Chen H, Jiang H, Morgan JA.

Non-natural cinnamic acid derivatives as substrates of cinnamate 4- hydroxylase.Phytochemistry. 2007 Feb; 68 (3) : 306-11. Epub 2006 Dec 1.

Dodhia VR, Fantuzzi A, Gilardi G. Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego. J Biol Inorg Chem. 2006 Oct;11 (7) : 903-16.

Hayashi K, Sakaki T, Kominami S, Inouye K, Yabusaki Y. Coexpression of genetically engineered fused enzyme between yeast NADPH-P450 reductase and human cytochrome P450 3A4 and human cytochrome b5 in yeast. Arch Biochem Biophys . 2000 Sep 1 ; 381 (1) : 164-70.

Katsuyama Y, Funa N, Miyahisa I, Horinouchi S.

Synthesis of unnatural flavonoids and stilbenes by exploiting the plant biosynthetic pathway in Escherichia coli. Chem Biol. 2007 Jun; 14 ( 6) : 613- 21.

Parikh A, Guengerich FP. Expression, purification, and characterization of a catalytically active human cytochrome P450 1A2 : rat NADPH-cytochrome P450 reductase fusion protein. Protein Expr Purif. 1997 Apr; 9 (3) : 346-54.

Potter GA, Patterson LH, Wanogho E, Perry PJ, Butler PC, Ijaz T, Ruparelia KC, Lamb JH, Farmer PB, Stanley LA, Burke MD. The cancer preventative agent resveratrol is converted to the anticancer agent piceatannol by the cytochrome P450 enzyme CYPlBl .Br J Cancer. 2002 Mar 4,-86 (5) :774

Roupe K, Halls S, Davies NM.

Determination and assay validation of pinosylvin in rat serum: application to drug metabolism and pharmacokinetics . J Pharm Biomed Anal. 2005 Jun 1/38(1) :148-54.

Sanoh S, Kitamura S, Sugihara K, Ohta S. Cytochrome P450 IAl/2 mediated metabolism of trans-stilbene in rats and humans .Biol Pharm Bull. 2002 Mar;25 (3) : 397-400.