Production of Stilbenoids

Info

Publication number: 20140206051
Type: Application
Filed: Oct 15, 2013
Publication Date: Jul 24, 2014
Applicant: Evolva SA (Reinach)
Inventors: Bo STENHUUS (Kobenhavn O), Hans Peter SMITS (Holte), Thomas DURHUUS (Kobenhavn NV), Michael KATZ (Malmo)
Application Number: 14/054,583

Abstract

A method for the production of a stilbenoid, such as resveratrol or pinosylvin, by fermenting plant material such a grape must using a yeast having a metabolic pathway producing said stilbenoid, separating a solids waste material from said fermentation and extracting said stilbenoid.

Description

Description

The present invention relates to the production of stilbenoids by extraction thereof from wine making waste.

A number of strains of yeast which have been genetically engineered to produce one or more stilebenoids such as resveratrol and pinosylvin have been described. Thus, 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). WO2006/089898 disclosed a Saccharomyces cerevisiae also having introduced therein a gene encoding a phenylalanine or tyrosine-ammonia lyase (PAL/TAL) and a gene encoding a cinnamate 4-hydroxylase (C4H). WO2006/125000 discloses oleaginous cells having resveratrol production capacity. WO2008/009728 discloses a pinosylvin producing Saccharomyces cerevisiae having introduced therein a PAL, 4-coumarate CoA-ligase or cinnamate-CoA ligase, a pinosylvin synthase.

The extraction of resveratrol from grape seed and skin (marc) of red wine grapes has been proposed (Australian Harvest—Press release).

Beeckwilder et al disclose resveratrol production by a genetically engineered yeast and detection of resveratrol in the liquid culture medium. It was stated that resveratrol accumulated in the medium rather than in the cells.

Pretorius disclosed the possibility of developing wine yeasts producing resveratrol but indicated that the chances of success were unknown.

Becker et al disclosed a genetically modified yeast which produced glycosylated resveratrol during fermentation of a culture medium containing coumaric acid, and discussed the possibility of fermenting grapes with such a modified yeast in the hope of producing wine containing more than a normal level of resveratrol. Glycosylated resveratrol production was demonstrated by extraction from yeast cells grown in culture medium.

Further sources of stilbenoids are desirable. There is now provided according to the present invention a method for the production of a stilbenoid, comprising extracting said stilbenoid from a solids waste material separated from a fermentation of plant material conducted using a yeast having a metabolic pathway producing said stilbenoid.

The term ‘solids waste material’ refers to a waste material containing undissolved solids, optionally with significant quantities of free liquid such that the waste material may be flowable or not, including pastes or slurries as well as embracing dry solids. ‘Waste material’ includes any material that is not the desired end product of the fermentation (which will typically be wine or beer of some form) and includes residual plant material mixed with yeast cells (live or dead).

The method may further comprise the preliminary steps of conducting said fermentation of plant material using a said yeast having a metabolic pathway producing said stilbenoid and separating a solids waste material from said fermentation. The solids waste material may comprise yeast solids and plant material solids.

Preferably, the fermentation is a fermentation of fruit must together with or separated from pommace or is a fermentation of pommace separated from fruit must. The fruit may for instance be grape, apple or pear. The fermentation may be a beer making fermentation, all forms of beer being included, whether obtained by the use of a top fermenting yeast or a bottom fermenting yeast.

Methods for extracting stilbenoids, including resveratrol and pinosylvin, are described in the above publications. Suitable solvents include esters such as ethyl acetate or a solvent as described in GB 0714671.5, i.e. an ester which preferably is of the general formula R⁶—COO—R⁷, and R⁶is H or an aliphatic straight or branched chain hydrocarbon moiety of from 1-6 carbon atoms and R′ 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. R⁷may have from 3 to 9 carbon atoms. R⁶may 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 C₆to C₁₆straight or branched chain alkane, e.g. a C_9-14alkane, e.g. a C 2 alkane. Preferably, said alkane is n-dodecane.

The stilbenoid producing yeast may be as described in any of the above publications or genetically engineered according to the principles or practice there described. In particular, it may be a resveratrol producing yeast as described generally or by way of example in WO2006/089898 or a pinosylvin producing yeast as described generally or by way of example in WO2008/009728.

Preferably therefore, the yeast 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 yeast.

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

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

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

Thus, the yeast 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 yeast 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 yeast from nucleic acid coding for said enzyme which is not native to the yeast.

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 yeast.

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 yeast.

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 yeast 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 ACC1 may be boosted to increase the pool of malonyl-CoA available in the metabolic pathway.

The yeast may be of the genus Saccharomyces and may be of the species Saccharomyces cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, or S. uvarum or others, especially any conventionally used in brewing or wine making.

The accompanying drawings show results obtained in the examples as follows:

FIG. 1 shows a divergent fusion fragment formed between a TEF2 promoter and TDH3 promoter;

FIG. 2 shows HPLC chromatograms of pulp extract obtained in Example 14 with Absorbance plotted against retention time;

FIG. 3 shows further chromatograms obtained in Example 14 showing UV spectra of pinosylvin peaks in waste stream extracts (pulp sample and sediment sample) and of a standard; and

FIG. 4 shows still HPLC chromatograms of sediment obtained in Example 14 with Absorbance plotted against retention time.

The invention will be illustrated by the following non-limiting examples.

EXAMPLES Example 1 Isolation of Genes Encoding PAL2, C4H, AR2, 4CL and VST1

Phenylalanine ammonia lyase (PAL2) (Cochrane et al., 2004) (SEQ ID NO 1), cinnamate 4-hydroxylase (C4H) (Mizutani et al, 1997) (SEQ ID NO 2), cytochrome P450 reductase (AR2)(Mizutani and Ohta, 1998) (SEQ ID NO 3), 4-coumarate:coenzymeA ligase (4CL) (Hamberger and Hahlbrock 2004; Ehlting et al., 1999) (SEQ ID NO 4) were isolated via PCR from A. thaliana cDNA (BioCat, Heidelberg, Germany) using the primers in table 1.

The codon optimized VST1 gene encoding Vitis vinifera (grapevine) resveratrol synthase (Hain et al., 1993) (SEQ ID NO 5) for expression in S. cerevisiae was synthesized by GenScript Corporation (Piscataway, N.J.). The synthetic VST1 gene was delivered inserted in E. coli pUC57 vector flanked by BamH1 and Xho1 restriction sites. The synthetic gene was purified from the pUC57 vector by BamH1/Xho1 restriction and purified from agarose gel using the QiaQuick Gel Extraction Kit (Qiagen).

TABLE 1 Primer for amplification of gene (Restriction sites are underlined) Gene 5-CG GAATTC CGTACG TA ATG GAT CAA ATC PAL2 GAA GCA ATG TT SEQ ID NO: 10 5-CG ACTAGT TTA GCA AAT CGG AAT CGG AGC PAL2 SEQ ID NO: 11 5-CG CTCGAG GCGGCCGC TAAAAT ATG GAC CTC C4H CTC TTG CTG GAG SEQ ID NO: 12 5-AGTAGAIGGAGTAGATGGAGTAGATGGAGTAGATGG C4H ACA GTT CCT TGG TTT CAT AAC G SEQ ID NO: 13 5-CCATCTACTCCATCTACTCCATCTACTCCATCTACT AR2 AGG AGA TCC GGT TCT GGG A SEQ ID NO: 14 5-CG GGTACCAT TTA CCA TAC ATC TCT AAG AR2 ATA TCT TCC SEQ ID NO: 15 5′ GCGAATTCTTATGACGACACAAGATGTGATAGTCAA 4CL TGAT SEQ ID NO: 16 5′ GCACTAGTATCCTAGTTCATTAATCCATTTGCTAG 4CL TCTTGC SEQ ID NO: 17

The coding sequence of tyrosine ammonia lyase (TAL) from Rhodobacter capsulatus (Kyndt et al., 2002; is codon optimized for expression in S. cerevisiae using the online service backtranslation tool at www.entelechon.com, yielding sequence SEQ ID NO: 6)

Example 2 Construction of a Yeast Vector for Galactose Induced Expression of PAL2 and C4H:AR2 Fusion Gene

The gene encoding PAL2 was amplified from cDNA from A. thaliana as template using forward primer 5-CG GAATTC CGTACG TA ATG GAT CAA ATC GAA GCA ATG TT-3 SEQ ID NO 29 and reverse primer 5-CG ACTAGT TTA GCA AAT CGG AAT CGG AGC-3 SEQ ID NO 30. The amplified PAL2 PCR-product was digested with EcoR1/Spe1 and ligated into EcoR1/Spe1 digested pESC-URA vector (Stratagene), resulting in vector pESC-URA-PAL2. Two different clones of pESC-URA-Pal2 were sequenced to verify the sequence of the cloned gene.

C4H was amplified using cDNA from A. thaliana as template using forward primer 5-CG CTCGAG GCGGCCGC TAAAAT ATG GAC CTC CTC TTG CTG GAG-3 SEQ ID NO 31 and reverse primer 5-AGTAGATGGAGTAGATGGAGTAGATGGAGTAGATGG ACA GTT CCT TGG TTT CAT AAC G-3 SEQ ID NO 32. AR2 was amplified using cDNA from A. thaliana as template using forward primer 5-CCATCTACTCCATCTACTCCATCTACTCCATCTACT AGG AGA TCC GGT TCT GGG A-3 SEQ ID NO 33 and reverse primer 5′-CG GGTACCAT TTA CCA TAC ATC TCT AAG ATA TCT TCC-3 SEQ ID NO 34. The amplified PCR products C4H and AR2 were used as templates for the creation of the fusion gene C4H:AR2 using the forward primer 5-CG CTCGAG GCGGCCGC TAAAAT ATG GAC CTC CTC TTG CTG GAG-3 SEQ ID NO 35 and the reverse primer 5-CG GGTACC AT TTA CCA TAC ATC TCT AAG ATA TCT TCC-3 SEQ ID NO 36.

The fusion gene C4H:AR2 gene was digested with XhoI/KpnI and ligated into XhoI/KpnI digested pESC-URA-PAL2. The resulting plasmid, pESC-URA-PAL2-C4H:AR2, contained the genes encoding PAL2 and C4H:AR2 under the control of the divergent galactose induced <=GAL1/GAL10=> promoters. The sequence of the gene encoding C4H:AR2 was verified by sequencing of two different clones of pESC-URA-PAL2-C4H:AR2.

Example 3 Construction of a Yeast Vector for Galactose Induced Expression of 4CL1 and VST1

The gene encoding 4CL was isolated as described in example 1. The amplified 4CL PCR-product was digested with EcoR1/Spe1 and ligated into EcoR1/Spe1 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 VST1 was isolated as described in example 1. The amplified synthetic VST1 gene was digested with BamH1/Xho1 and ligated into BamH1/Xho1 digested pESC-HIS-4CL. The resulting plasmid, pESC-HIS-4CL-VST1, contained the genes encoding 4CL and VST1 under the control of the divergent galactose induced <=GAL1/GAL10=> promoters. The sequence of the gene encoding VST1 was verified by sequencing of two different clones of pESC-HIS-4CL-VST1.

Example 4 Construction of Strong Constitutive Promoter Fragment TDH3

The 600 base pair TDH3 (GPD) promoter was amplified from S. cerevisiae genomic DNA using the forward primer 5′GC GAGCTC AGT TTA TCA TTA TCA ATA CTC GCC ATT TCA AAG SEQ ID NO: 18 containing a Sac1 restriction site and the reverse primer 5′-CG TCTAGA ATC CGT CGA AAC TAA GTT CTG GTG TTT TAA AAC TAA AA SEQ ID NO: 19 containing a Xba1 restriction site. The amplified TDH3 fragment was digested with Sac1/Xba1 and ligated into Sac1/Xba1 digested plasmid pRS416 (Sikorski and Hieter, 1989) as described previously (Mumberg et al, 1995) resulting in plasmid pRS416-TDH3.

Example 5 Construction of Constitutive Strong Promoter Fragment TEF2

The 400 base pair TEF2 promoter was amplified from S. cerevisiae genomic DNA using the forward primer 5′-GC GAGCTC ATA GCT TCA AAA TGT TTC TAC TCC TTT TTT ACT CTT SEQ ID NO: 20 containing a Sac1 restriction site and the reverse primer 5′-CG TCTAGA AAA CTT AGA TTA GAT TGC TAT GCT TTC TTT CTA ATG A SEQ ID NO: 21 containing a Xba1 restriction site. The amplified TEF2 fragment was digested with Sac1/Xba1 and ligated into Sac1/Xba1 digested plasmid pRS416 (Sikorski and Hieter, 1989) as described previously (Mumberg et al, 1995) resulting in plasmid pRS416-TEF2.

Example 6 Construction of Fused Divergent Constitutive TEF and TDH3 Promoter Fragment

A divergent fusion fragment (FIG. 1) 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′ TTGCGTATTGGGCGCTCTTCC GAG CTC AGT TTA TCA TTA TCA ATA CTC GC SEQ ID NO: 22 containing the underlined overhang for fusion PCR to TEF2 fragment and the reverse primer 5′ AT GGATCC TCT AGA ATC CGT CGA AAC TAA GTT CTG SEQ ID NO: 23 containing the underlined BamH1 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 Sad restriction site was reamplified from PRS416-TEF2 using the forward primer 5′ AT GAATTC TCT AGA AAA CTT AGA TTA GAT TGC TAT GCT TTC SEQ ID NO: 24 containing the underlined EcoR1 restriction site and the reverse primer 5′ TGA TAA TGA TAA ACT GAG CTC GGA AGA GCG CCC AAT ACG CAA AC 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 680 base pair TEF2 fragment and the 600 base pair TDH3 fragments were joined together (fused) using fusion PCR with the forward primer 5′ AT GAATTC TCT AGA AAA CTT AGA TTA GAT TGC TAT GCT TTC SEQ ID NO 37 and the reverse primer 5′ AT GGATCC TCT AGA ATC CGT CGA AAC TAA GTT CTG SEQ ID NO: 26, resulting in the divergent fragment <=TEF2/TDH3=> (Sequence ID NO 7).

Example 7 Construction of a Yeast Vector for Constitutive Expression of PAL2 and C4H:AR2 Fusion Gene

The vector pESC-URA-PAL2-C4H:AR2 with divergent galactose inducible promoters GAL1/GAL10 was sequentially digested with NotI and BsiWI to remove the GAL1/GAL10 promoters.

The divergent constitutive <=TEF2/TDH3=> promoter fragment (Example 6) was re-amplified with forward primer 5-GC CGTACG TCT AGA AAA CTT AGA TTA GAT TGC TAT GCT TTC-3 SEQ ID NO: 27 and reverse primer 5-ATT GCGGCCGC TCT AGA ATC CGT CGA AAC TAA GTT CTG-3 SEQ ID NO: 28. The resulting PCR product was sequentially digested with NotI and BsiWI and ligated into the above vector without the GAM/Gall° fragment. This resulted in a vector pESC-URA-TEF-PAL2-TDH3-C4H:AR2 with replaced promoters, from GAM/Gall° to TEF2/TDH3 Sequence ID NO 8).

Example 8 Construction of a Yeast Vector for Constitutive Expression of a TAL Gene

The vector pESC-URA-TAL with divergent galactose inducible promoters GAL1/GAL10 is sequentially digested with EcoRI and BamHI to remove the GAL1/GAL10 promoters.

The divergent constitutive <=TEF2/TDH3=> promoter fragment (Example 6) was sequentially digested with EcoR1 and BamH1 and ligated into the above BamHI/EcoRI linearized pESC-URA-TAL vector without the GAL1/GAL10 fragment. This resulted in a vector pesc-URA-TEF-TAL with replaced promoters, from GAL1/Ga110 to TEF2/TDH3.

Example 9 Construction of a Yeast Vector for Constitutive Expression Induced of 4CL and VST1

The vector pESC-HIS-4CL-VST1 with divergent galactose inducible promoters GAL1/GAL10 was sequentially digested with EcoR1 and BamH1 to remove the GAL1/GAL10 promoters.

The divergent constitutive <=TEF2/TDH3=> promoter fragment (Example 6) was sequentially digested with EcoR1 and BamH1 and ligated into the above linearized vector without the GAL1/GAL10 fragment. This resulted in a vector pesc-HIS-TEF2-4CL-TDH3-VST1 with replaced promoters, from GAL1/Gal10 to TEF2/TDH3 SEQ ID NO 9).

Example 10 Generation of a Strain with Constitutive Expression of the Phenylpropancid Pathway from Phenylalanine to Resveratrol in the Yeast S. Cerevisiae

The transformation of the yeast cell was conducted in accordance with methods known in the art, for instance by using lithium acetate transformation method (Gietz and Schiestl, 1991). S. cerevisiae strain FS01528 (CEN.PK MATa ura3 His3) was co-transformed with pESC-URA-TEF-PAL2-TDH3-C4H:AR2 (example 7) and pesc-HIS-TEF2-4CL-TDH3-VST1 (example 9), and the transformed strain was named FS09215. Transformants were selected on medium lacking uracil and histidine and streak purified on the same medium.

Example 11 Generation of a Strain with Constitutive Expression of the Phenylpropancid Pathway from Tyrosine to Resveratrol in the Yeast S. Cerevisiae

The transformation of the yeast cell is conducted in accordance with methods known in the art, for instance by using lithium acetate transformation method (Gietz and Schiestl, 1991). S. cerevisiae strain FS01528 (CEN.PK MATa ura3 His3) is co-transformed with pESC-URA-TEF-TAL (example 8) and pesc-HIS-TEF2-4CL-TDH3-VST1 (example 9), to obtain strain TEF2-TAL-TEF2-4CL-TDH3-VST1. Transformants are selected on medium lacking uracil and histidine and streak purified on the same medium.

Example 12 HPLC Determination of Stilbenoids, Phenylpropanoids and Ethanol

For quantitative analysis of cinnamic acid, trans-resveratrol and trans-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 2=306 nm. A Phenomenex (Torrance, Calif., USA) Luna 2.5 micrometer C18 (100×2.00 mm) column was used at 60° C. As mobile phase a non linear S-shaped gradient of acetonitrile and milliQ water (both containing 50 ppm trifluoroacetic acid) was used at a flow of 0.8 ml/min. The S-shaped gradient profile was from 10% to 100% acetonitrile in 5 minutes. The elution time was approximately 3.0 minutes for trans-resveratrol and 4.4 minutes trans-pinosylvin. Pure pinosylvin standard (>95% pure) was purchased from ArboNova (Turku, Finland) and pure trans-resveratrol standard was purchased from Sigma.

The grape-must was analysed for the content of ethanol by HPLC using an Aminex HPX-87H ion-exclusion column (Bio-Rad, Hercules, Calif.) at 60° C., with 5 mM H2SO4 as a mobile phase at a flow rate of 0.6 ml/min. The ethanol was detected by a refractometer (Shodex RI-71).

Example 13 Generation of Biomass

Yeast strains FS01201 (CEN.PK 113-7D wild type non modified control strain) was kept on YPD agar plates with 20 g/l glucose. FS09215 (genetically modified resveratrol producer from example 10) was kept on SC-HIS-URA agar plates with 20 g/l glucose.

The two yeast strains were grown in 10-16 500 ml shake flasks with 200 ml DELFT medium (Verduyn et al, 1992) containing 45 g/l glucose, 30 g/l ammonium sulphate, 14 g/l KH₂PO₄, and 1.5 g/l MgSO₄for 4 days at 30° C. and 150 rpm. A paste of wet weight cells was collected (harvested) by centrifugation at 3000 g for 5 minutes in 50 ml Sartorious tubes and discarding the supernatant after each round. After repetitive rounds of centrifugation 26 g wet weight was collected of strain FS01201 and 24 g wet weight of FS09215.

Example 14 Production of Red Wine from Grapes Using a S. Cerevisiae Strain with Constitutive Expression of the Phenylpropanoid Pathway from Phenylalanine to Resveratrol

Commercially available seedless “Crimson” grapes (product of Brazil), were used to produce two red wine's: a control wine produced by using strain FS01201 and a stilbenoid-enriched wine by using strain FS09215 as described in example 10. Said strain harbours a phenylpropanoid pathway comprising an oxygen-dependent cinnamate-4-hydroxylase (C4H) that converts cinnamic-acid in coumaric-acid. Hence, under the oxygen-deprived conditions that typically prevail in the anaerobic fermentation-process used for wine-making, said strain can only produce resveratrol if endogenous coumaric acid is present in the grape-pulp and grape-must. With a lack of sufficient oxygen, however, said strain has the potential of producing pinosylvin, that is derived from cinnamic acid.

Preparation of Starter Culture

A starter culture of strain FS01201 and FS09215 was prepared by crushing 2 kilo's of grapes and filtering the resulting grape pulp with a loose-woven cotton cloth (cheesecloth) and subsequently collecting the grape juice in a sterile open plastic bucket. An aliquot of 500 ml of grape juice was enriched with 40 grams of glucose and divided into two aliquots of 250 ml that were transferred to two sterile 500 ml-shakeflasks. The shakeflasks were inoculated with approximately 2 grams wet-weight of cells from either FS01201 or FS09215 as prepared according to example 13, and incubated for approximately 24 hours at room temperature. Activity of yeast was indicated by formation of CO₂resulting in a foam-layer.

Primary Pulp Fermentation

A pulp of grapes was prepared by disrupting 16 kilo's of grapes with a semi-professional kitchen blender. The resulting grape-pulp was enriched with approximately 2 coffee-spoons of “yeast nutrient” (stimulates yeast growth) and 3 coffee-spoons of “pecto-enzymes” (breaks down pectin in the grape skin). Both the pecto-enzymes and yeast nutrient were part of a commercially available wine-making kit. Said enriched grape-pulp was divided into two equal aliquots, approximately 7 to 8 litre each, and transferred into two 10 litre plastic round sterilized containers that were open at the top. A pulp-fermentation was initiated by adding the total amount of 250 ml of starter culture to the grape pulp, and mixing it well together with a large spoon; one bucket was inoculated with F501201, and the other with FS09215.

The progression of the pulp-fermentation was visually monitored on a daily basis, and the appearance of a foam-layer on the top, caused by CO₂formation, indicated that the fermentation was successfully ongoing. The CO₂formation caused the grape-pulp to float on top of the grape-juice, and therefore the pulp was mixed with a large spoon on a daily basis as well. Formation of foam ceased after 9 days, and the grape-pulp “fluidized”, which indicated that almost al grape-sugars were consumed and the pulp-fermentation was near its end. The grape pulp, therefore, was separated from the liquid fraction (containing the juice and the yeast, i.e. the grape-must) by using a cheesecloth. For each strain approximately 5.5 to 6 litre of grape-must was collected. The fermented juice was analyzed for the content of alcohol and stilbenoids. The FS01201- and FS09215 grape-must contained 84.01 g/l (i.e. 10.6 vol %) of ethanol and 79.65 g/l (i.e. 10.1 vol %) respectively. The development of ethanol formation during the pulp fermentation is listed in table 2 below.

TABLE 2 Ethanol formation in primary pulp fermentation FS01201 FS09215 g/l vol % g/l vol % Day 6 69.87 8.86 68.72 8.71 Day 7 83.69 10.61 81.8 10.37 Day 8 81.44 10.32 81.68 10.35 Day 9 84.01 10.65 79.65 10.10

Furthermore, no stilbenoids were found in either grape-must, however, low levels of cinnamic acid (1.05 mg/l) were determined in the FS09215 grape-must. For the FS01201 and FS09215 pulp-fermentation, a total of 1757 grams and 1780 grams wet-weight of grape-pulp was collected respectively. The grape pulp and approximately 1.5 litre of the remaining grape-must were stored at 4° C.

Secondary Grape-Must Fermentation

The fermentation was now continued with the grape-must. In order to enhance the alcohol percentage to a level that is usually found in commercial red wines (12- to 15 vol %), an aliquot of 3.5 litre of grape-must of either pulp fermentation was enriched with approximately 340 g of commercially available sugar (“Dansukker”, dissolved and heated in approximately 300 ml of water). The addition of the extra sugar caused a dilution of the grape-must, resulting in a slight reduction of ethanol titers. Said enriched grape-must was transferred to a 5 litre glass-bottle that was stoppered with a “water-lock” to enable release of CO2, but at the same time preventing contamination. After 4 days the ethanol concentration rose to 99.46 g/l (i.e. 12.6 vol %) and 102.78 g/l (i.e. 13.0 volt). To enhance the ethanol concentration even further, a second addition was made: 150 grams of sugar (“Dansukker”) was dissolved into 1400 ml of the previously stored non-enriched grape-must, which was then subsequently added to the fermentation glass-bottle. The total volume of the grape-must was now approximately 5 litres, and with that almost completely filled the glass bottle leaving no room for air in the top of the bottle. The addition of the extra sugar caused a dilution of the grape-must, resulting in a slight reduction of ethanol titers. After a further 8 days, foam formation (i.e. CO₂formation) ceased completely, indicating that the fermentation was finished and that all sugars were depleted. The final ethanol concentration was 116.06 g/l (i.e. 14.7 vol %) and 109.64 g/l (i.e. 13.9 vol %), as listed in table 3.

TABLE 3 Ethanol-, phenylpropanoid- and stilbenoid formation in secondary grape-must fermentation FS01201 FS09215 ethanol (g/l) ethanol (vol %) ethanol (g/l) ethanol (vol %) cinnamic acid (mg/l) pinosylvin (mg/l) Day 10 82.39 10.44 85.21 10.80 1.19 0.62 Day 11 91.52 11.60 91.72 11.62 1.51 0.62 Day 12 99.73 12.64 98.2 12.45 not analyzed not analyzed Day 13 99.46 12.61 102.78 13.03 not analyzed not analyzed Day 14 97.58 12.37 92.04 11.67 1.36 0.57 Day 15 98.21 12.45 96.06 12.17 not analyzed not analyzed Day 16 108.07 13.70 100.86 12.78 1.64 0.57 Day 17 109.45 13.87 104.22 13.21 1.72 0.60 Day 18 120.48 15.27 111.6 14.14 1.63 0.60 Day 20 121.79 15.44 111.84 14.17 1.76 0.57 Day 21 116.06 14.71 109.64 13.90 1.90 0.60

The reference strain FS01201 did neither produce phenylpropanoid-intermediates nor stilbenoids. The chromatograms of the grape-must of FS09215 contained a peak with a similar retention time as pinosylvin; indeed said peak with retention time of 4.4 minutes, displayed a UV-spectrum that was identical to pinosylvin. Similarly, the presence of cinnamic acid was confirmed as well. Quantification of the peaks indicated that the grape-must of strain FS09215 contained cinnamic acid and pinosylvin in final concentrations of 1.90 mg/l and 0.60 mg/l respectively (Table 3). Neither resveratrol nor coumaric acid could be detected, indicating that the activity of C4H was hampered by the anaerobic conditions. Furthermore, the lack of resveratrol and coumaric acid suggested that no endogenous coumaric acid was present in the grape-must of the “Crimson” grapes used for this experiment.

In both fermentations, sediment settled on the bottom, which likely composed of small-size particle grape-residues and yeast cells. Said sediment was isolated from the grape-must by siphoning, and approximately 300 ml could be collected for either fermentation. The sediments were stored at 4° C. until further analysis on stilbenoid content.

Analysis of Waste-Stream for the Presence of Stilbenoids

Approximately 125 grams of the grape pulp that was generated in the primary pulp fermentation was extracted over night with 30 ml of ethyl acetate (divided in three 50 ml Sartorious tubes) using a rotary unit at ambient temperature (24° C.). The extraction tubes were covered with aluminium foil to avoid any light induced degradation of stilbenoids. The following day the extraction mixture was centrifuged at 3800×g for 10 minutes, and the upper, yellow/greenish coloured, ethyl acetate was collected and pooled into one tube. From the initial 30 ml ethyl acetate, approximately 28 ml extract was collected for the FS1201 grape-pulp suspension, and 30 ml of ethyl acetate for the FS09215 grape-pulp suspension. Said ethyl acetate fractions were reduced in volume by evaporation for 2 hours, using a freeze dryer, until a dry residue was obtained.

The dry, dark coloured, residue was dissolved in 500 microlitre 50% ethanol which resulted in a solution that contained non-dissolved dark precipitates. The solution was whirly-mixed and centrifuged at 13000×g for 5 minutes and the supernatant was diluted 5-fold with 50% ethanol. Said procedure resulted in a clear yellowish solution that could be used for HPLC analysis.

The grape-pulp of the control strain FS01201 did neither contain stilbenoids nor cinnamic- or coumaric acid (FIG. 6).

The chromatogram of the grape-pulp of FS09215 contained a peak with a similar retention time as pinosylvin; indeed said peak with retention time of 4.4 minutes, displayed a UV-spectrum that was identical to pinosylvin (FIGS. 6 and 7). Similarly, the presence of cinnamic acid was confirmed as well. Quantification of the peaks indicated that the grape-pulp contained cinnamic acid and pinosylvin in concentrations of 1.96 mg/kg pulp and 1.94 mg/kg pulp respectively. The total amount of grape-pulp recovered was 1780 grams, and can be considered as a primary waste-stream generated with the production of 5 litre red wine. Therefore, the production of 5 litre of red wine led to a grape-pulp waste-stream containing in total 1.780*1.96=3.49 mg cinnamic acid and 1.780*1.94=3.45 mg pinosylvin. Hence, for 1 litre of red wine produced, 3.49/5=0.70 mg of cinnamic acid- and 3.45/5=0.69 mg pinosylvin could be recovered from the pulp waste-stream.

Approximately 20 ml of sediment that was generated in the secondary grape-must fermentation was extracted over-night with 10 ml of ethyl acetate in a 50 ml Sartorious tube, using a rotary unit at ambient temperature (24° C.). The extraction tubes were covered with aluminium foil to avoid any light induced degradation of stilbenoids. The following day the extraction mixture was centrifuged at 3800×g for 10 minutes, and the upper, yellowish/greenish coloured, ethyl acetate was collected and pooled into one tube. From the initial 10 ml of ethyl acetate approximately 8 ml extract was recovered for both the FS1201- and FS9215 sediment suspension. Said ethyl acetate fractions were reduced in volume by evaporation for 2 hours using a freeze dryer until a dry residue was obtained. The dry, dark coloured, residue was dissolved in 500 microlitre 50% ethanol which resulted in a solution that contained non-dissolved dark precipitates. The solution was whirly-mixed and centrifuged at 13000×g for 5 minutes and the supernatant was diluted 5-fold with 50% ethanol. Said procedure resulted in a clear yellowish solution that could be used for HPLC analysis.

The sediment of the control strain FS01201 did not contain stilbenoids nor cinnamic- nor coumaric acid (FIG. 8).

The chromatogram of the sediment of FS09215 contained a peak with a similar retention time as pinosylvin; indeed said peak with retention time of 4.4 minutes, displayed a UV-spectrum that was identical to pinosylvin (FIGS. 8 and 7). Similarly, the presence of cinnamic acid was confirmed as well.

Quantification of the peaks indicated that the sediment contained cinnamic acid and pinosylvin in concentrations of 7.28 mg/l sediment and 2.60 mg/L sediment respectively. The total amount of sediment recovered was approximately 300 ml, and can be considered as secondary waste-stream generated with the production of 5 litre red wine. Therefore, the production of 5 litre of red wine led to a sediment waste-stream containing in total 0.3*7.28=2.18 mg cinnamic acid and 0.3*2.60=0.78 mg pinosylvin. Hence, for 1 litre of red wine produced, 2.18/5=0.44 mg of cinnamic acid- and 0.78/5=0.16 mg of pinosylvin could be recovered from the sediment waste stream.

Hence, for the production of 1 litre of red wine a total waste-stream was produced from which a total amount of 0.70+0.44=1.14 mg cinnamic acid, and 0.69+0.16=0.85 mg pinosylvin could be recovered.

Example 15 Production of Red Wine from Grapes Using a S. Cerevisiae Strain with Constitutive Expression of the Phenylpropanoid Pathway from Tyrosine to Resveratrol

Commercially available seedless “Crimson” grapes (product of Brazil), are used to produce two red wine's: a control wine produced by using strain FS01201 and a stilbenoid enriched wine by using strain FS-TEF2-TAL-TEF2-4CL-TDH3-VST1 described in example 11. Said strain harbours a phenylpropanoid pathway comprising enzymes that do not use oxygen as substrate.

Hence, said strain can produce resveratrol under the oxygen-deprived conditions that typically prevail in the anaerobic fermentation-process used for wine-making.

Preparation of Starter Culture

A starter culture of strain FS01201 and FS-TEF2-TAL-TEF2-4CL-TDH3-VST1 is prepared by crushing 2 kilo's of grapes and filtering the resulting grape pulp with a loose-woven cotton cloth (cheesecloth) and subsequently collecting the grape juice in a sterile open plastic bucket. An aliquot of 500 ml of grape juice is enriched with 40 grams of glucose and divided into two aliquots of 250 ml that are transferred to two sterile 500 ml-shakeflasks. The shakeflasks are inoculated with approximately 2 grams wet-weight of either FS01201 or FS-TEF2-TAL-TEF2-4CL-TDH3-VST1 as prepared according to example 1, and incubated for approximately 24 hours at room temperature. Activity of yeast is indicated by formation of CO₂resulting in a foam-layer.

Primary Pulp Fermentation

A pulp of grapes is prepared by disrupting 16 kilo's of grapes with a semi-professional kitchen blender. The resulting grape-pulp is enriched with approximately 2 coffee-spoons of “yeast nutrient” (stimulates yeast growth) and 3 coffee-spoons of “pecto-enzymes” (breaks down pectin in the grape skin). Both the pecto-enzymes and yeast nutrient are part of a wine-making kit that is commercially available. Said enriched grape-pulp is divided into two equal aliquots, approximately 7 to 8 litre each) and transferred into a 10 litre plastic round sterilized container that is open at the top. A pulp-fermentation is initiated by adding the total amount of 250 ml of starter culture to the grape pulp, and mixing it well together with a large spoon; one bucket is inoculated with F501201, and the other with FS-TEF2-TAL-TEF2-4CL-TDH3-VST1.

The progression of the pulp-fermentation is visually monitored on a daily basis, and the appearance of a foam-layer on the top, caused by CO₂formation, indicates that the fermentation is successfully ongoing. The CO₂formation causes the grape-pulp to float on top of the grape-juice, and therefore the pulp is mixed with a large spoon on a daily basis as well. The pulp-fermentation is near its end when almost al grape-sugars are consumed, which is indicated by cessation of foam-formation and “fluidizing” of the grape-pulp. The grape pulp is then separated from the liquid fraction (containing the juice and the yeast, i.e. the grape-must) by using a cheesecloth. For each strain approximately 5.0 to 6 litres of grape-must is collected and analyzed for the content of alcohol and stilbenoids. The grape pulp and approximately 1.5 litres of the remaining non-enriched grape-must are stored at 4° C.

Secondary Grape—Must Fermentation

The fermentation is now continued with the grape-must. In order to enhance the alcohol percentage to a level that is usually found in commercial red wines (12- to 15 vol %), an aliquot of 3.5 litre of grape-must of either pulp fermentation is enriched with approximately 340 g of commercially available sugar (“Dansukker”, dissolved and heated in approximately 300 ml of water). The addition of the extra sugar causes a dilution of the grape-must, resulting in a slight reduction of ethanol titers. The enriched grape-must is transferred to a 5 litre glass-bottle that is stoppered with a “water-lock” to enable release of CO₂, but at the same time preventing contamination. When the ethanol concentration reaches a level in between 12- to 13 vol %., a second sugar-addition is made to enhance the ethanol concentration even further: 150 grams of sugar (“Dansukker”) is dissolved into 1400 ml of the previously stored non-enriched grape-must, which is then subsequently added to fermentation glass-bottle. The total volume of the fermentation broth is now approximately 5 litres, and with that almost completely fills the glass bottle leaving no room for air in the top of the bottle. The addition of the extra sugar causes a dilution of the grape-must, resulting in a slight reduction of ethanol titers. The fermentation is finished when all sugars are depleted, and is indicated by a complete cessation of foam formation (i.e. CO₂formation), and the final ethanol concentration is in between 14- to 15 vol %. For each strain the grape-must is analyzed for the content of alcohol and stilbenoids.

In both fermentations sediment settles on the bottom, which likely composes of small-sized particle grape-residues and yeast cells. Said sediment is isolated from the grape-must by siphoning. The sediments are stored at 4° C. until further analysis on stilbenoid content.

Analysis of Waste-Stream for the Presence of Stilbenoids

Approximately 125 grams of the grape pulp that is generated in the primary pulp fermentation is extracted over night with 30 ml of ethyl acetate (divided in three 50 ml Sartorious tubes) using a rotary unit at ambient temperature (24° C.). The extraction tubes are covered with aluminium foil to avoid any light induced degradation of stilbenoids. The following day the extraction mixture is centrifuged at 3800×g for 10 minutes, and the upper, yellow/greenish coloured, ethyl acetate is collected and pooled into one tube. Said ethyl acetate fractions are reduced in volume by evaporation for 2 hours, using a freeze dryer, until a dry residue is obtained. The dry, dark coloured, residue is dissolved in 500 microlitre 50% ethanol which results in a solution that contains non-dissolved dark precipitates. The solution is whirly-mixed and centrifuged at 13000×g for 5 minutes and the supernatant is diluted 5-fold with 50% ethanol. Said procedure results in a clear yellowish solution that can be used for HPLC analysis.

The grape-pulp can be considered as a primary waste-stream generated with the production of 5 litre wine, and the resveratrol that can be recovered from the pulp can be expressed in terms of production of 1 litre red-wine.

Approximately 20 ml of sediment that is generated in the secondary grape-must fermentation is extracted over night with 10 ml of ethyl acetate in a 50 ml Sartorious tube, using a rotary unit at ambient temperature (24° C.). The extraction tubes are covered with aluminium foil to avoid any light induced degradation of stilbenoids. The following day the extraction mixture is centrifuged at 3800×g for 10 minutes, and the upper, yellowish/greenish coloured, ethyl acetate is collected and pooled into one tube. Said ethyl acetate fractions are reduced in volume by evaporation for 2 hours, using a freeze-dryer, until a dry residue is obtained.

The dry, dark coloured, residue is dissolved in 500 microlitre 50% ethanol which results in a solution that contains non-dissolved dark precipitates. The solution is whirly-mixed and centrifuged at 13000×g for 5 minutes and the supernatant is diluted 5-fold with 50% ethanol. Said procedure results in a clear yellowish solution that can be used for HPLC analysis.

The sediment can be considered as a secondary waste-stream generated with the production of 5 litre wine, and resveratrol that can be recovered from the sediment can be expressed in terms of production of 1 litre red-wine.

Hence, for the production of 1 litre of red wine the total amount of resveratrol that can be recovered from the waste-stream can be found by summation of the amount of the resveratrol present in both the primary- and secondary waste-stream.

Example 16 Isolation of Genes Encoding SAM8, 4CL2 and VST1

The codon optimized SAM8 gene encoding Saccharotrix espaniensis Tyrosine ammonia lyase (Berner et al, 2006) (SEQ ID NO 38) for expression in S. cerevisiae was synthesized by GenScript Corporation (Piscataway, N.J.). The synthetic Sam8 gene was delivered inserted in E. coli pUC57 vector flanked by EcoRI and SpeI restriction sites. The synthetic gene was purified from the pUC57 vector by EcoRI/SpeI restriction 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 39) was isolated via PCR from A. thaliana cDNA (BioCat, Heidelberg, Germany) using the primers, Forward 5′GCGAATTCTTATGACGACACAAGATGTGATAGTCAATGAT SEQ ID NO 40 with the underlined restriction sequence for ECOR1, and Reverse 5′GCACTAGTATCCTAGTTCATTAATCCATTTGCTAGTCTTGC SEQ ID NO 41 with the underlined restriction site for SpeI.

The codon optimized VST1 gene encoding Vitis vinifera (grapevine) resveratrol synthase (Hain et al., 1993) (SEQ ID NO 42) for expression in S. cerevisiae was synthesized by GenScript Corporation (Piscataway, N.J.). The synthetic VST1 gene was delivered inserted in E. coli pUC57 vector flanked by BamH1 and Xho1 restriction sites. The synthetic gene was purified from the pUC57 vector by BamH1/Xho1 restriction and purified from agarose gel using the QiaQuick Gel Extraction Kit (Qiagen).

Example 17 Construction of a Yeast Vector for Galactose Induced Expression of SAM8

The EcoRI/SpeI digested SAM8 product, isolated as described in example 16, was ligated into EcoRI/SpeI digested pESC-URA vector (Stratagene), resulting in vector pESC-URA-SAM8. Two different clones of pESC-URA-SAM8 were sequenced to verify the sequence of the cloned gene.

Example 18 Construction of a Yeast Vector for Galactose Induced Expression of 4CL2 and VST1

The gene encoding 4CL2 was isolated using the primers as described in example 16. The amplified 4CL2 PCR-product was digested with EcoR1/Spe1 and ligated into EcoR1/Spe1 digested pESC-HIS vector (Stratagene), resulting in vector pESC-HIS-4CL2. Two different clones of pESC-HIS-4CL2 were sequenced to verify the sequence of the cloned gene.

The gene encoding VST1 was isolated as described in example 16. The amplified synthetic VST1 gene was digested with BamH1/Xho1 and ligated into BamH1/Xho1 digested pESC-HIS-4CL2. The resulting plasmid, pESC-HIS-4CL2-VST1, contained the genes encoding 4CL2 and VST1 under the control of the divergent galactose induced <=GAL1/GAL10=> promoters. The sequence of the gene encoding VST1 was verified by sequencing of two different clones of pESC-HIS-4CL2-VST1.

Example 19 Construction of Strong Constitutive Promoter Fragment TDH3

The 600 base pair TDH3 (GPD) promoter was amplified from S. cerevisiae genomic DNA using the forward primer 5′GC GAGCTC AGT TTA TCA TTA TCA ATA CTC GCC ATT TCA AAG SEQ ID NO 43 containing a Sad restriction site and the reverse primer 5′-CG TCTAGA ATC CGT CGA AAC TAA GTT CTG GTG TTT TAA AAC TAA AA SEQ ID NO 44 containing a Xba1 restriction site. The amplified TDH3 fragment was digested with Sac1/Xba1 and ligated into Sac1/Xba1 digested plasmid pRS416 (Sikorski and Hieter, 1989) as described previously (Mumberg et al, 1995) resulting in plasmid pRS416-TDH3.

Example 20 Construction of Constitutive Strong Promoter Fragment TEF2

The 400 base pair TEF2 promoter was amplified from S. cerevisiae genomic DNA using the forward primer 5′-GC GAGCTC ATA GCT TCA AAA TGT TTC TAC TCC TTT TTT ACT CTT SEQ ID NO 45 containing a Sac1 restriction site and the reverse primer 5′-CG TCTAGA AAA CTT AGA TTA GAT TGC TAT GCT TTC TTT CTA ATG A SEQ ID NO 46 containing a Xba1 restriction site. The amplified TEF2 fragment was digested with Sac1/Xba1 and ligated into Sac1/Xba1 digested plasmid pRS416 (Sikorski and Hieter, 1989) as described previously (Mumberg et al, 1995) resulting in plasmid pRS416-TEF2.

Example 21 Construction of Fused Divergent Constitutive TEF and TDH3 Promoter Fragment

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′ TTGCGTATTGGGCGCTCTTCC GAG CTC AGT TTA TCA TTA TCA ATA CTC GC SEQ ID NO 47 containing the underlined overhang for fusion PCR to TEF2 fragment and the reverse primer 5′ AT GGATCC TCT AGA ATC CGT CGA AAC TAA GTT CTG SEQ ID NO 48 containing the underlined BamH1 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 Sad restriction site was reamplified from PRS416-TEF2 using the forward primer 5′ AT GAATTC TCT AGA AAA CTT AGA TTA GAT TGC TAT GCT TTC SEQ ID NO 49 containing the underlined EcoR1 restriction site and the reverse primer 5′ TGA TAA TGA TAA ACT GAG CTC GGA AGA GCG CCC AAT ACG CAA AC SEQ ID NO 50 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 680 base pair TEF2 fragment and the 600 base pair TDH3 fragments were joined together (fused) using fusion PCR with the forward primer 5′ AT GAATTC TCT AGA AAA CTT AGA TTA GAT TGC TAT GCT TTC SEQ ID NO 51 and the reverse primer 5′ AT GGATCC TCT AGA ATC CGT CGA AAC TAA GTT CTG SEQ ID NO 52, resulting in the divergent fragment <=TEF2/TDH3=> (SEQ ID NO 53).

Example 22 Construction of a Yeast Vector for Constitutive Expression of Sam8

The vector pESC-URA-Sam8 with divergent galactose inducible promoters GAL1/GAL10 was sequentially digested with EcoRI and BamHI to remove the GAL1/GAL10 promoters.

The divergent constitutive <=TEF2/TDH3=> promoter fragment (Example 21) was re-amplified with forward primer 5′ AT GGATCC TCT AGA AAA CTT AGA TTA GAT TGC TAT GCT TTC SEQ ID NO 54 and reverse primer 5′AT GAATTC TCTAGA ATC CGT CGAAACTAAGTTCTGG SEQ ID NO 55.

The resulting PCR product was sequentially digested with ECORI and BamH1 and ligated into the above ECORI/BamHI digested vector (pESC-URA-Sam8) without the GAL1/Gal10 fragment. This resulted in a vector pESC-URA-TDH3-Sam8 with replaced promoters, from GAL1/Gal10 to TEF2/TDH3 (SEQ ID NO 56).

Example 23

Construction of a yeast vector for constitutive expression induced of 4CL2 and VST1

The vector pESC-HIS-4CL2-VST1 with divergent galactose inducible promoters GAL1/GAL10 was sequentially digested with EcoR1 and BamH1 to remove the GAL1/GAL10 promoters.

The divergent constitutive <=TEF2/TDH3=> promoter fragment (Example 21) was sequentially digested with EcoR1 and BamH1 and ligated into the above linearized vector without the GAL1/GAL10 fragment. This resulted in a vector pesc-HIS-TEF2-4CL2-TDH3-VST1 with replaced promoters, from GAL1/Gal10 to TEF2/TDH3 (SEQ ID NO 57).

Example 24 Generation of Strain with Constitutive Expression of the Pathway to Resveratrol in the Yeast S. Cerevisiae

The transformation of the yeast cell was conducted in accordance with methods known in the art, for instance by using lithium acetate transformation method (Gietz and Schiestl, 1991). S. cerevisiae strain FS01528 (CEN.PK MATa ura3 His3) was co-transformed with pESC-URA-TDH3-SaM8 (example 22) and pesc-HIS-TEF2-4CL2-TDH3-VST1 (example 23), and the transformed strain was named FS-SAM8-4CL2-VST1. Transformants were selected on medium lacking uracil and histidine and streak purified on the same medium.

Example 25 Generation of Biomass

Batch fermentation of FS-SAM8-4CL2-VST1 were carried out in order to generate wet biomass for inoculation of the wine fermentation. The fermentation was carried out in a Sartorius Biostat B plus fermentor under aerobic conditions. The working volume was 1 L, agitation was 1000 rpm, and air flow was set to 1.5 vvm. Temperature and pH were 30° C. and 5.5, respectively. The fermentation was inoculated to an initial OD of 0.0005. The composition is described in the following:

Media:

Compound Concentration [g/L] Glucose 165 Urea 22.72 KH2PO4 30 MgSO4 5 Vitamin solution 10 mL* Trace metal solution 10 mL* Antifoam 100 μL

Vitamin solution

Concentration [g/L] Biotin 0.05 Calcium panthotenate 1 Nicotinic acid 1 Myo-inositol 25 Thiamine HCl 1 Pyridoxal HCl 1 Para-aminobenzoic acid 0.2

Trace metal solution

Concentration [g/L] EDTA 15 ZnSO4 × 7H2O 4.5 MnCl2 × 2H2O 1 CoCl2 × 6H2O 0.3 CuSO4 × 5H2O 0.3 Na2MoO4 × 2H2O 0.4 CaCl2 × 2H2O 4.5 FeSO4 × 7H2O 3 H3BO3 1 KI 0.1

At the end of the fermentation, when all glucose was depleted, the biomass was harvested into Falcon tubes by centrifugation. Four 50 mL sterile Falcon tubes were used and the cells were harvested using 5 consecutive centrifuge runs at 4° C. with 4000 rpm for five minutes. The centrifuge used was a Satorius Sigma 3-16K including a swing out rotor with for buckets. The supernatant was each discarded after each run. The four tubes contained approximately 70 g of wet biomass, which was used as inoculum for the wine fermentation.

Example 26 Resveratrol Production by Fermenting Wine

Objective: To evaluate the relative efficacy of producing resveratrol by wine fermentation using wine juice concentrate as plant material compared to commercially available wine yeast,

Two identical wine making kits procured from Winexpert Incorporated of Canada were used as the basis of making a comparison between the commercially available yeast Saccharomyces bayanus (Lalvin EC1118), the control strain, for wine making versus FS-SAM8-4CL2-VST1. The kit used was the “Selection Original—Barolo Style.” This style utilizes the Nebbiolo red grape as the basis of the grape juice concentrate. This concentrate is preserved with suphur dioxide, citric acid, malic acid, tartaric acid, and diammonium phosphate. Also supplied in the kit are single packages of a premeasured amount of bentonite for use as a clarifying agent, potassium metabisuphite as a stabilizer, and oak chips used for flavoring. The package of oak chips was not used in this experiment for either fermentation. Kit designated Lot 07318080147 was used for fermentation of Saccharomyces bayanus (Lalvin EC1118) and Lot 07318080172 was for fermentation of FS-SAM8-4CL2-VST1.

Each of two kits was treated similarly except for the inoculum. For the wine fermentation using the control strain five grams of yeast (Lalvin EC1118 of Lallemand Inc.) were used, whereas for FS-SAM8-4CL2-VST1 18 g of wet biomass, which was approximately 5 g/L dry weight, was used as inoculum.

Before inoculation, all equipment and containers being used were first sanitized with a solution of metabisulfite (˜50 grams in 4 liters). Spring water (“Deerpark”) was used for all solutions. Two liters of warm water (˜300° C.) were added to a clean 30 liter plastic carboy container and stirred vigorously while slowly sprinkling in the contents of the bentonite package until fully wetted and dispersed; for approximately one minute. The grape juice concentrate (15 L) were filled into the containers. The package of grape juice concentrate was washed with 4 liters, which was afterwards added to container. The final container volume was adjusted to 23 liters with cool. The oak chip package intended to be used as a flavoring enhancer was not used in either the control or treatment groups. With the juice solution at about room temperature (˜20.0° C.), the package of Lalvin yeast was sprinkled on top of the control juice solution and FS-SAM8-4CL2-VST1 was poured into solution. Each container (primary fermentor) was covered with an air-lock. The wine was fermented for 50 days. Samples of wine and mash were taken at the end of the fermentation and placed into a sealed plastic cup and stored frozen until analyses.

Example 27 Extraction of Resveratrol from Wine-Mash

Two duplicate samples of the mash from the control- and treatment groups were evaluated for their content of stilbenoids, hereafter referred to as control A, control B, treatment A and treatment B. Aliquots of 50 ml of either samples, containing a mixture of mash and wine, were centrifuged for 10 minutes at 3500XG at 10° C., after which the supernatant was discarded. Hereafter, the wet weight content of mash was 12.57 g for control A, 14.03 g for control B, 11.63 g for treatment A and 12.52 g for treatment B. Next, 10 ml of 99% ethyl acetate was added and whirly-mixed at room temperature on an automated whirly mixer for 2 hours at 2500 rpm. Then samples were centrifuged for 10 minutes at 3500×g at 10° C., and the upper, yellow/reddish coloured, ethyl acetate was collected reduced in volume by evaporation in a freeze-dryer. After approximately 2 hours a dry reddish residue was obtained, which was carefully resuspended in 120 μl 20% ethanol and resulted in a solution that contained non-dissolved dark precipitates. The solution was, therefore, whirly-mixed and centrifuged at 13000×g for 5 minutes. The supernatant now consisted of a clear reddish 20%-ethanol solution, which was diluted 100-fold further in two steps of 10-fold; the first dilution step was rendered in 20%-ethanol whereas Millipore water was used for the subsequent second 10-fold dilution step. Samples were then ready to be analyzed by HPLC.

Example 28 HPLC Determination of Stilbenoids and Phenylpropanoids

For quantitative analysis of coumaric acid, cinnamic acid, trans-resveratrol and trans-pinosylvin, samples were subjected to separation by high-performance liquid chromatography (HPLC), using a HPLC-system from Dionex, prior to UV-diode-array detection at 1=306 nm. A Phenomenex (Torrance, Calif., USA) Gemini C6-Phenyl, 3 micron (100×3.00 mm) column was used at 35° C. The method consisted of a linear gradient of methanol and millipore water (both containing 50 ppm trifluoroacetic acid), at a flow rate of 0.5 ml/min. The gradient profile was linear from 20% methanol to 100% methanol over 20 min. The elution times were 7.5 min. for coumaric acid, 10.1 min. for trans-resveratrol, 11.8 min. for cinnamic acid and 14.0 min for pinosylvin.

Example 29 Concentration of Resveratrol in the Mash

The chromatograms of both the control group and the treatment group contained all a peak with a similar retention time as resveratrol (9.9 minutes) and with an UV spectrum that resembled the UV spectrum of resveratrol. Quantification of the peak indicated that the resveratrol content in the mash was 0.33 mg/kg for control A, 0.48 mg/kg for control B, giving an average of 0.41 mg/kg for the control group. The mash of treatment A contained 0.80 mg/kg and treatment B contained 0.73 mg/kg, giving an average resveratrol content of 0.77 mg/kg for the treatment group. Hence, on average, the mash of the treatment group contained 89% more resveratrol than the mash of the control group.

It can, therefore, be concluded that the use of resveratrol-producing yeast in a wine fermentation process has led to a substantial enrichment of the resveratrol in the mash.

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

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Claims

1. A method for the production of a mixture comprising stilbenoids, comprising extracting resveratrol or pinosylvin from a solids waste material separated from a fermentation of plant material conducted using a genetically modified yeast strain having a metabolic pathway producing the mixture comprising stilbenoids.

2. The method of claim 1, further comprising the preliminary steps of conducting said fermentation of plant material using the genetically modified yeast strain having a metabolic pathway producing the mixture comprising stilbenoids and separating a solids waste material from said fermentation.

3. The method of claim 1, wherein the fermentation is a fermentation of fruit must together with or separated from pommace.

4. The method of claim 1, wherein the fermentation is a fermentation of pommace separated from fruit must.

5. The method of claim 2, wherein the fruit is grape, apple or pear.

6. The method of claim 1, wherein the fermentation is a beer making fermentation.

7. The method of claim 1, wherein the genetically modified yeast strain is genetically modified Saccharomyces yeast.

8. The method of claim 1, further comprising the step of recovering resveratrol or pinosylvin.