Patent Publication Number: US-9834793-B2

Title: Metabolically engineered cells for the production of resveratrol or an oligomeric or glycosidically-bound derivative thereof

Description:
This application is a continuation of U.S. application Ser. No. 12/081,571, filed Apr. 17, 2008 (now U.S. Pat. No. 9,040,269), which is a continuation in part of U.S. application Ser. No. 11/816,847, filed on Aug. 22, 2007 (now U.S. Pat. No. 8,895,287), which is the U.S. national stage of International Application No. PCT/EP2006/060154, filed on Feb. 21, 2006, which claims priority to Great Britain Patent Application No. 0503657.9 filed on Feb. 22, 2005, the disclosures of each of which are explicitly incorporated by reference in their entirety. 
    
    
     FIELD OF THE INVENTION 
     This invention relates generally to the production of the polyphenol resveratrol or an oligomeric or glycosidically bound derivative thereof such as its β-glucoside piceid using microbial cells. Furthermore, it relates to the use of naturally occurring or recombinant micro-organisms that produce resveratrol or such a derivative for production of food, feed and beverages. 
     BACKGROUND OF THE INVENTION 
     Production of chemicals from micro-organisms has been an important application of biotechnology. Typically, the steps in developing such a bio-production method may include 1) selection of a proper micro-organism host, 2) elimination of metabolic pathways leading to by-products, 3) deregulation of desired pathways at both enzyme activity level and the transcriptional level, and 4) overexpression of appropriate enzymes in the desired pathways. In preferred aspect, the present invention has employed combinations of the steps above to redirect carbon flow from phenylalanine or tyrosine through enzymes of the plant phenylpropanoid pathway which supplies the necessary precursor for the desired biosynthesis of resveratrol. 
     Resveratrol (or 3,4,5-trihydroxystilbene) is a phytophenol belonging to the group of stilbene phytoalexins, which are low-molecular-mass secondary metabolites that constitute the active defense mechanism in plants in response to infections or other stress-related events. Stilbene phytoalexins contain the stilbene skeleton (trans-1,2-diphenylethylene) as their common basic structure: that may be supplemented by addition of other groups as well (Hart and Shrimpton, 1979, Hart, 1981). Stilbenes have been found in certain trees (angio-sperms, gymnosperms), but also in some herbaceous plants (in species of the Myrtaceae, Vitaceae and Leguminosae families). Said compounds are toxic to pests, especially to fungi, bacteria and insects. Only few plants have the ability to synthesize stilbenes, or to produce them in an amount that provides them sufficient resistance to pests. 
     The synthesis of the basic stilbene skeleton is pursued by stilbene synthases. So far, two enzymes have been designated as a stilbene synthase; pinosylvine synthase and resveratrol synthase. To date, the groundnut ( Arachis hypogaea ) resveratrol synthase has been characterised in most detail, such that most of the properties are known (Schoppner and Kindl, 1984). Substrates that are used by stilbene synthases are malonyl-CoA, cinnamoyl-CoA or coumaroyl-CoA. These substances occur in every plant because they are used in the biosynthesis of other important plant constituents as well such as flavonoids, flower pigments and lipids. Resveratrol ( FIG. 1  trans-form) consists of two closely connected phenol rings and belongs therefore to the polyphenols. While present in other plants, such as  eucalyptus , spruce, and lily, and in other foods such as mulberries and peanuts, resveratrol&#39;s most abundant natural sources are  Vitis vinifera , - labrusca , and - muscadine  ( rotundifolia ) grapes, which are used to make wines. The compound occurs in the vines, roots, seeds, and stalks, but its highest concentration is in the skin (Celotti et al., 1996), which contains 50-100 μg/g. (Jang et al. 1997). During red wine vinification the grape skins are included in the must, in contrast to white wine vinification, and therefore resveratrol is found in small quantities in red wine only. Resveratrol has, besides its antifungal properties, been recognized for its cardioprotective- and cancer chemopreventive activities; it acts as a phytoestrogen, an inhibitor of platelet aggregation (Kopp et al, 1998; Gehm et al 1997; Lobo et al 1995), and an antioxidant (Jang et al., 1997; Huang 1997). These properties explain the so-called French Paradox, i.e. the wine-drinking French have a low incidence of coronary heart disease despite a low-exercise, high-fat diet. Recently it has been shown that resveratrol can also activate the SIR2 gene in yeast and the analogous human gene SIRT1, which both play a key role in extending life span. Ever since, attention is very much focused on the life-span extending properties of resveratrol (Hall, 2003, Couzin, 2004). 
     American health associations, such as the Life Extension Foundation, are promoting the vast beneficial effects of this drug, and thereby propelling the ideal conditions for a successful commercialisation. Present production processes rely mostly upon extraction of resveratrol, either from the skin of grape berries, or from Knot weed. This is a labour intensive process and generates low yield which, therefore, prompts an incentive for the development of novel, more efficient and high-yielding production processes. 
     In plants, the phenylpropanoid pathway is responsible for the synthesis of a wide variety of secondary metabolic compounds, including lignins, salicylates, coumarins, hydroxycinnamic amides, pigments, flavonoids and phytoalexins. Indeed formation of resveratrol in plants proceeds through the phenylpropanoid pathway. The amino acid L-phenylalanine is converted into trans-cinnamic acid through the non-oxidative deamination by L-phenylalanine ammonia lyase (PAL) ( FIG. 2 ). Next, trans-cinnamic acid is hydroxylated at the para-position to 4-coumaric acid (4-hydroxycinnamic acid) by cinnamate-4-hydroxylase (C4H), a cytochrome P450 monooxygenase enzyme, in conjunction with NADPH:cytochrome P450 reductase (CPR). The 4-coumaric acid, is subsequently activated to 4-coumaroyl-CoA by the action of 4-coumarate-CoA ligase (4CL). Finally, resveratrol synthase (VST) catalyses the condensation of a phenylpropane unit of 4-coumaroyl-CoA with malonyl CoA, resulting in formation of resveratrol. 
     Recently, a yeast was disclosed that could produce resveratrol from 4-coumaric acid that is found in small quantities in grape must (Becker et al. 2003). The production of 4-coumaroyl-CoA, and concomitant resveratrol, in laboratory strains of  S. cerevisiae , was achieved by co-expressing a heterologous coenzyme-A ligase gene, from hybrid poplar, together with the grapevine resveratrol synthase gene (vst1). The other substrate for resveratrol synthase, malonyl-CoA, is already endogenously produced in yeast and is involved in de novo fatty-acid biosynthesis. The study showed that cells of  S. cerevisiae  could produce minute amounts of resveratrol, either in the free form or in the glucoside-bound form, when cultured in synthetic media that was supplemented with 4-coumaric acid. 
     However, said yeast would not be suitable for a commercial application because it suffers from low resveratrol yield, and requires addition of 4-coumaric acid, which is only present in few industrial media. In order to facilitate and broaden the application of resveratrol as both a pharmaceutical and neutraceutical, it is therefore highly desirable to obtain a yeast that can produce resveratrol directly from glucose, without addition of 4-coumaric acid. 
     A recent study (Ro and Douglas, 2004) describes the reconstitution of the entry point of the phenylpropanoid pathway in  S. cerevisiae  by introducing PAL, C4H and CPR from Poplar. The purpose was to evaluate whether multienzyme complexes (MECs) containing PAL and C4H are functionally important at this entry point into phenylpropanoid metabolism. By feeding the recombinant yeast with [3H]-phenylalanine it was found that the majority of metabolized [3H]-phenylalanine was incorporated into 4-[3H]-coumaric acid, and that phenylalanine metabolism was highly reduced by inhibiting C4H activity. Moreover, PAL-alone expressers metabolized very little phenylalanine into cinnamic acid. When feeding [3H]-phenylalanine and [14C]-trans-cinnamic acid simultaneously to the triple expressers, no evidence was found for channeling of the endogenously synthesized [3H]-trans-cinnamic acid into 4-coumaric acid. Therefore, efficient carbon flux from phenylalanine to 4-coumaric acid via reactions catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast, and sheer biochemical coupling of PAL and C4H seems to be sufficient to drive carbon flux into the phenylpropanoid pathway. In yet another study (Hwang et al., 2003) production of plant-specific flavanones by  Escherichia coli  was achieved through expression of an artificial gene cluster that contained three genes of a phenyl propanoid pathway of various heterologous origins; PAL from the yeast  Rhodotorula rubra,  4CL from the actinomycete  Streptomyces coelicolor , and chalcone synthase (CHS) from the licorice plant  Glycyrrhiza echinata . These pathways bypassed C4H, because the bacterial 4CL enzyme ligated coenzyme A to both trans-cinnamic acid and 4-coumaric acid. In addition, the PAL from  Rhodotorula rubra  uses both phenylalanine and tyrosine as the substrates. Therefore,  E. coli  cells containing the gene clusters and grown on glucose, produced small amounts of two flavanones, pinocembrin (0.29 g/l) from phenylalanine and naringenin (0.17 g/l) from tyrosine. In addition, large amounts of their precursors, 4-coumaric acid and trans-cinnamic acid (0.47 and 1.23 mg/liter respectively), were accumulated. Moreover, the yields of these compounds could be increased by addition of phenylalanine and tyrosine. 
     Whereas the enzyme from dicotylic plants utilizes only phenylalanine efficiently, several studies indicated that PAL from monocotylic plants, and some micro-organisms, utilizes tyrosine as well (Rösler et al., 1997). In such reactions the enzyme activity is designated tyrosine ammonia lyase (TAL,  FIG. 3 ). Conversion of tyrosine by TAL results in the direct formation of 4-coumaric acid without the intermediacy of C4H and CPR. Mostly both activities reside on the same polypeptide and have very similar catalytic efficiencies, in spite of large differences in Km and turnover number. However, most PAL/TAL enzymes from plants prefer phenylalanine rather than tyrosine. The level of TAL activity is mostly lower than PAL activity, but the magnitude of this difference varies over a wide range. For example, the parsley enzyme has a Km for phenylalanine of 15-25 μM and for tyrosine 2.0-8.0 mM with turnover numbers 22 s −1  and 0.3 s −1  respectively. In contrast, the maize enzyme has a Km for phenylalanine only 15-fold higher than for tyrosine, and turnover numbers about 10-fold higher. Moreover, in the red yeasts,  Rhodotorula glutinis  ( Rhodosporidium toruloides ) and - rubra , the TAL catalytic activity is close to the PAL catalytic activity with a ratio of TAL/PAL of approximately 0.58. It is believed that the PAL enzyme in these yeasts degrades phenylalanine as a catabolic function and the trans-cinnamic acid formed is converted to benzoate and other cellular materials, whereas in plants it is thought to be merely a regulatory enzyme in the biosynthesis of lignin, isoflavonoids and other phenylpropanoids. 
     Recently, an open reading frame was found in the bacterium  Rhodobacter capsulatus  that encodes a hypothetical biosynthetic tyrosine ammonia lyase (TAL) that is involved in the biosynthesis of the chromophore of the photoactive yellow protein (Kyndt et al., 2002). This was the first time that a PAL-homologous gene was found in bacteria. The TAL gene was isolated and overproduced in  Escherichia coli . The Km and kcat values for the conversion of tyrosine to 4-coumaric acid were 15.6 μM and 27.7 s −1  respectively, and for conversion of L-phenylalanine to trans-cinnamic acid were 1277 μM and 15.1 s −1  respectively. As a consequence of the smaller Km and a slightly larger kcat, the enzyme shows a strong preference for tyrosine over L-phenylalanine, with a catalytic efficiency (Km/kcat) for tyrosine of approximately 150-fold larger than for phenylalanine. The kinetic studies established that tyrosine, and not L-phenylalanine, is the natural substrate of the enzyme under physiological conditions. Very recently a study described the heterologous coexpression of phenylalanine ammonia lyase, cinnamate-4-hydroxylase, 4-coumarate-Coa-ligase and chalcone synthase, for the production of flavonoids in  E. coli  (Watts et al., 2004). The simultaneous expression of all four genes, however, was not successful because of a nonfunctional cinnamate-4-hydroxylase. The substitution of phenylalanine ammonia lyase and cinnamate-4-hydroxylase by a new tyrosine ammonia lyase that was cloned from  Rhodobacter sphaeroides , could, however, solved the problem and led to high-level production of the flavonone naringenin. Furthermore, said tyrosine ammonia lyase from  Rhodobacter sphaeroides  is also used for heterologous production of 4-coumaric acid (i.e. para-hydroxycinnamic acid) in  Escherichia coli  (US-A-2004059103). Evenmore, further methods for development of a biocatalyst for conversion of glucose into 4-coumaric acid are described. US-A-2004023357 discloses a tyrosine ammonia lyase from the yeast  Trichosporon cutaneum  for the production of coumaric acid in  Escherichia coli  and  Saccharomyces cerevisiae . US-A-2001053847 describes the incorporation of the wild type PAL from the yeast  Rhodotorula glutinis  into  E. coli , underlining the ability of the wildtype PAL to convert tyrosine directly to 4-coumaric acid. Moreover, there is also exemplification of incorporation of the wildtype PAL from the yeast  Rhodotorula glutinis , plus a plant C4H and CPR into  E. coli  and  S. cerevisiae . Also described is the development of a biocatalyst through mutagenesis of the wild type yeast PAL  Rhodotorula glutinis  with enhanced TAL activity (U.S. Pat. No. 6,521,748). Neither of the aforementioned patents claim the incorporation of 4CL and VST for the production of resveratrol. 
     Recently, evidence was shown that the filamentous fungi  A. oryzae  contained the enzyme chalcone synthase (CHS) that is normally involved in the biosynthesis of flavonoids, such as naringenin, in plants (Seshime et al., 2005). Indeed it was also shown that  A. oryzae  contained the major set of genes responsible for phenylpropanoid-flavonoid metabolism, i.e PAL, C4H and 4CL. However, there is no evidence that  A. oryzae  contained a stilbene synthase such as resveratrol synthase. 
     The present invention now provides a micro-organism 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. Such a micro-organism may be naturally occurring and may be isolated by suitable screening procedures, but more preferably is genetically engineered. 
     Preferably, said resveratrol or derivative is produced in a reaction catalysed by an enzyme in which endogenous malonyl-CoA is a substrate, and preferably said resveratrol is produced from 4-coumaroyl-CoA. 
     Said resveratrol or derivative is preferably produced from 4-coumaroyl-CoA by a resveratrol synthase which is preferably expressed in said micro-organism from nucleic acid coding for said enzyme which is not native to the micro-organism. 
     Generally herein, unless the context implies otherwise, references to resveratrol include reference to oligomeric or glycosidically bound derivatives thereof, including particularly piceid. 
     Thus, in certain preferred embodiments, said resveratrol synthase is a resveratrol synthase (EC 2.3.1.95) from a plant belonging to the genus of  Arachis , e.g.  A. glabatra, A. hypogaea , a plant belonging to the genus of  Rheum , e.g.  R. tataricum , a plant belonging to the genus of  Vitus , e.g.  V. labrusca, V. riparaia, V. vinifera , or any one of the genera  Pinus, Piceea, Lilium, Eucalyptus, Parthenocissus, Cissus, Calochortus, Polygonum, Gnetum, Artocarpus, Nothofagus, Phoenix, Festuca, Carex, Veratrum, Bauhinia  or  Pterolobium.    
     Preferably, said 4-coumaric acid is produced from trans-cinnamic acid, suitably by an enzyme in a reaction catalysed by said enzyme in which oxygen is a substrate, NADH or NADPH is a cofactor and NAD +  or NADP +  is a product. 
     Thus, said 4-coumaric acid may be produced from trans-cinnamic acid by a cinnamate 4-hydroxylase, which preferably is expressed in said micro-organism from nucleic acid coding for said enzyme which is not native to the micro-organism. 
     In certain preferred embodiments, including those referred to in the paragraphs above, said cinnamate-4-hydroxylase is a cinnamate-4-hydroxylase (EC 1.14.13.11) from a plant or a micro-organism. The plant may belong to the genus of  Arabidopsis , e.g.  A. thaliana , a plant belonging to the genus of  Citrus , e.g.  C. sinensis, C. x paradisi , a plant belonging to the genus of  Phaseolus , e.g.  P. vulgaris , a plant belonging to the genus of  Pinus , e.g.  P. taeda , a plant belonging to the genus of  Populus , e.g.  P. deltoides, P. tremuloides, P. trichocarpa , a plant belonging to the genus of  Solanum , e.g.  S. tuberosum , a plant belonging to the genus of  Vitus , e.g.  Vitus vinifera , a plant belonging to the genus of  Zea , e.g.  Z. mays , or other plant genera e.g.  Ammi, Avicennia, Camellia, Camptotheca, Catharanthus, Glycine, Helianthus, Lotus, Mesembryanthemum, Physcomitrella, Ruta, Saccharum, Vigna . The micro-organism might be a fungus belonging to the genus  Aspergillus , e.g.  A. oryzae.    
     Preferably, said 4-coumaric acid is produced from tyrosine in a reaction catalysed by an enzyme in which ammonia is produced and suitably, said 4-coumaric acid is produced from tyrosine by a L-phenylalanine ammonia lyase or a tyrosine ammonia lyase, e.g. tyrosine ammonia lyase (EC 4.3.1.5) from yeast or bacteria. Suitably, the tyrosine ammonia lyase is from the yeast  Rhodotorula rubra  or from the bacterium  Rhodobacter capsulatus.    
     Optionally, said tyrosine ammonia lyase is expressed in said micro-organism from nucleic acid coding for said enzyme which is not native to the micro-organism. 
     Alternatively, said trans-cinnamic acid may be produced from L-phenylalanine in a reaction catalysed by an enzyme in which ammonia is produced and suitably said trans-cinnamic acid is formed from L-phenylalanine by a phenylalanine ammonia lyase. 
     In certain preferred embodiments, said L-phenylalanine ammonia lyase is a L-phenylalanine ammonia lyase (EC 4.3.1.5) from a plant or a micro-organism. The plant may belong to the genus of  Arabidopsis , e.g.  A. thaliana , a plant belonging to the genus of  Brassica , e.g.  B. napus, B. rapa , a plant belonging to the genus of  Citrus , e.g.  C. reticulata, C. clementinus, C. limon , a plant belonging to the genus of  Phaseolus , e.g.  P. coccineus, P. vulgaris , a plant belonging to the genus of  Pinus , e.g.  P. banksiana, P. monticola, P. pinaster, P. sylvestris, P. taeda , a plant belonging to the genus of  Populus , e.g.  P. balsamifera, P. deltoides, P. Canadensis, P. kitakamiensis, P. tremuloides , a plant belonging to the genus of  Solanum , e.g.  S. tuberosum , a plant belonging to the genus of  Prunus , e.g.  P. avium, P. persica , a plant belonging to the genus of  Vitus , e.g.  Vitus vinifera , a plant belonging to the genus of  Zea , e.g.  Z. mays  or other plant genera e.g.  Agastache, Ananas, Asparagus, Bromheadia, Bambusa, Beta, Betula, Cucumis, Camellia, Capsicum, Cassia, Catharanthus, Cicer, Citrullus, Coffea, Cucurbita, Cynodon, Daucus, Dendrobium, Dianthus, Digitalis, Dioscorea, Eucalyptus, Gallus, Ginkgo, Glycine, Hordeum, Helianthus, Ipomoea, Lactuca, Lithospermum, Lotus, Lycopersicon, Medicago, Malus, Manihot, Medicago, Mesembryanthemum, Nicotiana, Olea, Oryza, Pisum, Persea, Petroselinum, Phalaenopsis, Phyllostachys, Physcomitrella, Picea, Pyrus, Quercus, Raphanus, Rehmannia, Rubus, Sorghum, Sphenostylis, Stellaria, Stylosanthes, Triticum, Trifolium, Triticum, Vaccinium, Vigna, Zinnia . The micro-organism might be a fungus belonging to the genus  Agaricus , e.g.  A. bisporus , a fungus belonging to the genus  Aspergillus , e.g.  A. oryzae, A. nidulans, A. fumigatus , a fungus belonging to the genus  Ustilago , e.g.  U. maydis , a bacterium belonging to the genus  Rhodobacter , e.g.  R. capsulatus , a yeast belonging to the genus  Rhodotorula , e.g.  R. rubra.    
     Suitably, said L-phenylalanine ammonia lyase is expressed in said micro-organism from nucleic acid coding for said enzyme which is not native to the micro-organism. 
     Preferably, 4-coumaroyl-CoA is formed in a reaction catalysed by an enzyme in which ATP and CoA are substrates and ADP is a product and suitably 4-coumaroyl-CoA is formed in a reaction catalysed by a 4-coumarate-CoA ligase. 
     Said 4-coumarate-CoA ligase may be a 4-coumarate-CoA ligase (EC 6.2.1.12) from a plant, a micro-organism or a nematode. The plant may belong to the genus of  Abies , e.g.  A. beshanzuensis, B. firma, B. holophylla , a plant belonging to the genus of  Arabidopsis , e.g.  A. thaliana , a plant belonging to the genus of  Brassica , e.g.  B. napus, B. rapa, B. oleracea , a plant belonging to the genus of  Citrus , e.g.  C. sinensis , a plant belonging to the genus of  Larix , e.g.  L. decidua, L. gmelinii, L. griffithiana, L. himalaica, L. kaempferi, L. laricina, L. mastersiana, L. occidentalis, L. potaninii, L. sibirica, L. speciosa , a plant belonging to the genus of  Phaseolus , e.g.  P. acutifolius, P. coccineus , a plant belonging to the genus of  Pinus , e.g.  P. armandii P. banksiana, P. pinaster , a plant belonging to the genus of  Populus , e.g.  P. balsamifera, P. tomentosa, P. tremuloides , a plant belonging to the genus of  Solanum , e.g.  S. tuberosum , a plant belonging to the genus of  Vitus , e.g.  Vitus vinifera , a plant belonging to the genus of  Zea , e.g.  Z. mays , or other plant genera e.g.  Agastache, Amorpha, Cathaya, Cedrus, Crocus, Pestuca, Glycine, Juglans, Keteleeria, Lithospermum, Lolium, Lotus, Lycopersicon, Malus, Medicago, Mesembryanthemum, Nicotiana, Nothotsuga, Oryza, Pelargonium, Petroselinum, Rhyscomitrella, Picea, Prunus, Pseudolarix, Pseudotsuga, Rosa, Rubus, Ryza, Saccharum, Suaeda, Thellungiella, Triticum, Tsuga . The micro-organism might be a filamentous fungi belonging to the genus  Aspergillus , e.g.  A. flavus, A. nidulans, A. oryzae, A. fumigatus , a filamentous fungus belonging to the genus  Neurospora , e.g.  N. crassa , a fungus belonging to the genus  Yarrowia , e.g.  Y. lipolytica , a fungus belonging to the genus of  Mycosphaerella , e.g.  M. graminicola , a bacterium belonging to the genus of  Mycobacterium , e.g.  M. bovis, M. leprae, M. tuberculosis , a bacterium belonging to the genus of  Neisseria , e.g.  N. meningitidis , a bacterium belonging to the genus of  Streptomyces , e.g.  S. coelicolor , a bacterium belonging to the genus of  Rhodobacter , e.g.  R. capsulatus , a nematode belonging to the genus  Ancylostoma , e.g.  A. ceylanicum , a nematode belonging to the genus  Caenorhabditis , e.g.  C. elegans , a nematode belonging to the genus  Haemonchus , e.g.  H. contortus , a nematode belonging to the genus  Lumbricus , e.g.  L. rubellus , a nematode belonging to the genus  Meloidogyne , e.g.  M. hapla , a nematode belonging to the genus  Strongyloidus , e.g.  S. rattii, S. stercoralis , a nematode belonging to the genus  Pristionchus , e.g.  P. pacificus.    
     Optionally, a NADPH:cytochrome P450 reductase (CPR) has been recombinantly introduced into said micro-organism. This may be a plant CPR introduced into a non-plant micro-organism. Alternatively, a native NADPH:cytochrome P450 reductase (CPR) has been overexpressed in said micro-organism. 
     In certain preferred embodiments, including those referred to in the paragraphs above, said NADPH:cytochrome P450 reductase is a NADPH:cytochrome P450 reductase (EC 1.6.2.4) from a plant belonging to the genus of  Arabidopsis , e.g.  A. thaliana , a plant belonging to the genus of  Citrus , e.g.  C. sinensis, C. x paradisi , a plant belonging to the genus of  Phaseolus , e.g.  P. vulgaris , a plant belonging to the genus of  Pinus , e.g.  P. taeda , a plant belonging to the genus of  Populus , e.g.  P. deltoides, P. tremuloides, P. trichocarpa , a plant belonging to the genus of  Solanum , e.g.  S. tuberosum , a plant belonging to the genus of  Vitus , e.g.  Vitus vinifera , a plant belonging to the genus of  Zea , e.g.  Z. mays , or other plant genera e.g.  Ammi, Avicennia, Camellia, Camptotheca, Catharanthus, Glycine, Helianthus, Lotus, Mesembryanthemum, Physcomitrella, Ruta, Saccharum, Vigna.    
     Whilst the micro-organism may be naturally occurring, preferably at least one copy of at least one genetic sequence encoding a respective enzyme in said metabolic pathway has been recombinantly introduced into said micro-organism. 
     Additionally or alternatively to introducing coding sequences coding for a said enzyme, one may provide one or more expression signals, such as promoter sequences, not natively associated with said coding sequence in said organism. Thus, optionally, at least one copy of a genetic sequence encoding a tyrosine ammonia lyase is operatively linked to an expression signal not natively associated with said genetic sequence in said organism, and/or at least one copy of a genetic sequence encoding a L-phenylalanine ammonia lyase is operatively linked to an expression signal not natively associated with said genetic sequence in said organism. 
     Optionally, at least one copy of a genetic sequence encoding cinnamate 4-hydroxylase, whether native or not, is operatively linked to an expression signal not natively associated with said genetic sequence in said organism. 
     Optionally, at least one copy of a genetic sequence encoding a 4-coumarate-CoA ligase, whether native or not, is operatively linked to an expression signal not natively associated with said genetic sequence in said organism. 
     Optionally, at least one copy of a genetic sequence encoding a resveratrol synthase, whether native or not, is operatively linked to an expression signal not natively associated with said genetic sequence in said organism. 
     Expression signals include nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Such sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. 
     In certain aspects the invention provides a metabolically engineered micro-organism having an operative metabolic pathway in which a first metabolite is transformed into a second metabolite in a reaction catalysed by a first enzyme, said reaction step producing ammonia, and in which said second metabolite is transformed into a third metabolite in a reaction catalysed by a second enzyme, in which oxygen is a substrate, NADPH or NADH is a cofactor and NADP +  or NAD +  is a product, and in which said third metabolite is transformed into a fourth metabolite in a reaction catalysed by a third enzyme in which ATP and CoA is a substrate, and ADP is a product, and in which said fourth metabolite is transformed into a fifth metabolite in a reaction catalysed by a fourth enzyme in which endogenous malonyl-CoA is a substrate. 
     The present invention also provides a metabolically engineered micro-organism having an operative metabolic pathway in which a first metabolite is transformed into a said third metabolite catalysed by a first enzyme, said reaction step producing ammonia, without the involvement of said second enzyme, and in which said third metabolite is transformed into a said fourth metabolite in a reaction catalysed by a said third enzyme in which ATP and CoA is a substrate, and ADP is a product, and in which said fourth metabolite is transformed into a said fifth metabolite in a reaction catalysed by a said fourth enzyme in which endogenous malonyl-CoA is a substrate. 
     The micro-organisms described above include ones 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. 
     They include also ones 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. 
     In the present context the term “micro-organism” relates to microscopic organisms, including bacteria, microscopic fungi, including yeast. 
     More specifically, the micro-organism may be a fungus, and more specifically a filamentous fungus belonging to the genus of  Aspergillus , e.g.  A. niger, A. awamori, A. oryzae, A. nidulans , a yeast belonging to the genus of  Saccharomyces , e.g.  S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum , a yeast belonging to the genus  Kluyveromyces , e.g.  K. lactis K. marxianus  var.  marxianus, K. thermotolerans , a yeast belonging to the genus  Candida , e.g.  C. utilis C. tropicalis, C. albicans, C. lipolytica, C. versatilis , a yeast belonging to the genus  Pichia , e.g.  P. stipidis, P. pastoris, P. sorbitophila , or other yeast genera, e.g.  Cryptococcus, Debaromyces, Hansenula, Pichia, Yarrowia, Zygosaccharomyces  or  Schizosaccharomyces . Concerning other micro-organisms a non-exhaustive list of suitable filamentous fungi is supplied: a species belonging to the genus  Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, Trichoderma.    
     Concerning bacteria a non-exhaustive list of suitable bacteria is given as follows: a species belonging to the genus  Bacillus , a species belonging to the genus  Escherichia , a species belonging to the genus  Lactobacillus , a species belonging to the genus  Lactococcus , a species belonging to the genus  Corynebacterium , a species belonging to the genus  Acetobacter , a species belonging to the genus  Acinetobacter , a species belonging to the genus  Pseudomonas , etc. 
     The preferred micro-organisms of the invention may be  S. cerevisiae, A. niger, A. oryzae, E. coli, L. lactis  or  B. subtilis.    
     The constructed and engineered micro-organism can be cultivated using commonly known processes, including chemostat, batch, fed-batch cultivations, etc. 
     Thus, the invention includes a method for producing resveratrol or an oligomeric or glycosidically-bound derivative thereof comprising contacting a non-plant cell with a carbon substrate in the substantial absence of an external source of 4-coumaric acid, said cell having the capacity to produce resveratrol or an oligomeric or glycosidically-bound derivative thereof under the conditions, in which the micro-organism may be selected from the group consisting of fungi and bacteria, especially yeast. 
     Said carbon substrate is optionally selected from the group of fermentable carbon substrates consisting of monosaccharides, oligosaccharides and polysaccharides, e.g. glucose, fructose, galactose, xylose, arabinose, mannose, sucrose, lactose, erythrose, threose, and/or ribose. Said carbon substrate may additionally or alternatively be selected from the group of non-fermentable carbon substrates including ethanol, acetate, glycerol, and/or lactate. Said non-fermentable carbon substrate may additionally or alternatively be selected from the group of amino acids and may be phenylalanine and/or tyrosine. 
     In an alternative aspect, the invention includes a method for producing resveratrol or an oligomeric or glycosidically-bound derivative thereof through heterologous expression of nucleotide sequences encoding phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate-CoA ligase and resveratrol synthase and also a method for producing resveratrol through heterologous expression of nucleotide sequences encoding tyrosine ammonia lyase, 4-coumarate-CoA ligase and resveratrol synthase. 
     The invention as described above has allowed the production of yeast cells producing high levels of resveratrol. Accordingly, the invention includes a micro-organism composition comprising micro-organism cells and at least 0.4 μg/g resveratrol on a dry weight basis produced in said micro-organism cells, preferably comprising at least 0.5 μg/g of said resveratrol, more preferably at least 200 μg/g. The stated level of resveratrol can be found in the yeast cells themselves. The composition may essentially consist of said yeast cells. 
     The resveratrol producing microorganisms described above and the pinosylvin producing organisms described in WO2008/009728 could desirably be improved to produce higher yields by redirecting the flux through the metabolism of the microorganism. 
     One option is to increase the amount malonyl-CoA available for further conversion into pinosylvin and resveratrol or other stilbenoids. Increasing the amount of malonoyl-CoA will have a positive effect on the production of all stilbenes of the type given in formula I 
     
       
         
         
             
             
         
       
     
     R1, R2, R3, R4, and R5 independently are either —H or —OH because malonyl-CoA is responsible for the upper ring. The stilbene that is produced depends on the other organic acid component involved, where cinnamic acid gives pinosylvin and coumaric acid gives resveratrol. Caffeic acid will give piceatannol. 
     By increasing the amount of available malonyl-CoA the yield of stilbenoid can be increased. A first method involves overexpression of ACC1 to create the increased supply. 
     Thus, the invention further includes a recombinant micro-organism having an operative metabolic pathway in which one or more stilbenes according to the general formula I: 
                         
are formed from a precursor optionally hydroxy-substituted phenyl-2-propenoic acid or ester thereof and malonyl-CoA by the action of a stilbene synthase, wherein the amount of malonyl-CoA available for use in said pathway has been increased by providing more than a native expression level of an enzyme catalysing the reaction
 
ATP+acetyl-CoA+HCO3−=ADP+phosphate−+malonyl-CoA.
 
     Preferably, said more than native expression level of said enzyme has been provided by replacing a native promoter of a gene expressing said enzyme with a promoter providing a higher level of expression. For instance, said native promoter is replaced with a strong constitutive yeast promoter. The strong constitutive promoter may be the promoter of one of the yeast genes TDH3, ADH1, TPI1, ACT1, GPD, TEF1, TEF2, and PGI. The promoter may optionally be native to the yeast in which stilbenoid production is to be produced. 
     Alternatively or additionally, said more than native expression level of said enzyme has been provided by recombinantly introducing into said micro-organism at least one exogenous genetic sequence encoding a said enzyme. This may be an acetyl coenzymeA carboxylase (ACC1—EC No. 6.4.1.2). 
     A second and independent strategy which also increases the yield of the stilbenes is overexpression of CPR. 
     Thus, the micro-organism may be recombinantly engineered to produce more than a native amount of a cytochrome P450 reductase (CPR). This may be by replacing a native promoter of a gene expressing said CPR with a promoter providing a higher level of expression, for instance with a strong constitutive yeast promoter such as the promoter of one of the yeast genes TDH3, ADH1, TPI1, ACT1 GPD, TEF1, TEF2, and PGI, which optionally may be native to the yeast itself. 
     The micro-organism may comprise recombinantly introduced genes expressing a phenylalanine ammonia lyase, a cinnamate 4-hydroxylase and/or a coumarate-CoA ligase or appropriate enzymes for other stilbenes. 
     Effect of Overexpressing ACC1 
     Acetyl CoenzymeA carboxylase (ACC1 EC-Number 6.4.1.2) generates malonyl-CoA according to the below reaction: 
     [ACC1 Reaction]
 
ATP+acetyl-CoA+HCO3−=ADP+phosphate−+malonyl-CoA
 
     By overexpressing ACC1 more malonyl-CoA is built up and this extra pool of malonyl-CoA is expected to generate more stilbenoids since the stilbene synthase reaction requires malonyl-CoA as building block for stilbene synthesis according to the reactions below: 
     [Resveratrol Synthase Reaction EC-Number 2.3.1.95]
 
3 malonyl-CoA+4-coumaroyl-CoA=4CoA+3,4′,5-trihydroxystilbene+4CO2
 
[Stilbene Synthase Reaction]
 
3 malonyl-CoA+cinnamoyl-CoA=4CoA+3,5-dihydroxystilbene+4CO2
 
[General for any Hydroxyl Stilbene Synthase]
 
3 malonyl-CoA+hydroxycinnamoyl-CoA=4CoA+hydroxystilbene+4CO2
 
     Other appropriate organic acids substituting for 4-coumaroyl-CoA produce other stilbenoids. 
     Effect of Overexpressing CPR 
     Hydroxylases, such as cinnamate 4-hyroxylase (1.14.13.11), are cytochrome P450 monooxygenases that catalyse the insertion of one atom of oxygen into an organic substrate while the other oxygen atom is reduced to water. This reaction requires NADPH according to the below reaction for the hydroxylation of cinnamic acid:
 
trans-cinnamic acid+NADPH+H + +O 2 =4-hydroxycinnamic acid+NADP + +H 2 O
 
     The active site of cytochrome P450 hydroxylases contains a heme iron center. The iron is tethered to the protein via a thiolate ligand derived from a cysteine residue. In general the mechanism is as follows: 
     1. The resting state of the protein is as oxidized Fe3+. 
     2. Binding of the substrate, cinnamic acid, initiates electron transport and oxygen binding. 
     3. Electrons are supplied to the p450 hydroxylase by another protein, either cytochrome P450 reductase (CPR), ferredoxins, or cytochrome b5 to reduce the heme iron. 
     4. Molecular oxygen is bound and split by the now reduced iron. 
     5. An iron-bound oxidant, oxidizes the substrate to an alcohol or an epoxide, regenerating the resting state of the p450 hydroxylase. 
     As described above CPR act as an electron carrier and donor for the NADPH dependent cytochrome P450 hydroxylase reaction. Thus by overexpressing CPR more electrons (NADPH) are generated for the NADPH dependent hydroxylation leading to more coumaric acid, and as a consequence more coumaric acid leads to more resveratrol by the resveratrol pathway. Similar considerations apply in the production of other stilbenoids. 
     Resveratrol or an oligomeric or glycosidically-bound derivative thereof or other stilbenoids so produced may be used as a nutraceutical in a dairy product or a beverage such as beer. 
     Resveratrol produced according to the invention may be cis-resveratrol or trans-resveratrol, but it is to be expected that the trans- form will normally predominate, as with other stilbenoids. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       To assist in the ready understanding of the above description of the invention reference has been made to the accompanying drawings in which: 
         FIG. 1  shows the chemical structure of trans-resveratrol; 
         FIG. 2  shows the phenylpropanoid pathway utilising phenylalanine ammonia lyase acting on L-phenylalanine; and 
         FIG. 3  shows the alternative pathway utilising phenylalanine ammonia lyase acting on L-tyrosine. 
         FIG. 4  shows the HPLC-chromatograms of extracts of  S. cerevisiae  strains FSSC-PALC4H4CLVST, FSSC-TAL4CLVST, grown on 100 g/l galactose. A chromatogram of 60 nanogram of pure resveratrol is included. 
         FIG. 5  shows the UV absorption spectrum for pure trans-resveratrol and trans-resveratrol produced by  S. cerevisiae  strain FSSC-PALC4H4CLVST, grown on 100 g/l galactose. 
         FIG. 6  shows the HPLC-chromatograms of extracts from  E. coli  strains FSEC-TAL4CLVST and FSEC-control, grown on 50 g/l glucose. 
         FIG. 7  shows the HPLC-chromatograms of extracts from  E. coli  strains FSEC-TAL4CLVST and FSEC-control, grown on 50 g/l glucose with addition of 20 mg/l coumaric acid. The UV absorption spectrum for trans-resveratrol produced in strain FSEC-TAL4CLVST is included. 
     
    
    
     The invention will be further described and illustrated by the following non-limiting examples. 
     EXAMPLES 
     Example 1 
     Isolation of Genes Encoding PAL, TAL, C4H, CPR, 4CL, and VST 
     Phenylalanine ammonia lyase (PAL2) (Cochrane et al., 2004; SEQ ID NO: 1, 2), cinnamate 4-hydroxylase (C4H) (Mizutani et al., 1997; SEQ ID NO: 3, 4) and 4-coumarate:CoenzymeA ligase (4CL1) (Hamberger and Hahlbrock 2004; Ehlting et al., 1999; SEQ ID NO: 5, 6) were isolated via PCR from  A. thaliana  cDNA (BioCat, Heidelberg, Germany) using the primers in table 1. PAL2 and 4CL1 were chosen amongst several  A. thaliana  homologues due to favourable kinetic parameters towards cinnamic acid and coumaroyl-CoA, respectively (Cochrane et al., 2004; Hamberger and Hahlbrock 2004; Ehlting et al., 1999). 
     The coding sequence of resveratrol synthase (VST) from  Rheum tataricum  (Samappito et al., 2003; SEQ ID NO: 7, 8) and tyrosine ammonia lyase (TAL) from  Rhodobacter capsulatus  (Kyndt et al., 2002; SEQ ID NO: 11, 12) were codon optimized for expression in  S. cerevisiae  using the online service backtranslation tool at www.entelechon.com, yielding sequence SEQ ID NO: 9, 10 and SEQ ID NO: 13, 14 respectively. Oligos for the synthetic gene assembly were constructed at MWG Biotech and the synthetic gene was assembled by PCR using a slightly modified method protocol of from Martin et al. (2003) described below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Primers and restriction sites for the amplification of genes 
               
            
           
           
               
               
               
               
            
               
                 Primer for amplification of gene* 
                   
                 Restriction 
                 Restriction 
               
               
                 (Restriction sites are underlined) 
                 Gene 
                 site: primer 
                 site: vector 
               
               
                   
               
               
                 5′-CG GAATTC TCATGGATCAAATCGAAGCAATGTT 
                 PAL2 
                 EcoRl 
                 EcoR1 
               
               
                   
               
               
                 5′-CG ACTAGT TTAGCAAATCGGAATCGGAGC 
                 PAL2 
                 Spe1 
                 Spe1 
               
               
                   
               
               
                 5′-CG CTCGAG AT ATGGACCTCCTCTTGCTGGA 
                 C4H 
                 Xhol 
                 Xho1 
               
               
                   
               
               
                 5′-CG GGTACC TTAACAGTTCCTTGGTTTCATAAC 
                 C4H 
                 Kpn1 
                 Kpn1 
               
               
                   
               
               
                 5′-GC TCTAGA CCT ATGGCGCCACAAGAACAAGCAGTTT  
                 4CL1 
                 Xba1 
                 Spe1 
               
               
                   
               
               
                 5′-GC GGATCC CCT TCACAATCCATTTGCTAGTTT TGCC 
                 4CL1 
                 BamH1 
                 BglII 
               
               
                   
               
               
                 5′-CC  GGATCC AAATGGCCCCAGAAGAGAGCAGG 
                 VST 
                 BamH1 
                 BamH1 
               
               
                   
               
               
                 5′-CG  CTCGAG TTAAGTGATCAATGGAACCGAAGACAG 
                 VST 
                 Xhol 
                 Xho1 
               
               
                   
               
               
                 5′-CC GAATTC CCATGACCCTGCAATCTCAAACAGCTAAAG 
                 TAL 
                 EcoR1 
                 EcoRl 
               
               
                   
               
               
                 5′-CC ACTAGT TTAAGCAGGTGGATCGGCAGCT 
                 TAL 
                 Spel 
                 Spe1 
               
               
                   
               
               
                 5′-CC CTCGAG ATCATGCCGTTTGGAATAGACAACACCGA  
                 CPR1 
                 Xho1 
                 Xho1 
               
               
                   
               
               
                 5′-CC AAGCTT ATCGGGCTGATTACCAGACATCTTCTTG 
                 CPR1 
                 HindIII 
                 HindIII 
               
               
                   
               
               
                 5′-CC GGATCC CCATGTCCTCTTCTTCTTCTTCGTCAAC 
                 AR2 
                 Bamh1 
                 Bamh1 
               
               
                   
               
               
                 5′-CC CTCGAG GTGAGTGTGTGGCTTCAATAGTTT CG 
                 AR2 
                 Xho1 
                 Xho1 
               
               
                   
               
               
                 *SEQ ID Nos 19-32 
               
            
           
         
       
     
     Primers from MWG for the assembly of the synthetic gene were dissolved in milliQ-water to a concentration of 100 pmole/μl. An aliquot of 5 μl of each primer was combined in a totalmix and then diluted 10-fold with milliQ water. The gene was assembled via PCR using 5 μl diluted totalmix per 50 μl as template for fusion DNA polymerase (Finnzymes). The PCR programme was as follows: Initial 98° C. for 30 s., and then 30 cycles with 98° C. for 10 s., 40° C. for 1 min. and 72° C. at 1 min./1000 basepairs, and a final 72° C. for 5 min. From the resulting PCR reaction, 20 μl was purified on 1% agarose gel. The result was a PCR smear and the regions around the wanted size were cut out from agarose gel and purified using the QiaQuick Gel Extraction Kit (Qiagen). A final PCR with the outer primers (for TAL and VST) in table 1 rendered the required TAL and VST genes. Point mutations were corrected using either the Quickchange site directed mutagenesis II kit (Stratagene, La Jolla, Calif.), or using PCR from overlapping error free DNA stretches from several different  E. coli  subclones. 
     NADPH:Cytochrome P450 reductase (CPR) from  A. thaliana  (AR2) (Mizutani and Ohta, 1998; SEQ ID NO: 17, 18) and from  S. cerevisiae  (CPR1) (Aoyama et al., 1978; SEQ ID NO: 15, 16), were isolated from  A. thaliana  cDNA (BioCat, Heidelberg, Germany) and  S. cerevisae  genomic DNA, respectively, using the primers in table 1. 
     Example 2 
     Construction of a Yeast Vector for Expression of PAL 
     The gene encoding PAL, isolated as described in example 1, was reamplified by PCR using forward- and reverse primers, with 5′ overhangs containing EcoR1 and Spe1 restriction sites (table 1). The amplified PAL PCR product was digested with EcoR1/Spe1 and ligated into EcoR1/Spe1 digested pESC-URA vector (Stratagene), resulting in vector pESC-URA-PAL. The sequence of the gene was verified by sequencing of two different clones. 
     Example 3 
     Construction of a Yeast Vector for Expression of PAL and C4H 
     The gene encoding C4H, isolated as described in example 1, was amplified by PCR using the forward- and reverse primers, with 5′ overhangs containing Xho1 and Kpn1 restriction sites. The amplified C4H PCR-product was digested with Xho1/Kpn1 and ligated into similarly digested pESC-URA-PAL vector. The resulting plasmid, pESC-URA-PAL-C4H, contained the genes encoding PAL and C4H under the control of the divergent GAL1/GAL10 promoter. The sequence of the gene encoding C4H was verified by sequencing of two different clones. 
     Example 4 
     Construction of a Yeast Vector for Expression of 4CL 
     The gene encoding 4CL was isolated as described in example 1. The amplified 4CL PCR-product was digested with Xba1/BamH1 and ligated into Spe1/BglII digested pESC-TRP vector (Stratagene), resulting in vector pESC-TRP-4CL. Two different clones of pESC-TRP-4CL were sequenced to verify the sequence of the cloned gene. 
     Example 5 
     Construction of a Yeast Vector for Expression of 4CL and VST 
     The gene encoding VST was isolated as described in example 1. The amplified synthetic VST gene was digested with BamH1/Xho1 and ligated into BamH1/Xho1 digested pESC-TRP-4CL (example 4). The resulting plasmid, pESC-TRP-4CL-VST, contained the genes encoding 4CL and VST under the control of the divergent GAL1/GAL10 promoter. The sequence of the gene encoding VST was verified by sequencing of two different clones of pESC-TRP-4CL-VST. 
     Example 6 
     Construction of a Yeast Vector for Expression of TAL 
     The gene encoding TAL was isolated as described in example 1. The amplified synthetic TAL gene was digested with EcoR1/Spe1 and ligated into EcoR1/Spe1-digested pESC-URA vector. The resulting plasmid, pESC-URA-TAL, contained the gene encoding for TAL under the control of the divergent GAL1/GAL10 promoter. The sequence was verified by sequencing of two different clones of pESC-URA-TAL. 
     Example 7 
     Construction of a Yeast Vector for Overexpression of  S. cerevisiae  Endogenous CPR 
     The gene encoding CPR from  S. cerevisiae  (CPR1) was isolated as described in example 1. The amplified CPR1 gene was digested with Xho1/HindIII and ligated into Xho1/HindIII-digested pESC-LEU vector (Stratagene), resulting in vector pESC-LEU-CPR1. The sequence was verified by sequencing of two different clones of pESC-LEU-CPR1. 
     Example 8 
     Construction of a Yeast Vector for Overexpression of  A. thaliana  CPR (AR2) 
     The gene encoding CPR from  A. thaliana  (AR2) was isolated as described in example 1. The amplified AR2 gene was digested with BamH1/Xho1 and ligated into BamH1/Xho1 digested pESC-LEU vector (Stratagene), resulting in vector pESC-LEU-AR2. 
     The sequence was verified by sequencing of two different clones of pESC-LEU-AR2. 
     Example 9 
     Expression of the Pathway to Resveratrol in the Yeast  S. cerevisiae  Using PAL, C4H, 4CL and VST 
     Yeast strains containing the appropriate genetic markers were transformed with the vectors described in examples 2, 3, 4, 5, 6, 7 and 8, separately or in combination. The transformation of the yeast cell was conducted in accordance with methods known in the art, for instance, by using competent cells or by electroporation (see, e.g., Sambrook et al., 1989). Transformants were selected on medium lacking uracil and/or tryptophan and streak purified on the same medium. 
       S. cerevisiae  strain CEN.PK 113-5D (MATa ura3) was transformed separately with the vector pESC-URA-PAL (example 2), yielding the strain FSSC-PAL, and with pESC-URA-PAL-C4H (example 3), resulting in the strain FSSC-PALC4H.  S. cerevisiae  strain FS01267 (MATa trp1 ura3) was co-transformed with pESC-URA-PAL-C4H and pESC-TRP-4CL (example 4), and the transformed strain was named FSSC-PALC4H4CL. The same strain was also co-transformed with pESC-URA-PAL-C4H and pESC-TRP-4CL-VST (example 5), resulting in the strain FSSC-PALC4H4CLVST. 
     Example 10 
     Expression of the Pathway to Resveratrol in  S. cerevisiae  Using TAL, 4CL and VST 
       S. cerevisiae  strain CEN.PK 113-5D (MATa ura3) was transformed separately with the vector pESC-URA-TAL (example 6), yielding the strain FSSC-TAL.  S. cerevisiae  strain FS01267 (MATa trp1 ura3) was co-transformed with pESC-URA-TAL (example 6) and pESC-TRP-4CL (example 4), and the transformed strain was named FSSC-TAL4CL. The same strain was also co-transformed with pESC-URA-TAL and pESC-TRP-4CL-VST (example 5), resulting in the strain FSSC-TAL4CLVST. Transformants were selected on medium lacking uracil and or tryptophan and streak purified on the same medium. 
     Example 11 
     Expression of the Pathway to Resveratrol in  S. Cerevisiae  with Overexpressed Endogenous CPR 
       S. cerevisiae  strain FS01277 (MATa ura3 leu2 trp1) was co-transformed with vectors pESC-URA-PAL-C4H (example 3), pESC-TRP-4CL (example 4), and pESC-LEU-CPR1 (example 7). The transformed strain was named FSSC-PALC4H4CLVSTCPR. Transformants were selected on medium lacking uracil and/or tryptophan and streak purified on the same medium. 
     Example 12 
     Expression of the Pathway to Resveratrol in  S. cerevisiae  with Overexpressed  A. thaliana  CPR (AR2) 
       S. cerevisiae  strain FS01277 (MATa ura3 leu2 trp1) was co-transformed with vectors pESC-URA-PAL-C4H (example 3), pESC-TRP-4CL (example 4), and pESC-LEU-AR2 (example 8). The transformed strain was named FSSC-PALC4H4CLVSTAR2. Transformants were selected on medium lacking uracil and or tryptophan and streak purified on the same medium. 
     Example 13 
     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 200 ml defined mineral medium (Verduyn et al, 1992) that contained vitamins, trace elements, 5 g/l glucose and 40 g/l or 100 g/l galactose. The 500 ml stoppered shake flasks were incubated for three days at 30° C. and 160 rpm. 
     Example 14 
     Extraction of Resveratrol 
     Cells were harvested by centrifugation 5000 g for 5 minutes. An aliquot of 50 ml of supernatant was extracted once with 20 ml ethyl acetate. The ethyl acetate was freeze dried and the dry product redissolved in 0.7 ml methanol and filtered into HPLC vials. 
     The cell pellet from 200 ml medium was dissolved in 1 to 2 ml water and divided into 3 fastprep tubes and broken with glass beads. The crude extracts from the three tubes were pooled into 10 ml 100% methanol in a 50 ml sartorius tube and extracted on a rotary chamber for 48 hours in a dark cold room at 4° C. After 48 hours the cell debris was removed via centrifugation for 5 min. at 5000 g and the methanol was removed by freeze-drying overnight. The dried residue was redissolved in 1 ml phosphate-citrate buffer pH 5.4 and 10 units beta-glucosidase from almonds was added (Sigma) to release resveratrol from putatively glucoside-bound forms. The mixture was incubated for three hours at 37° C. and then extracted twice with 1 ml ethyl acetate. The combined ethyl acetate was freeze dried and the dry residue was redissolved in 0.7 ml methanol and filtered into HPLC vials. 
     Example 15 
     Analysis of Resveratrol 
     Thin Layer Chromatography 
     A method based upon thin layer chromatography that enabled the quick separation of cinnamic, coumaric and resveratrol on the same TLC-plate was developed for quick screening analysis. An aliquot of 1 ml culture containing both cells and supernatant were extracted with 500 microliter ethyl acetate and centrifuged for 30 s. at 13000 rpm with a microcentrifuge. The ethyl acetate was dried and redissolved in methanol. The extracts were analyzed on Silica G plates (0.2 mm Alugram SIL G/UV 254 , Macherey-Nagel) containing a fluorescent indicator. The mobile phase was a mixture of chloroform, ethyl acetate and formic acid (25:10:1). 
     HPLC 
     For quantitative analysis of cinnamic acid, coumaric acid, and resveratrol, 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, Calif., USA) Luna 3 micrometer C18 (100×2.00 mm) column was used at 40° C. As mobile phase a gradient of acetonitrile and milliq water (both containing 50 ppm trifluoroacetic acid) was used at a flow of 0.4 ml/min. The gradient profile was linear from 15% acetonitrile to 100% acetonitrile over 20 min. The elution times were approximately 3.4 min. for coumaric acid, 5.5 min. for free trans-resveratrol and 6.8 min. for cinnamic acid. 
     Pure resveratrol standard was purchased from Cayman chemical company, whereas pure coumaric acid and cinnamic acid standards were purchased from and Sigma. 
     Results 
     Strains FSSC-PALC4H4CLVST and FSSC-TAL4CLVST, were cultivated on 100 g/l galactose as described in example 13, and analyzed for their content of intracellular resveratrol according to example 14 and 15. Additionally, a control strain FSSC-control was included that contained the empty vectors pESC-URA and pESC-TRP only. The HPLC-analysis showed that strains FSSC-PALC4H4CLVST and FSSC-TAL4CLVST contained a component with a retention time of 5.5 min. that was identical to trans-resveratrol ( FIG. 4 ). Said result was confirmed by the UV absorption spectra that were similar to the absorption spectrum of pure trans-resveratrol ( FIG. 5 ) as well, with a λ max  of approximately 306 nm. 
     The results, therefore, demonstrated the presence of an active phenyl-propanoid pathway in  S. cerevisiae  that led to in vivo production of trans-resveratrol. The production of resveratrol can most likely be improved by cultivating the strains under well-defined growth conditions in batch- and continuous cultures, and/or optimizing the expression/activities of the individual enzymes. 
     Example 16 
     Construction of a Bacterial Vector for Expression of TAL in  Escherichia coli    
     The gene encoding TAL, isolated as described in Example 1, was reamplified by PCR from the plasmid pESC-URA-TAL (example 6) using the forward primer 5′-CCG CTCGAG CGG ATG ACC CTG CAA TCT CAA ACA GCT AAA G-3′ SEQ ID NO 33 and the reverse primer 5′-GC GGATCC TTA AGC AGG TGG ATC GGC AGC T-3′ SEQ ID NO 34 with 5′ overhangs containing the restriction sites XhoI and BamHI, respectively. The introduction of restriction sites at the 5′ and  3 ′ ends of the gene allowed ligation of the restricted PCR product into a pET16b vector (Novagen), digested with XhoI and BamHI to yield pET16b-TAL. The pET16b vector contained both the ampicillin resistance gene, and the T7 promoter. Hence, above procedure resulted in a vector with an antibiotic selection marker that contained the gene encoding for TAL under the control of the T7 promoter. The sequence of the gene encoding TAL was verified by sequencing of one clone of pET16b-TAL. 
     Example 17 
     Construction of a Bacterial Vector for Expression of 4CL and VST in  Escherichia coli    
     The gene encoding VST, isolated as described in example 1, was cut out with the restriction enzymes BamHI and XhoI from the digested plasmid pESC-TRP-4CL-VST (example 5), which contains the genes encoding 4CL and VST. The VST gene was ligated into a pET26b vector (Novagen), containing the kanamycin resistance gene, digested with BamHI and SalI to yield pET26b-VST. The restriction enzymes XhoI and SalI have compatible ends, which enabled proper ligation. The pET26b vector contained both the kanamycin resistance gene, and the T7 promoter. Hence, above procedure resulted in a vector with an antibiotic selection marker that contained the gene encoding for VST under the control of the T7 promoter. The gene encoding for 4CL, isolated as described in example 1, was reamplified by PCR from the plasmid pESC-URA-4CL-VST (example 5) using the forward primer 5′-TG CCATGG CA ATGGCGCCAC AAGAACAAGC AGTTT-3′ SEQ ID NO 35 and the reverse primer 5′-GC GGATCC CCT TCA CAA TCC ATT TGC TAG TTT TGCC-3′ SEQ ID NO 36 with 5′ overhangs containing the restriction sites NcoI and BamHI, respectively. The introduction of restriction sites at the 5′ and 3′ ends of the gene allowed ligation of the restricted PCR product into a pET16b vector (Novagen) digested with NcoI and BamHI. The resulting plasmid, pET16b-4CL, contained the gene encoding for 4CL under the control of the T7 promoter. Both the T7 promoter and the gene encoding for 4CL were reamplified as one fragment by PCR from the plasmid pET16b-4CL using the forward primer 5′-TT GCGGCCGC AAA TCT CGA TCC CGC GAA ATT AAT ACG-3′ SEQ ID NO 37 and the reverse primer 5′-CG CTCGAG CCT TCA CAA TCC ATT TGC TAG TTT TGCC-3′ SEQ ID NO 38 with 5′ overhangs, containing the restriction sites NotI and XhoI, respectively. The introduction of restriction sites at the 5′ and 3′ ends of the DNA fragment allowed ligation of the restricted PCR product into the plasmid pET26b-VST that was digested with NotI and XhoI before ligation. The resulting plasmid, pET26b-VST-4CL, contained the two genes 4CL and VST that each were under control of an individual T7 promoter. 
     Example 18 
     Expression of the Pathway to Resveratrol in  Escherichia Coli , Using TAL, 4CL and VST 
     The transformation of the bacterial cell was conducted in accordance with methods known in the art, for instance, by using competent cells or by electroporation (see, e.g., Sambrook et al., 1989). The  E. coli  strain BL21 (DE3) (Novagen) was co-transformed with the two vectors pET16b-TAL (example 16) and pET26b-VST-4CL (Example 17), resulting in strain FSEC-TAL4CLVST. In addition,  E. coli  strain BL21 (DE3) was co-transformed with the two empty vectors pET16b (Novagen) and pET26b (Novagen), resulting in strain FSEC-control, which was used as a control strain. Transformants were selected on Luria-Bertani (LB) medium with 100 μg/ml ampicillin and 60 μg/ml kanamycin. 
     Example 19 
     Fermentation with Recombinant  Escherichia coli  Strains in Shake Flasks 
     Pre-cultures of  Escherichia coli  BL21 (DE3) were grown in glass tubes at 160 rpm and 37° C. in 7 ml of LB medium containing 100 μg/ml ampicillin and 60 μg/ml kanamycin. Exponentially growing precultures were used for inoculation of 500 ml baffled shake flasks that contained 200 ml LB medium supplemented with 50 g/l glucose, 5 g/l K 2 HPO 4 , 80 μg/ml ampicilin and 50 μg/ml kanamycin, which were incubated at 160 rpm and 37° C. After 5 hours, isopropyl β-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM, as an inducer of the T7 promoter that was in front of each of the three genes TAL, 4CL and VST. After an incubation period of 48 hours at 37° C., the cells were harvested and subjected to extraction procedures and analysed for the presence of produced resveratrol. 
     Example 20 
     Extraction and Analysis of Resveratrol in  Escherichia coli    
     Extraction and analysis was performed using the methods as described in example 14 and 15. 
     Results 
     Strain FSEC-TAL4CLVST and FSEC-control, were cultivated on 50 g/l glucose as described in example 19, and analyzed for their content of intracellular resveratrol according to example 14 and 15. The HPLC-analysis showed that strain FSEC-TAL4CLVST did contain considerable amounts of a component with a retention time of 3.4 min., which is identical to coumaric acid ( FIG. 6 ). However, the extract did not contain a component that eluted at the same time as trans-resveratrol. Said result, therefore, indicated that the tyrosine ammonia lyase (TAL) was active indeed, but did not lead to production of detectable amounts of resveratrol. The lack of resveratrol formation, however, could be the result of; i) a non-functional coumarate-CoA ligase (4CL); ii) a non-functional resveratrol synthase (VST); iii) too low levels of coumaric acid, caused by either non-optimal cultivation conditions, or non-optimal expression/activity of TAL, or branching of coumaric acid into other products. To evaluate said hypotheses, the strains were grown on similar media as described in example 19 but now in the presence of 20 mg/l of coumaric acid. The subsequent HPLC-analysis of extracts of FSEC-TAL4CLVST indeed showed a cluster of peaks around the same retention time as trans-resveratrol, which was not observed in extracts of FS-control ( FIG. 6 ). Indeed, the UV absorption spectrum of the peak with a retention time of 5.5 min. was similar to the spectrum of pure trans-resveratrol ( FIG. 7 ), whereas no such spectrum could be obtained for peaks in the control strain. The results, therefore, strongly suggest the presence of an active phenylpropanoid pathway in  Escherichia coli , which can lead to production of resveratrol. Most likely the production of resveratrol without addition of coumaric acid can be achieved by cultivating the strains under well-defined growth conditions in batch- and continuous cultures, and/or optimizing the expression/activities of the individual enzymes. 
     Example 21 
     Construction of a Bacterial Vector for Expression of PAL and C4H in  Lactococcus lactis    
     The plasmid pSH71 and derivatives thereof, which is used in the following examples, is a bifunctional shuttle vector with multiple origins of replication from  Escherichia coli  and  Lactococcus lactis . With that, the host range specificity traverses  Escherichia coli  and other species of lactic acid bacteria. Though transformations in  Lactococcus lactis  usually proceed without problems, putative difficult transformations in other species of lactic acid bacteria can, therefore, be overcome by using  Escherichia coli  as an intermediate host for the construction of recombinant plasmids. The plasmid contains one or more marker genes to allow the microorganism that harbour them to be selected from those which do not. The selection system that is used for  Lactococcus lactis  is based upon dominant markers, e.g. resistance against erythromycin and chloramphenicol, but systems based upon genes involved in carbohydrate metabolism, peptidases and food grade markers, have also been described. In addition, the plasmid contains promoter- and terminator sequences that allow the expression of the recombinant genes. Suitable promoters are taken from genes of  Lactococcus lactis  e.g. lacA. Furthermore, the plasmid contains suitable unique restriction sites to facilitate the cloning of DNA fragments and subsequent identification of recombinants. 
     In the examples below the plasmid contains either the erythromycine resistance gene, designated as pSH71-ERY r , or the chloramphenicol resistance gene, designated as pSH71-CM r    
     The gene encoding PAL, isolated as described in example 1, is reamplified by PCR from the plasmid pESC-URA-PAL-C4H (example 3), using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the gene allows ligation of the restricted PCR product into a digested pSH71-ERY r  vector that contains the lacA promoter from  Lactococcus lactis . The resulting plasmid, pSH71-ERY r -PAL, contains the gene encoding PAL under the control of the lacA promoter from  Lactococcus lactis . The gene encoding C4H, isolated as described in example 1, is reamplified by PCR from the plasmid pESC-URA-PAL-C4H (example 3) using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the gene allows ligation of the restricted PCR product into a digested pSH71-CM r  vector to yield pSH71-CM r -C4H. The lacA promoter and the gene encoding C4H are reamplified as one fragment by PCR from the plasmid pSH71-CM r -C4H using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmid pSH71-ERY r -PAL. The resulting plasmid, pSH71-ERY r -PAL-C4H, contains the genes encoding PAL and C4H that are each under the control of an individual lacA promoter. The sequence of the genes encoding PAL and C4H is verified by sequencing of two different clones of pSH71-ERY r -PAL-C4H. 
     Example 22 
     Construction of a Bacterial Vector for Expression of TAL in  Lactococcus lactis    
     The gene encoding for TAL, isolated as described in example 1, is reamplified by PCR from the plasmid pESC-URA-TAL (example 6) using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the gene allows ligation of the restricted PCR product into a digested pSH71-ERY r  vector. The resulting plasmid, pSH71-ERY r -TAL, contains the gene encoding for TAL under the control of the lacA promoter from  Lactococcus lactis . The sequence of the gene encoding for TAL is verified by sequencing of two different clones of pSH71-ERY r -TAL. 
     Example 23 
     Construction of a Bacterial Vector for Expression of 4CL and VST in  Lactococcus lactis    
     The gene encoding 4CL, isolated as described in example 1, is reamplified by PCR from the plasmid pESC-TRP-4CL-VST (example 5), using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the gene allows ligation of the restricted PCR product into a digested pSH71-CM r  vector. The resulting plasmid, pSH71-CM r -4CL, contains the gene encoding for 4CL under the control of the lacA promoter from  Lactobacillus lactis . The gene encoding VST, isolated as described in example 1, is reamplified by PCR from the plasmid pESC-TRP-4CL-VST (example 5) using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the gene allows ligation of the restricted PCR product into a digested pSH71-ERY r  vector. The resulting plasmid, pSH71-ERY r -VST, contains the gene encoding VST under the control of the lacA promoter from  Lactococcus lactis . The lacA promoter and the gene encoding VST are reamplified as one fragment by PCR from the plasmid pSH71-ERY r -VST using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmid pSH71-CM r -4CL. The resulting plasmid, pSH71-CM r -4CL-VST, contains the genes encoding 4CL and VST that are each under the control of their individual lacA promoter. 
     The sequence of the genes encoding 4CL and VST is verified by sequencing of two different clones of pSH71-CM r -4CL-VST. 
     Example 24 
     Expression of the Pathway to Resveratrol in  Lactococcus lactis    
       Lactococcus lactis  strains are transformed with the vectors described in examples 21, 22 and 23, separately or in combination. The transformation of the bacterial cell is conducted in accordance with methods known in the art, for instance, by using competent cells or by electroporation (see, e.g., Sambrook et al., 1989). Transformants are selected on medium containing the antibiotics erythromycin and chloramphenicol and streak purified on the same medium. 
       Lactococcus lactis  strain MG1363 is transformed separately with the vector pSH71-ERY r -TAL (example 22), yielding the strain FSLL-TAL; with pSH71-ERY r -PAL-C4H (example 21), yielding the strain FSLL-PALC4H and with pSH71-CM r -4CL-VST (example 23), yielding strain FSLL-4CLVST. In addition,  Lactococcus lactis  strain MG1363 is co-transformed with pSH71-ERY r -TAL (example 22) and pSH71-CM r -4CL-VST (example 23), and the transformed strain is named FSLL-TAL4CLVST. The same strain is also co-transformed with pSH71-ERY r -PAL-C4H (example 21), and pSH71-CM r -4CL-VST (example 23), resulting in the strain FSLL-PALC4H4CLVST. 
     Example 25 
     Fermentation with Recombinant  Lactococcus lactis  Strains in Fermentors 
     The recombinant yeast strains can be grown in fermenters operated as batch, fed-batch or chemostat cultures. 
     Batch and Fed-Batch Cultivations 
     The microorganism is grown in a baffled bioreactor with a working volume of 1.5 liters under anaerobic, aerobic or microaerobic conditions. All cultures are incubated at 30° C., at 350 rpm. A constant pH of 6.6 is maintained by automatic addition of 10 M KOH. Cells are grown on lactose in defined MS10 medium supplemented with the following components to allow growth under aerobic conditions: MnSO 4  (1.25×10 −5  g/l), thiamine (1 mg/l), and DL-6,8-thioctic acid (2.5 mg/l). The lactose concentration is, for example 50 g/l. The bioreactors are inoculated with cells from precultures grown at 30° C. in shake flasks on the medium described above buffered with threefold-higher concentrations of K 2 HPO 4  and KH 2 PO 4 . Anaerobic conditions are ensured by flushing the medium with N 2  (99.998% pure) prior to inoculation and by maintaining a constant flow of 50 ml/min of N 2  through the headspace of the bioreactor during cultivation. The bioreactors used for microaerobic and aerobic cultivation are equipped with polarographic oxygen sensors that are calibrated with air (DOT, 100%) and N 2  (DOT, 0%). Aerobic conditions are obtained by sparging the bioreactor with air at a rate of 1 vvm to ensure that the DOT is more than 80%. During microaerobic experiments the DOT is kept constant 5% by sparging the reactor with gas composed of a mixture of N 2  and atmospheric air, at a rate of 0.25 vvm. 
     Chemostat Cultures 
     In chemostat cultures the cells can be grown in, for example, 1-L working-volume Applikon laboratory fermentors at 30° C. and 350 rpm. The dilution rate (D) can be set at different values, e.g. at 0.050 h −1 , 0.10 h −1 , 0.15 h −1 , or 0.20 h −1 . The pH is kept constant, e.g at 6.6, by automatic addition of 5 M KOH, using the growth medium described above, supplemented with antifoam (50 μl/l). The concentration of lactose can be set at different values, e.g. is 3.0 g/l 6.0 g/l, 12.0 g/l, 15.0 g/l or 18.0 g/l. The bioreactor is inoculated to an initial biomass concentration of 1 mg/l and the feed pump is turned on at the end of the exponential growth phase. 
     An anaerobic steady state is obtained by introducing 50 ml/min of N 2  (99.998% pure) into the headspace of the bioreactor. Different anoxic steady states can obtained by sparging the reactor with 250 ml/min of gas composed of N 2  (99.998% pure) and atmospheric air at various ratios. The oxygen electrode is calibrated by sparging the bioreactor with air (100% DOT) and with N 2  (0% DOT). 
     For all conditions, the gas is sterile filtered before being introduced into the bioreactor. The off gas is led through a condenser cooled to lower than −8° C. and analyzed for its volumetric content of CO 2  and O 2  by means of an acoustic gas analyser. 
     Cultivations are considered to be in steady state after at least 5 residence times, and if the concentrations of biomass and fermentation end products remain unchanged (less than 5% relative deviation) over the last two residence times. 
     Example 26 
     Extraction and Analysis of Resveratrol in  Lactococcus lactis    
     Extraction and analysis is performed using the methods as described in examples 14 and 15. 
     Example 27 
     Construction of a Fungal Vector for Expression of PAL and C4H in Species Belonging to the Genus  Aspergillus    
     The plasmid that is used in the following examples, is derived from pARp1 that contains the AMA1 initiating replication sequence from  Aspergillus nidulans , which also sustains autonomous plasmid replication in  A. niger  and  A. oryzae  (Gems et al., 1991). Moreover, the plasmid is a shuttle vector, containing the replication sequence of  Escherichia coli , and the inherent difficult transformations in  Aspergillus niger  and  Aspergillus oryzae  can therefore overcome by using  Escherichia coli  as an intermediate host for the construction of recombinant plasmids. The plasmid contains one or more marker genes to allow the microorganism that harbour them to be selected from those which do not. The selection system can be either based upon dominant markers e.g. resistance against hygromycin B, phleomycin and bleomycin, or heterologous markers e.g amino acids and the pyrG gene. In addition the plasmid contains promoter- and terminator sequences that allow the expression of the recombinant genes. Suitable promoters are taken from genes of  Aspergillus nidulans  e.g. alcA, glaA, amy, niaD, and gpdA. Furthermore, the plasmid contains suitable unique restriction sites to facilitate the cloning of DNA fragments and subsequent identification of recombinants. 
     The plasmid used in the following examples contains the strong constitutive gpdA-promoter and auxotropic markers, all originating from  Aspergillus nidulans ; the plasmid containing the gene methG that is involved in methionine biosynthesis, is designated as pAMA1-MET; the plasmid containing the gene hisA that is involved in histidine biosynthesis, is designated as pAMA1-HIS. 
     The gene encoding PAL, isolated as described in example 1, is reamplified by PCR from the plasmid pESC-URA-PAL-C4H (example 3), using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the gene allows ligation of the restricted PCR product into a digested pAMA1-MET vector that contains the gpdA promoter from  Aspergillus nidulans . The resulting plasmid, pAMA1-MET-PAL contains the gene encoding PAL under the control of the gpdA promoter from  Aspergillus nidulans.    
     The gene encoding C4H, isolated as described in example 1, is reamplified by PCR from the plasmid pESC-URA-PAL-C4H (example 3) using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the gene allows ligation of the restricted PCR product into a digested pAMA1-HIS vector to yield pAMA1-HIS-C4H. The gpdA promoter and the gene encoding C4H are reamplified as one fragment by PCR from the plasmid pAMA1-HIS-C4H using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmid pAMA1-MET-PAL. The resulting plasmid, pAMA1-MET-PAL-C4H, contains the genes encoding PAL and C4H that are each under the control of an individual pgdA promoter from  Aspergillus nidulans . The sequence of the genes encoding PAL and C4H is verified by sequencing of two different clones of pAMA1-MET-PAL-C4H. 
     Example 28 
     Construction of a Fungal Vector for Expression of TAL in Species Belonging to the Genus  Aspergillus    
     The gene encoding for TAL, isolated as described in example 1, is reamplified by PCR from the plasmid pESC-URA-TAL (example 6) using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the gene allows ligation of the restricted PCR product into a digested pAMA1-MET vector. The resulting plasmid, pAMA1-MET-TAL, contains the gene encoding for TAL under the control of the gpdA promoter from  Aspergillus nidulans . The sequence of the gene encoding for TAL is verified by sequencing of two different clones of pAMA1-MET-TAL. 
     Example 29 
     Construction of a Fungal Vector for Expression of 4CL and VST in Species Belonging to the Genus  Aspergillus    
     The gene encoding 4CL, isolated as described in example 1, is reamplified by PCR from the plasmid pESC-TRP-4CL-VST (example 5), using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the gene allows ligation of the restricted PCR product into a digested pAMA1-HIS vector that contains the gpdA promoter from  Aspergillus nidulans . The resulting plasmid, pAMA1-HIS-4CL contains the gene encoding 4CL under the control of the gpdA promoter from  Aspergillus nidulans . The gene encoding VST, isolated as described in example 1, is reamplified by PCR from the plasmid pESC-TRP-4CL-VST (example 5) using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the gene allows ligation of the restricted PCR product into a digested pAMA1-MET vector to yield pAMA1-MET-VST. The gpdA promoter and the gene encoding VST are reamplified as one fragment by PCR from the plasmid pAMA1-MET-VST using forward- and reverse primers, with 5′ overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5′ and 3′ ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmid pAMA1-HIS-4CL. The resulting plasmid, pAMA1-HIS-4CL-VST, contains the genes encoding 4CL and VST that are each under the control of an individual pgdA promoter from  Aspergillus nidulans . The sequence of the genes encoding 4CL and VST is verified by sequencing of two different clones of pAMA1-HIS-4CL-VST. 
     Example 30 
     Expression of the Pathway to Resveratrol in  Aspergillus niger    
       Aspergillus niger  strains are transformed with the vectors described in examples 27, 28 and 29, separately or in combination. The transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989). Transformants are selected on minimal medium lacking methionine and/or histidine. 
     A strain of  Aspergillus niger  that is auxotrophic for histidine and methionine, for instance, strain FGSC A919 (see http://www.fgsc.net), is transformed separately with the vector pAMA1-MET-TAL (example 28), yielding the strain FSAN-TAL; with pAMA1-MET-PAL-C4H (example 27), yielding the strain FSAN-PALC4H and with pAMA1-HIS-4CL-VST (example 29), yielding strain FSAN-4CLVST. In addition,  Aspergillus niger  strain FGSC A919 is co-transformed with pAMA1-MET-TAL (example 28) and pAMA1-HIS-4CL-VST (example 29), and the transformed strain is named FSAN-TAL4CLVST. The same strain is also co-transformed with pAMA1-MET-PAL-C4H (example 27), and pAMA1-HIS-4CL-VST (example 29), resulting in the strain FSAN-PALC4H4CLVST. 
     Example 31 
     Expression of the Pathway to Resveratrol in  Aspergillus oryzae    
     A strain of  Aspergillus oryzae  that contains a native set of genes encoding for PAL, C4H and 4CL (Seshime et al., 2005) and that is auxotrophic for methionine, is transformed with the vector pAMA1-MET-VST (example 29), yielding the strain FSAO-VST. The transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989). Transformants are selected on minimal medium lacking methionine. 
     Example 32 
     Fermentation with Recombinant Strains of  Aspergillus niger  and  Aspergillus oryzae  in Fermentors 
     The recombinant yeast strains can be grown in fermenters operated as batch, fed-batch or chemostat cultures. 
     Batch and Fed-Batch Cultivations 
     The microorganism is grown in a baffled bioreactor with a working volume of 1.5 liters under aerobic conditions. All cultures are incubated at 30° C., at 500 rpm. A constant pH of 6.0 is maintained by automatic addition of 10 M KOH, and aerobic conditions are obtained by sparging the bioreactor with air at a rate of 1 vvm to ensure that the DOT is more than 80%. Cells are grown on glucose in defined medium consisting of the following components to allow growth in batch cultivations: 7.3 g/l (NH 4 ) 2 SO 4 , 1.5 g/l KH 2 PO 4 , 1.0 g/l MgSO 4 .7H 2 O, 1.0 g/l NaCl, 0.1 g/l CaCl 2 .2H 2 O, 0.1 ml/l Sigma antifoam, 7.2 mg/l ZnSO 4 .7H 2 O, 1.3 mg/l CuSO 4 .5H 2 O, 0.3 mg/l NiCl 2 .6H 2 O, 3.5 mg/l MnCl 2 .4H 2 O and 6.9 mg/l FeSO 4 .7H 2 O. The glucose concentration is, for example, 10- 20-, 30-, 40- or 50 g/l. To allow growth in fed-batch cultivations the medium is composed of: 7.3 g/l (NH 4 ) 2 SO 4 , 4.0 g/l KH 2 PO 4 , 1.9 g/l MgSO 4 .7H 2 O, 1.3 g/l NaCl, 0.10 g/l CaCl 2 .2H 2 O, 0.1 ml/l Sigma antifoam, 7.2 mg/l ZnSO 4 .7H 2 O, 1.3 mg/l CuSO 4 .5H 2 O, 0.3 mg/l NiCl 2 .6H 2 O, 3.5 mg/l MnCl 2 .4H 2 O and 6.9 mg/l FeSO 4 .H 2 O in the batch phase. The reactor is then fed with, for example, 285 g/kg glucose and 42 g/kg (NH 4 ) 2 SO 4 . 
     Free mycelium from a pre-batch is used for inoculating the batch- and fed-batch cultures. A spore concentration of 2.10 9  spores/l is used for inoculation of the pre-batch culture at pH 2.5. Spores are obtained by propagation of freeze-dried spores onto 29 g rice to which the following components are added: 6 ml 15 g/l sucrose, 2.3 g/l (NH 4 ) 2 SO 4 , 1.0 g/l KH 2 PO 4 , 0.5 g/l MgSO 4 .7H 2 O, 0.50 g/l NaCl, 14.3 mg/l ZnSO 4 .7H 2 O, 2.5 mg/CuSO 4 .5H 2 O, 0.50 mg/l NiCl 2 .6H 2 O, and 13.8 mg/l FeSO 4 .7H 2 O. The spores are propagated at 30° C. for 7-14 days to yield a black layer of spores on the rice grains and are harvested by adding 100 ml of 0.1% Tween 20 in sterile water. For all conditions, the gas is sterile filtered before being introduced into the bioreactor. The off gas is led through a condenser cooled to lower than −8° C. and analyzed for its volumetric content of CO 2  and O 2  by means of an acoustic gas analyser. 
     Chemostat Cultures 
     In chemostat cultures the cells can be grown in, for example, 1.5-L working-volume Biostat B laboratory fermentors at 30° C. and 500 rpm. A constant pH of 6.0 is maintained by automatic addition of 10 M KOH, and aerobic conditions are obtained by sparging the bioreactor with air at a rate of 1 vvm to ensure that the DOT is more than 80%. The dilution rate (D) can be set at different values, e.g. at 0.050 h −1 , 0.10 h −1 , 0.15 h −1 , or 0.20 h −1 . The pH is kept constant, e.g at 6.6, by automatic addition of 10 M KOH, using a minimal growth medium with the following components: 2.5 g/l (NH 4 ) 2 SO 4 , 0.75 g/l KH 2 PO 4 , 1.0 g/l MgSO 4 .7H 2 O, 1.0 g/l NaCl, 0.1 g/l CaCl 2 .2H 2 O, 0.1 ml/l Sigma antifoam, 7.2 mg/l ZnSO 4 .7H 2 O, 1.3 mg/l CuSO 4 .5H 2 O, 0.3 mg/l NiCl 2 .6H 2 O, 3.5 mg/l MnCl 2 .4H 2 O and 6.9 mg/l FeSO 4 .7H 2 O. The concentration of glucose can be set at different values, e.g. is 3.0 g/l 6.0 g/l, 12.0 g/l, 15.0 g/l or 18.0 g/l. The bioreactor is inoculated with free mycelium from a pre-batch culture as described above, and the feed pump is turned on at the end of the exponential growth phase. 
     For all conditions, the gas is sterile filtered before being introduced into the bioreactor. The off gas is led through a condenser cooled to lower than −8° C. and analyzed for its volumetric content of CO 2  and O 2  by means of an acoustic gas analyser. 
     Cultivations are considered to be in steady state after at least 5 residence times, and if the concentrations of biomass glucose and composition of the off-gas remain unchanged (less than 5% relative deviation) over the last two residence times. 
     Example 33 
     Extraction and Analysis of Resveratrol in  Aspergillus niger  and  Aspergillus oryzae    
     Extraction and analysis is performed using the methods as described in examples 14 and 15. 
     Example 34 
     Over-Expression of Native Yeast Genes by Gene Targeting Method 
     Over-expression of native yeasts genes with constitutive yeast promoters is carried out by means of a promoter-replacement method based on a linear, PCR-generated gene-targeting substrate and using  K. lactis  URA3 as a recyclable marker described previously (Erdeniz et al, 1997). This method includes the generation of an intermediate yeast strain, where the  Kluyveromyces lactis  URA3 marker gene is integrated in combination with two copies of the strong constitutive promoter sequence as a direct repeat on each side of the marker gene. The marker gene is then looped out through recombination mediated by the direct repeat, an event which is selected for by plating the intermediate strain on medium containing 5-fluoroorotic acid (5-FOA), which is toxic to cells expressing the URA3 gene. The result is a yeast strain, in which the native promoter has been replaced with the strong constitutive promoter. Integration of the above described promoter sequence and marker gene is directed to the correct location in the genome by means of PCR-generated target sequences. 
     The above described gene-targeting substrate can be constructed by means of multiple rounds of fusion-PCR. However, to avoid introduction of PCR-generated mutations, it is beneficial to use a bi-partite or even a quadruple gene-targeting substrate (Erdeniz et al, 1997). 
     Example 35 
     Over-Expression of Native Yeast Genes by Bi-Partite Gene Targeting Substrate Method 
     For example to overexpress a gene with the strong ADH1 promoter, this promoter has been introduced into intermediate working vectors on either side of  K. lactis  URA3, resulting in the vectors pWAD1, pWAD2, (WO/2005/118814). With these vectors as templates, fragments can be amplified that contain (in the 5′ to 3′ direction) 1) the ADH1 coupled to two thirds of  K. lactis  URA3 towards the 5′ end, using the primers AD-fw and Int3′, and 2) two thirds of  K. lactis  URA3 towards the 3′ end coupled to the ADH1, using the primers Int5′ and AD-rv. Target sequences corresponding to a 300-500 bp sequence upstream of the gene to be overexpressed and a 300-500 bp starting with ATG of the gene to be overexpressed, are amplified from genomic yeast DNA using suitable primers. The reverse primer used for amplification of the upstream target sequence contains a 5′ overhang that allows fusion to fragment 1 described above. The forward primer used for amplification of the target sequence starting with ATG contains a 5′ overhang that allows fusion with fragment 2 described above. Following fusion by PCR of the upstream target sequence with fragment 1, and fusion by PCR of fragment 2 with the target sequence starting with ATG, the two linear substrates as shown in  FIG. 6  are ready for transformation. 
     Example 36 
     Construction of a Strain Overexpressing Native  S. cerevisiae  NADP-Cytochrome P450 Reductase 
     The native promoter of  S. cerevisae  NADP-cytochrome P450 reductase CPR1 gene (encoded by YHR042W) was replaced with the constitutive  S. cerevisiae  alcohol dehydrogenase ADH1 promoter via chromosomal promoter exchange using the “bi-partite” PCR-based allele replacement method as described in example 34 and 35. Primers A and B were used to generate fragment CPR1-UP (Table 1) via PCR at a melting temperature of 56° C. using  S. cerevisiae  genomic DNA as template. Primers C and D were then used to generate fragment CPR1-S via PCR at a melting temperature of 56° C. using  S. cerevisiae  genomic DNA as template. Fragments AD1 (k1URA 3′ end fused to promoter ADH1) and AD2 (promoter ADH1 fused to K1URA 5′-end) were generated via PCR using primers AD-fw and Int3′ and Int5′ and AD-ry at a melting temperature of 56° C. and 56° C., respectively. Plasmid pWAD1 was used as template for generation of fragment AD1 and plasmid pWAD2 was used for generating fragment AD2. Fragments CPR_UP were then fused to fragment AD2 using fusion PCR with primers A and Int3′ at a melting temperature of 56° C. resulting in fusion fragment 1 (bi-partite substrate 1). A second fusion PCR was used to fuse fragments AD1 and CPR-S with Int5′ and primer D at a melting temperature of 56° C. resulting in fusion fragment 2 (bi-partite substrate 2). 
     Fusion fragments 1 and 2 (bi-partite substrates 1 and 2) were purified on agarose gel and used for co-transformation of  S. cerevisiae  strain FS01528 (Mata, ura3 his3) and the transformants were plated on SC-URA plates and incubated for 2-4 days at 30° C. Transformants were streak purified on SC-ura plates and incubated another 2 days at 30° C. and then plated onto 5-FOA (5-fluoroorotic acid) plates. After incubation for 2 days at 30° C. “pop-out” colonies appeared, which were streak purified on a new 5-FOA-plate and incubated another 2 days at 30° C. and then finally transferred to a rich medium plate YPD. The resulting colonies were analyzed for the presence of fragment of size 1700-1800 base pairs using yeast colony PCR with primers A and AD-rev and a melting temperature at 55° C. and an elongation time of 1 minute and 45 seconds. One of the positive colonies from the colony PCR containing the new replaced ADH1 promoter in front of the CPR1 gene was named FSpADH1-CPR (Mata ura3 his3 pADH1-CPR1) strain. 
     Table 1 Primers and fragments used in the “bi-partite” PCR-based allele replacement method to exchange native  S. cerevisiae  CPR1 promoter with  S. cerevisiae  ADH1 promoter 
     
       
         
           
               
            
               
                 Primers 
               
               
                 A 5′-GTATTCTATATCCACGCCTGCAAAC 
               
               
                   
               
               
                 B 5′- 
               
               
                 AGTACATACAGGGAACGTCCCTACAGGAACGCAAACTTAAGCTAC 
               
               
                   
               
               
                 C 5′- 
               
               
                 GCATACAATCAACTATCTCATATACAATGCCGTTTGGAATAGACAACACC 
               
               
                   
               
               
                 D 5′-GCTTCCGCATTACAAATAAAGTCTTCAA 
               
               
                   
               
               
                 AD-fw  
               
               
                 5′-GGACGTTCCCTGTATGTACTAGGGGGATCGAAGAAATGATGG 
               
               
                   
               
               
                 Int3′ 5′-GAGCAATGAACCCAATAACGAAATC 3′ 
               
               
                   
               
               
                 Int5′ 5′-CTTGACGTTCGTTCGACTGATGAGC 3′ 
               
               
                   
               
               
                 AD-rv 5′-TGTATATGAGATAGTTGATTGTATGC 
               
               
                   
               
               
                 Fragments 
               
               
                 CPR-UP generated from primers A and B 
               
               
                 (CPR1 gene fragment upstream of start 
               
               
                 codon (ATG)) 
               
               
                   
               
               
                 CPR-S generated from primers C and D 
               
               
                 (CPR1 gene fragment containing start 
               
               
                 codon (ATG)) 
               
               
                   
               
               
                 AD1 (ADH1 promoter coupled to two thirds 
               
               
                 of  K . lactis  URA3 towards the 5′ end 
               
               
                 generated from primers AD-fw and Int3′) 
               
               
                   
               
               
                 AD2 (Two thirds of  K . lactis  URA3 
               
               
                 towards the 3′ end coupled to the ADH1 
               
               
                 promoter. Generated from primers Int5′ 
               
               
                 and AD-rv) 
               
               
                   
               
               
                 Fusion fragment 1 (CPR-UP fragment fused 
               
               
                 to AD2 fragment) 
               
               
                   
               
               
                 Fusion fragment 2 (AD1 fragment fused to 
               
               
                 CPR-S fragment) 
               
            
           
         
       
     
     Example 37 
     Construction of a Strain Overexpressing Native  S. cerevisiae  ACC1 Gene 
     The yeast gene ACC1, encoding acetyl-CoA carboxylase, was overexpressed with the strong constitutive yeast TPI1 promoter as described previously (WO 2005/118814). This was done by replacing the native ACC1 promoter with the TPI1 promoter, using a slightly modified promoter-replacement method based on the bipartite gene-targeting method (Example 1 and 2). One part of the bipartite substrate consisted of two thirds (towards the 3′ end) of  K. lactis  URA3, fused to the TPI1 promoter sequence and a target sequence corresponding to the beginning of ACC1. The second part of the bipartite substrate consisted of a target sequence upstream of ACC1, fused to the TPI1 promoter sequence and two thirds (towards the 5′ end) of  K. lactis  URA3. Following transformation with the bipartite substrate and selection on medium lacking uracil, transformants were obtained in which the native promoter had been knocked out and replaced with two copies of the TPI1 promoter sequence as a direct repeat on either side of the  K. lactis  URA3 marker gene. A second recombination event, resulting in looping out of the selection marker, was selected for by replating transformants on medium containing 5′-fluoroorotic acid (5-FOA), which is toxic to cells expressing the URA3 gene. This resulted in a strain, in which the native ACC1 promoter had been replaced with the TPI1 promoter. 
     In order to construct part 1 of the bipartite substrate, two thirds (towards the 3′ end) of  K. lactis  ura3 was amplified from the plasmid pWJ716 using the primers 5′ CTTGACGTTCGTTCGACTGATGAGC 3′ and 5′ CTGGAATTCGATGATGTAGTTTCTGG 3′ (Table 2). Moreover, the TPI1 promoter sequence was amplified from genomic yeast DNA using the primers 5′ CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTC 3′ and 5′ TTTTTGATTAAAATTAAAAAAACTTTTTAGTTTATGTATGTGTTTTTTG 3′ and a downstream targeting sequence, consisting of the beginning of the ACC1 gene (i.e., the first 553 bp of the gene) was amplified from genomic yeast DNA using the primers 5′ AGTTTTTTTAATTTTAATCAAAAAATGAGCGAAGAAAGCTTATTCGAGTC 3′ and 5′ CACCTAAAGACCTCATGGCGTTACC 3′. These three fragments were fused to each other in two rounds of PCR. First, the TPI1 promoter sequence was fused to the downstream targeting sequence, using the primers 5′ CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTC 3′ and 5′ CACCTAAAGACCTCATGGCGTTACC 3′. The resulting product was then fused to the fragment containing two thirds (towards the 3′ end) of  K. lactis  URA3. The resulting fragment, 3′ 2/3  K. lactis  URA3-pTPI1-DOWN (ACC1) was part 1 of the bipartite gene targeting substrate. 
     In order to construct part 2 of the bipartite substrate, two thirds (towards the 5′-end) of  K. lactis  URA3 was amplified from the plasmid pWJ716 using the primers 5′ CGGTCTGCATTGGATGGTGGTAAC 3′ and 5′ GAGCAATGAACCCAATAACGAAATC 3′ (Table 2). The TPI1 promoter sequence was amplified from genomic yeast DNA using the primers 5′ CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTC 3′ and 5′ CACCATCCAATGCAGACCGTTTTAGTTTATGTATGTGTTTTTTG 3′, and a target sequence upstream of ACC1 was amplified from genomic DNA using primers 5′ TGTTCTGCTCTCTTCAATTTTCCTTTC 3′ and 5′ CTGGAATTCGATGATGTAGTTTCTAATTTTCTGCGCTGTTTCG 3′. These three fragments were fused in two rounds of PCR. First, the upstream targeting sequence was fused to the TPI1 promoter sequence, using the primers 5′ TGTTCTGCTCTCTTCAATTTTCCTTTC 3′ and 5′ CACCATCCAATGCAGACCGTTTTAGTTTATGTATGTGTTTTTTG 3′. The resulting fragment was then fused to the fragment containing two thirds (towards the 5′ end) of  K. lactis  URA3, resulting in the fragment UP (ACC1)-pTPI1-5′ 2/3  K. lactis  URA3, which constituted part 2 of the bipartite gene targeting substrate. 
     Yeast strain FS01372 (MATa ura3 trp1 PADH1-FAS1 pADH1-FAS2) was transformed with the linear substrates UP (ACC1)-pTPI1-5′ 2/3  K. lactis  URA3 and 3′ 2/3  K. lactis  URA3-pTPI1-DOWN (ACC1). Transformants were selected and streak-purified on medium lacking uracil and were then transferred to plates containing 5-FOA. Pop-out recombinants were streak-purified on 5-FOA-containing medium. The resulting strain was named FS01392 and had the genotype MATa ura3 trp1 pTPI1-ACC1 PADH1-FAS1 pADH1-FAS2). The correct integration of the TPI1 promoter was checked by colony PCR. 
     Table 2 Primers and fragments used in the “bi-partite” PCR-based allele replacement method to exchange native  S. cerevisiae  ACC1 promoter with the strong constitutive  S. cerevisiae  TPI1 promoter 
     
       
         
           
               
            
               
                 Primers 
               
               
                 A 5′-CTTGACGTTCGTTCGACTGATGAGC 
               
               
                   
               
               
                 B 5′-CTGGAATTCGATGATGTAGTTTCTGG 
               
               
                   
               
               
                 C 5′-CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTC 
               
               
                   
               
               
                 D 5′- 
               
               
                 TTTTTGATTAAAATTAAAAAAACTTTTTAGTTTATGTATGTGTTTTTTG 
               
               
                   
               
               
                 E 5′- 
               
               
                 AGTTTTTTTAATTTTAATCAAAAAATGAGCGAAGAAAGCTTATTCGAGTC 
               
               
                   
               
               
                 F 5′-CACCTAAAGACCTCATGGCGTTACC 
               
               
                   
               
               
                 G 5′-CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTC 
               
               
                 (G = C) 
               
               
                   
               
               
                 H 5′-CACCTAAAGACCTCATGGCGTTACC 
               
               
                 (H = F) 
               
               
                   
               
               
                 I 5′-CGGTCTGCATTGGATGGTGGTAAC 
               
               
                   
               
               
                 J 5′-GAGCAATGAACCCAATAACGAAATC 
               
               
                   
               
               
                 K 5′-CTACATCATCGAATTCCAGCTACGTATGGTCATTTCTTCTTC 
               
               
                   
               
               
                 L 5′-CACCATCCAATGCAGACCGTTTTAGTTTATGTATGTGTTTTTTG 
               
               
                   
               
               
                 M 5′-TGTTCTGCTCTCTTCAATTTTCCTTTC 
               
               
                   
               
               
                 N 5′-CTGGAATTCGATGATGTAGTTTCTAATTTTCTGCGCTGTTTCG 
               
               
                   
               
               
                 O 5′-TGTTCTGCTCTCTTCAATTTTCCTTTC 
               
               
                 (O = M) 
               
               
                   
               
               
                 P 5′-CACCATCCAATGCAGACCGTTTTAGTTTATGTATGTGTTTTTTG 
               
               
                 (P = L) 
               
               
                   
               
               
                 Fragments 
               
               
                   Klactis URA3 sequence generated with primers 
               
               
                 A and B 
               
               
                   
               
               
                 TPI1 promoter sequence generated with primers 
               
               
                 C and D 
               
               
                   
               
               
                 ACC1 downstream sequence generated with 
               
               
                 primers E and F 
               
               
                   
               
               
                 pTPI1-Down(ACC1) fusion generated from primers 
               
               
                 G and H 
               
               
                   
               
               
                   Klactis URA3-pTPI1-DOWN(ACC1) = part 1 of the 
               
               
                 bipartite substrate generated from primers 
               
               
                 A and H 
               
               
                   
               
               
                   Klactis URA3 sequence generated with primers 
               
               
                 I and J 
               
               
                   
               
               
                 TPI1 promoter sequence generated with primers 
               
               
                 K and L 
               
               
                   
               
               
                 ACC1 upstream sequence generated with primers 
               
               
                 M and N 
               
               
                   
               
               
                 UP(ACC1)-pTPI1 fusion generated from primers 
               
               
                 O and P 
               
               
                   
               
               
                 UP(ACC1)-pTPI1-5′ Klactis URA3 = part 2 of the 
               
               
                 bipartite substrate generated with primers 
               
               
                 M and J 
               
            
           
         
       
     
     Example 38 
     Deletion of Native Yeast Genes by Gene Targeting Method 
     Gene deletions were performed by a similar method as for gene overexpressions (Example 1) by means of homologous recombination using PCR-generated targeting substrates and the  K. lactis  URA3 gene as a selectable marker, essentially as described in Erdeniz, N., Mortensen, U. H., Rothstein, R. (1997) Genome Res. 7:1174-83. Information on primer design for fusion PCR can be found in the same publication. Generally, fusion of DNA fragments was made possible by using primers with appropriately designed 5′ overhangs for amplification of the original DNA fragments. In all cases, PCR-generated fragments were excised from a 1% agarose gel and purified before proceeding with fusion PCR. 
     Transformants were generally selected on -URA plates, and pop-out of the  K. lactis  URA3 marker gene was selected for by plating on 5-FOA medium (5-fluoroorotic acid, 750 mg/l). Correct gene deletions were verified by PCR, using primers on both sides of the deleted gene. Generally, PCR-verification of gene deletions was performed by means of colony-PCR. For colony-PCR, a small amount of cells was dispersed in 10 μl H 2 O and was placed at −80° C. for approximately 30 min, followed by 15 min. incubation at 37° C. The cell suspension was then used as template for PCR. 
     Example 39 
     Generation of Strain with Deleted Isocitrate Dehydrogenase, IDH1 
     The Native Yeast Gene IDH1 
                    SEQ ID NO: 59       ATGCTTAACAGAACAATTGCTAAGAGAACTTTAGCCACTGCCGCTCAGGC               GGAACGCACCCTACCCAAGAAGTATGGCGGTCGTTTCACCGTCACTTTGA               TACCTGGTGACGGTGTTGGGAAAGAAATCACTGATTCAGTGAGAACCATT               TTTGAGGCTGAAAATATCCCGATCGACTGGGAAACTATAAACATTAAGCA               AACAGATCATAAGGAAGGCGTCTATGAAGCTGTTGAGTCTCTAAAGAGAA               ATAAGATTGGTCTTAAGGGGCTATGGCACACTCCTGCTGACCAAACAGGT               CACGGTTCACTAAACGTTGCTTTGCGTAAACAACTAGATATCTACGCCAA               TGTGGCCCTTTTCAAATCCTTGAAGGGTGTCAAGACTAGAATTCCAGACA               TAGATTTGATTGTCATTAGAGAAAACACGGAGGGTGAGTTCTCAGGCCTG               GAACATGAATCCGTCCCTGGTGTAGTGGAATCTTTGAAAGTTATGACTAG               ACCTAAGACAGAAAGGATCGCCAGATTTGCCTTTGACTTCGCCAAGAAAT               ACAACAGAAAGTCTGTCACAGCTGTGCATAAGGCAAATATCATGAAGTTA               GGTGACGGTCTGTTCAGAAATATAATAACTGAAATTGGCCAAAAAGAATA               TCCTGATATTGACGTATCGTCCATCATTGTCGACAATGCCTCCATGCAGG               CGGTGGCCAAACCTCATCAATTTGATGTCCTAGTTACCCCTTCAATGTAC               GGTACCATCTTAGGCAACATTGGCGCTGCTTTGATCGGTGGTCCAGGATT               GGTGGCAGGTGCCAACTTTGGCAGGGACTATGCTGTCTTCGAACCAGGTT               CCAGACATGTTGGTTTAGATATTAAAGGCCAAAATGTGGCTAACCCAACT               GCCATGATCCTTTCCTCCACGTTAATGTTGAACCATTTGGGTTTGAATGA               ATATGCTACTAGAATCTCAAAGGCAGTTCATGAAACGATCGCAGAAGGTA               AGCATACCACTAGAGATATTGGTGGTTCCTCTTCTACTACTGACTTCACG               AATGAAATCATCAACAAATTATCTACCATGTAA            
encoded by YNL037c is deleted using a quadruple gene targeting substrate according to the following procedure:
 
     A target sequence upstream of IDH1 gene is amplified from genomic DNA by PCR using the primers IDH1-up-fw and IDH1-up-ry and is fused to the two thirds of the  K. lactis  URA3 gene to the 5′ end by PCR. Furthermore a target sequence corresponding to the downstream region of IDH1 is amplified from genomic DNA using the primers IDH1-D-fw and IDH1-d-rv. The downstream target sequence is fused to the two thirds of the  K. lactis  URA3 gene to the 3′ end by PCR. 
     The yeast strain FS01528 (MATa ura3 his3) is transformed with the two linear fusion substrates described above containing the upstream target region and the downstream target region of the gene to be deleted fused to either two thirds of the  K. lactis  URA3 gene. Transformants are selected on medium lacking uracil and are streak-purified on the same medium. Transformants are transferred to plates containing 5-FOA. Pop-out recombinants are streak-purified on 5-FOA-containing medium. The resulting strain has the genotype (MATa ura3 his3 IDH1Δ). Correct deletion of the IDH1 gene is verified by PCR using the primers IDH1-up-fw and IDH1-D-rv. 
     Example 40 
     Mating of Cells, Sporulation, Tetrad Dissection and Tetrad Scoring (Analysis) 
     Methods for combining genetic features by crossing of strains used in the examples are well known and are, e.g., described in: Adams, A., Gottschling, D. E., Kaiser, C. A., and Stearns, T. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1997). Typically, strains of opposite mating types were allowed to mate, diploids were selected and transferred to sporulation medium (20 g/l potassium acetate, 1 g/l glucose, 2.5 g/l yeast extract, pH 7.0) and were allowed to sporulate at 30° C. for approximately 3 days. The asci were dissected on a YPD plate using a Singer MSM microscope and micromanipulator dissection microscope. The mating types of the resulting tetrads were scored by replica-plating to a lawn of cells with either a or alpha mating type, incubating at 30° C. to allow mating, replica-plating to sporulation medium, and visualizing sporulation by illuminating plates under a 302 nm UV-light source. Auxotrophic markers were scored by replica plating to drop-out plates. Genetic modifications that could not be scored by phenotype were scored by colony-PCR. In general, the same primer sets that were used for verification of genomic integrations or knockouts were also used for colony-PCR scoring of tetrads. 
     Example 41 
     Isolation of Genes Encoding TAL, PAL, C4H, 4CL, and VST1 
     Tyrosine ammonia lyase (TAL) was isolated from  Rhodobacter capsulatus  by codon optimization for expression in  S. cerevisiae  and was further assembled as a synthetic gene as described above. 
     The isolation of phenylalanine ammonia lyase (PAL2), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoenzymeA ligase (4CL1) described above. 
     4-Coumarate:CoenzymeA Ligase (4CL2) 
                    (SEQ ID NO: 60)       ATGACGACACAAGATGTGATAGTCAATGATCAGAATGATCAGAAACAGTG               TAGTAATGACGTCATTTTCCGATCGAGATTGCCTGATATATACATCCCTA               ACCACCTCCCACTCCACGACTACATCTTCGAAAATATCTCAGAGTTCGCC               GCTAAGCCATGCTTGATCAACGGTCCCACCGGCGAAGTATACACCTACGC               CGATGTCCACGTAACATCTCGGAAACTCGCCGCCGGTCTTCATAACCTCG               GCGTGAAGCAACACGACGTTGTAATGATCCTCCTCCCGAACTCTCCTGAA               GTAGTCCTCACTTTCCTTGCCGCCTCCTTCATCGGCGCAATCACCACCTC               CGCGAACCCGTTCTTCACTCCGGCGGAGATTTCTAAACAAGCCAAAGCCT               CCGCGGCGAAACTCATCGTCACTCAATCCCGTTACGTCGATAAAATCAAG               AACCTCCAAAACGACGGCGTTTTGATCGTCACCACCGACTCCGACGCCAT               CCCCGAAAACTGCCTCCGTTTCTCCGAGTTAACTCAGTCCGAAGAACCAC               GAGTGGACTCAATACCGGAGAAGATTTCGCCAGAAGACGTCGTGGCGCTT               CCTTTCTCATCCGGCACGACGGGTCTCCCCAAAGGAGTGATGCTAACACA               CAAAGGTCTAGTCACGAGCGTGGCGCAGCAAGTCGACGGCGAGAATCCGA               ATCTTTACTTCAACAGAGACGACGTGATCCTCTGTGTCTTGCCTATGTTC               CATATATACGCTCTCAACTCCATCATGCTCTGTAGTCTCAGAGTTGGTGC               CACGATCTTGATAATGCCTAAGTTCGAAATCACTCTCTTGTTAGAGCAGA               TACAAAGGTGTAAAGTCACGGTGGCTATGGTCGTGCCACCGATCGTTTTA               GCTATCGCGAAGTCGCCGGAGACGGAGAAGTATGATCTGAGCTCGGTTAG               GATGGTTAAGTCTGGAGCAGCTCCTCTTGGTAAGGAGCTTGAAGATGCTA               TTAGTGCTAAGTTTCCTAACGCCAAGCTTGGTCAGGGCTATGGGATGACA               GAAGCAGGTCCGGTGCTAGCAATGTCGTTAGGGTTTGCTAAAGAGCCGTT               TCCAGTGAAGTCAGGAGCATGTGGTACGGTGGTGAGGAACGCCGAGATGA               AGATACTTGATCCAGACACAGGAGATTCTTTGCCTAGGAACAAACCCGGC               GAAATATGCATCCGTGGCAACCAAATCATGAAAGGCTATCTCAATGACCC               CTTGGCCACGGCATCGACGATCGATAAAGATGGTTGGCTTCACACTGGAG               ACGTCGGATTTATCGATGATGACGACGAGCTTTTCATTGTGGATAGATTG               AAAGAACTCATCAAGTACAAAGGATTTCAAGTGGCTCCAGCTGAGCTAGA               GTCTCTCCTCATAGGTCATCCAGAAATCAATGATGTTGCTGTCGTCGCCA               TGAAGGAAGAAGATGCTGGTGAGGTTCCTGTTGCGTTTGTGGTGAGATCG               AAAGATTCAAATATATCCGAAGATGAAATCAAGCAATTCGTGTCAAAACA               GGTTGTGTTTTATAAGAGAATCAACAAAGTGTTCTTCACTGACTCTATTC               CTAAAGCTCCATCAGGGAAGATATTGAGGAAGGATCTAAGAGCAAGACTA               GCAAATGGATTAATGAACTAG            
was isolated via PCR from  A. thaliana  cDNA (BioCat, Heidelberg, Germany) using the forward primer 5′-GCGAATTCTTATGACGACA CAAGATGTGATAGTCAATGAT-3′ containing an EcoR1 restriction site and reverse primer 5′-GCACTAGTATCCTAGTTCATTAATCCATTT GCTAGTCTTGCT-3′ containing a Spe1 restriction site.
 
     The VST1 gene encoding  Vitis vinifera  (grapevine) resveratrol synthase (Hain et al, 1993) was synthesized by GenScript Corporation (Piscataway, N.J.). The amino acid sequence: 
                        (SEQ ID NO: 63)                               1   MASVEEFRNA QRAKGPATIL AIGTATPDHC VYQSDYADYY FRVTKSEHMT                    51   ELKKKFNRIC DKSMIKKRYI HLTEEMLEEH PNIGAYMAPS LNIRQEIITA               101   EVPRLGRDAA LKALKEWGQP KSKITHLVFC TTSGVEMPGA DYKLANLLGL               151   ETSVRRVMLY HQGCYAGGTV LRTAKDLAEN NAGARVLVVC SEITVVTFRG               201   PSEDALDSLV GQALFGDGSS AVIVGSDPDV SIERPLFQLV SAAQTFIPNS               251   AGAIAGNLRE VGLTFHLWPN VPTLISENIE KCLTQAFDPL GISDWNSLFW               301   IAHPGGPAIL DAVEAKLNLE KKKLEATRHV LSEYGNMSSA CVLFILDEMR               351   KKSLKGEKAT TGEGLDWGVL FGFGPGLTIE TVVLHSVPTV TN**            
was used as template to generate a synthetic gene optimized for expression in  S. cerevisiae :
 
     
       
         
           
               
            
               
                 ATGGCATCCGTAGAGGAGTTCAGAAATGCACAGAGGGCAAAAGG 
               
               
                   
               
               
                 TCCAGCAACCATATTGGCTATTGGAACAGCCACCCCTGATCACTGTGTTT 
               
               
                   
               
               
                 ATCAATCTGATTACGCTGATTACTATTTCAGAGTAACTAAAAGTGAACAT 
               
               
                   
               
               
                 ATGACAGAACTTAAGAAAAAGTTTAATAGAATTTGTGATAAATCTATGAT 
               
               
                   
               
               
                 AAAGAAAAGATACATACATCTAACTGAAGAAATGTTAGAGGAACATCCAA 
               
               
                   
               
               
                 ATATAGGTGCATATATGGCACCATCTTTGAATATTAGACAAGAAATCATA 
               
               
                   
               
               
                 ACAGCCGAGGTACCTAGACTAGGTAGAGACGCAGCCTTGAAAGCTTTAAA 
               
               
                   
               
               
                 GGAATGGGGACAACCAAAATCTAAGATTACACATTTGGTTTTCTGTACAA 
               
               
                   
               
               
                 CTTCCGGTGTCGAAATGCCAGGTGCTGATTATAAACTAGCAAACCTATTG 
               
               
                   
               
               
                 GGATTAGAGACCTCTGTTAGAAGAGTTATGTTGTATCATCAAGGTTGTTA 
               
               
                   
               
               
                 CGCCGGAGGTACAGTGCTTAGAACTGCTAAGGATTTGGCAGAAAATAACG 
               
               
                   
               
               
                 CCGGTGCTAGGGTTTTAGTCGTCTGCAGTGAAATCACTGTCGTAACTTTC 
               
               
                   
               
               
                 AGAGGTCCATCAGAAGATGCTCTAGACAGTTTGGTCGGACAAGCATTGTT 
               
               
                   
               
               
                 TGGCGATGGATCTTCCGCCGTAATTGTAGGCAGCGATCCTGATGTGTCCA 
               
               
                   
               
               
                 TTGAAAGACCACTATTTCAATTAGTTTCTGCTGCTCAAACTTTTATTCCA 
               
               
                   
               
               
                 AATTCCGCCGGTGCCATAGCAGGAAACTTGAGAGAAGTTGGTTTGACTTT 
               
               
                   
               
               
                 TCATTTGTGGCCTAATGTCCCAACCTTAATTTCAGAAAACATCGAAAAAT 
               
               
                   
               
               
                 GCTTAACTCAAGCCTTTGACCCATTGGGCATAAGCGACTGGAACTCATTG 
               
               
                   
               
               
                 TTTTGGATTGCTCATCCAGGTGGTCCAGCAATTTTAGACGCAGTGGAGGC 
               
               
                   
               
               
                 AAAACTAAACTTAGAGAAGAAAAAGTTGGAAGCTACAAGACACGTTCTAT 
               
               
                   
               
               
                 CAGAGTATGGCAACATGAGCTCTGCCTGCGTTTTATTCATTCTAGATGAG 
               
               
                   
               
               
                 ATGAGGAAGAAGTCTTTAAAGGGTGAAAAAGCCACAACCGGAGAAGGTTT 
               
               
                   
               
               
                 AGATTGGGGTGTTCTATTTGGTTTCGGTCCTGGCTTAACAATTGAGACAG 
               
               
                   
               
               
                 TGGTGTTACACTCTGTTCCAACTGTCACTAACTAATGA 
               
            
           
         
       
     
     (SEQ ID NO: 64). 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 42 
     Construction of a Yeast Vector for Expression of TAL 
     Plasmid, pESC-URA-TAL, containing the gene encoding tyrosine ammonia lyase, TAL, under the control of the divergent GAL1/GAL10 promoter was constructed as described above for PAL. 
     Example 43 
     Construction of a Yeast Vector for Expression of 4CL 
     The gene encoding 4CL1 and 4CL2 were isolated as described above. The amplified 4CL1 PCR-product was digested with Xba1/BamH1 and ligated into Spe1/BglII digested pESC-TRP vector (Stratagene), resulting in vector pESC-TRP-4CL. 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-TRP-4CL1 and pESC-HIS-4CL2 were sequenced to verify the sequence of the cloned gene. 
     Example 44 
     Construction of a Yeast Vectors for Expression of 4CL and VST 
     The gene encoding VST from  Vitis vinifera  (grape) was isolated as described above. The purified BamH1/Xho1 digested VST gene fragment was ligated into BamH1/Xho1 digested pESC-HIS-4CL2 plasmid or pESC-trp-4CL1 plasmid (example 15). The resulting plasmids, pESC-HIS-4CL2-VST and pESC-TRP-4CL1-VST contained the genes encoding 4CL1, 4CL2 and VST under the control of the divergent GAL1/GAL10 promoter. The sequence of the gene encoding VST was verified by sequencing of two different clones of pESC-HIS-4CL2-VST and pESC-TRP-4CL1-VST. 
     Example 45 
     Expression of the PAL-Pathway to Resveratrol in the Yeast  S. cerevisiae  Using PAL, C4H, 4CL and VST 
     Yeast strains containing the appropriate genetic markers were transformed with the vectors described in examples 36 and 38. The transformation of the yeast cell was conducted in accordance with methods known in the art, for instance, by using competent cells or by electroporation (see, e.g., Sambrook et al., 1989). 
       S. cerevisiae  strain FS01267 (MATa ura3 trp1) was co-transformed with the vectors pESC-URA-PAL-C4H and pESC-TRP-4CL1-VST, resulting in the strain FSSC-PALC4H4CL1VST. 
       S. cerevisiae  strain FS01528 (MATa ura3 his3) was co-transformed with the vectors pESC-URA-PAL-C4H and pESC-HIS-4CL2-VST, resulting in the strain FSSC-PALC4H4CL2VST. 
     Transformants were selected on medium lacking uracil and tryptophan or uracil and histidine and streak purified on the same medium. 
     Example 46 
     Expression of the TAL-Pathway to Resveratrol in  S. cerevisiae  Using TAL, 4CL and VST 
       S. cerevisiae  strain FS01528 (MATa ura3 his3) was co-transformed with pESC-URA-TAL (example 42) and pESC-HIS-4CL2-VST (example 44), and the transformed strain was named FSSC-TAL4CL2VST. Transformants were selected on medium lacking uracil and histidine and streak purified on the same medium. 
     Example 47 
     Expression of the PAL-Pathway to Resveratrol in  S. cerevisiae  Strain Overexpressing Native  S. cerevisiae  NADP-Cytochrome P450 Reductase 
     FSpADH1-CPR (Mata ura3 his3 pADH1-CPR1) (Example 36) was co-transformed with the vectors pESC-URA-PAL-C4H and pESC-HIS-4CL2-VST, resulting in the strain FSSC-PALC4H4CL2VST-pADH1CPR1 (Mata ura3 his3 pADH1-CPR1, pESC-URA-PAL-C4H, pESC-HIS-4CL2-VST). 
     Example 48 
     Expression of the PAL-Pathway to Resveratrol in  S. cerevisiae  Strain Overexpressing Native  S. cerevisiae  ACC1 Gene 
     FS01392 (MATa ura3 trp1 pTPII-ACC1 PADH1-FAS1 pADH1-FAS2) (example 37) was co-transformed with the vectors pESC-URA-PAL-C4H and pESC-TRP-4CL1-VST, resulting in the strain FS01392-PAL. 
     As a control the strain FS01372 (MATa ura3 trp1 pTPII-ACC1 PADH1-FAS1 pADH1-FAS2)(Example 37) was also co-transformed with the vectors pESC-URA-PAL-C4H and pESC-TRP-4CL1-VST, resulting in the strain FS01372-PALcon. 
     Example 49 
     Expression of the TAL-Pathway to Resveratrol in  S. cerevisiae  Strain Overexpressing Native  S. cerevisiae  ACC1 Gene 
     FS01392 (MATa ura3 trp1 pTPII-ACC1 PADH1-FAS1 pADH1-FAS2) (example 37) was co-transformed with the vectors pESC-URA-TAL and pESC-TRP-4CL1-VST, resulting in the strain FS01392-TAL. 
     As a control the strain FS01372 (MATa ura3 trp1 pTPI1-ACC1 PADH1-FAS1 pADH1-FAS2)(Example 37) was also co-transformed with the vectors pESC-TAL and pESC-TRP-4CL1-VST, resulting in the strain FS01372-TALcon. 
     Example 50 
     HPLC Analysis of Hydroxyl Stilbenes 
     For quantitative analysis of cinnamic acid, coumaric acid, pinosylvin and resveratrol, cell free supernatant 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, Calif., USA) Luna 3 micrometer C18 (100×2.00 mm) column was used at 40° C. As mobile phase a gradient of acetonitrile and milliq water (both containing 50 ppm trifluoroacetic acid) was used at a flow of 0.4 ml/min. The gradient profile was linear from 15% acetonitrile to 100% acetonitrile over 20 min. The elution times were approximately 3.4 min. for coumaric acid, 5.5 min. for free trans-resveratrol and 6.8 min. for cinnamic acid. The elution time was approximately 8.8-8.9 minutes for trans-pinosylvin. 
     Pure pinosylvin standard (&gt;95% pure) was purchased from ArboNova (Turku, Finland). Pure resveratrol standard was purchased from Cayman chemical company, whereas pure coumaric acid and cinnamic acid standards were purchased from Sigma. 
     Example 51 
     Shake Flask Cultivations of Strain Overexpressing CPR 
     The metabolically engineered recombinant yeast strain with overexpressed CPR, FSSC-PALC4H4CL2VST-pADH1CPR1 (example 19), was inoculated to an initial optical density of 0.1 and grown in 100 ml defined mineral medium (Verduyn et al, 1992) that contained vitamins, trace elements, 3 g/l glucose and 40 g/l galactose for induction of the PAL-pathway genes. The control strain FSSC-PALC4H4CL2VST (example 17) was inoculated in the same way in a second shake flask for control comparison. 
     The 500 ml stoppered shake flasks were incubated for three days at 30° C. and 110 rpm. At 72 hours 1 ml samples were taken out from the cultivations, cells were removed by 1 minute centrifugation (13000 rpm, micro centrifuge), and the cell free supernatant was analyzed with HPLC. 
     The engineered strain overexpressing CPR produced 12.0 mg/l resveratrol compared to the control strain that produced 1.0 mg/l resveratrol after 72 hours cultivation. 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                   
                 Resveratrol 
               
               
                   
                 Strain 
                 (mg/l) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Control 
                 1.0 
               
               
                   
                 FSSC-PALC4H4CL2VST 
               
               
                   
                 Overexpressed CPR 
                 12.0 
               
               
                   
                 FSSC-PALC4H4CL2VST-pADH1CPR1 
               
               
                   
                   
               
            
           
         
       
     
     Example 52 
     Shake Flask Cultivations of Strains Overexpressing ACC1 
     The metabolically engineered recombinant yeast strains with overexpressed ACC1, FS01392-PAL (example 20a) and FS01392-TAL (example 20b), were inoculated to an initial optical density of 0.1 and grown in 100 ml defined mineral medium (Verduyn et al, 1992) that contained, vitamins, trace elements, 3 g/l glucose and 40 g/l galactose for induction of the PAL-pathway genes. After 24 hours 50 mg coumaric acid (Sigma) precursor dissolved in 1 ml 70% ethanol was added corresponding to a concentration of 500 mg/l coumaric acid in the shake flasks. 
     The control strains, FS01372-PALcon (example 20a) and FS01372-TAlcon (example 20b), were inoculated and grown in the same way in a second shake flask for control comparison. 
     The 500 ml stoppered shake flasks were incubated for three days at 30° C. and 110 rpm. At 68 hours 1 ml samples were taken out from the cultivations, cells were removed by 1 minute centrifugation (13000 rpm, micro centrifuge), and the cell free supernatant was analyzed with HPLC. 
     The engineered strain FS01392-PAL (overexpressing ACC1 and the PAL-pathway genes produced) 119 mg/l resveratrol and its control strain FS01372-PALcon produced 104 mg/l resveratrol, corresponding to a 14% increase in the engineered strain. 
     The engineered strain FS01392-TAL (overexpressing ACC1 and the TAL-pathway genes produced) 99.5 mg/l resveratrol and its control strain FS01372-TALcon produced 69 mg/l resveratrol, corresponding to a 44% increase in the engineered strain. 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                   
                 Resveratrol 
               
               
                   
                 Strain 
                 (mg/l)* 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 FS01392-PAL 
                 119.0 
               
               
                   
                 Overexpressed ACC1 
               
               
                   
                 Control-PAL 
                 104.0 
               
               
                   
                 FS01372-PALcon 
               
               
                   
                 FS01392-TAL 
                 99.5 
               
               
                   
                 Overexpressed ACC1 
               
               
                   
                 Control-TAL 
                 69.0 
               
               
                   
                 FS01372-TALcon 
               
               
                   
                   
               
               
                   
                 *In these experiments 500 mg/l coumaric acid was added to the shake flasks. 
               
            
           
         
       
     
     Example 53 
     Resveratrol Content of Genetically Engineered Yeast Cells 
     The resveratrol content of yeast cells genetically engineered to produce resveratrol as described in Example 9 was determined. Levels of from 0.44 to 0.53 μg/g were found. 
     Example 53 
     Determination of Intracellular and Extracellular Levels of Stilbenoids in a Continuous Culture of PALCPR 
     The yeast strain with overexpressed CPR, FSSC-PALC4H4CL2VST-pADH1CPR1 (see Example 47) was grown in a carbon-limited continuous culture with a working volume of 1 liter. The culture was fed with a defined medium according to Verduyn et al. (1992), containing: 5.0 g/L (NH 4 ) 2 SO 4 ; 3.0 g/L KH 2 PO 4 ; 0.5 g/L MgSO 4 .7H2O; trace metals and vitamins and 5 g/l glucose and 35 g/l galactose as the growth-limiting nutrients. Antifoam (300 μl/L, Sigma A-8436) was added to avoid foaming. The carbon source was autoclaved separately from the mineral medium and afterwards added to the fermentor. In addition, the vitamin and trace metal solutions were added to the fermentor by sterile filtration following autoclavation and cooling of the medium. The fermentor system was from Sartorius BBI systems and consisted of a baffled 3-liter reactor vessel with 1 liter working volume equipped with Biostat B Plus controller. The reactor vessel was equipped with two Rushton turbines which were rotating at either 1000 rpm, the temperature was kept at 30±1° C., and the pH was kept at 5.5±0.2 by automatic addition of 2M KOH. The gasflow was controlled by a mass flow controller and was set to 1.5 vvm (1.5 l/min). The off-gas was led through a cooled condenser, and was analyzed for O 2  and CO 2  (Model 1308, Innova, Denmark). An initial batch culture with 35 g/l galactose was started by inoculation of the culture with 10 ml of an exponential growing shakeflask culture containing 5 g/l glucose and 35 g/l galactose. The batch cultivation was switched to a continuous mode by feeding the same medium continuously to the reactor. The dilution rate was controlled on a constant level basis, aiming at D=0.050 h −1 . The continuous culture was regarded to be in steady state when both the dilution rate and off-gas signal had not changed for at least five residence times, and when the metabolite concentrations in two successive samples taken at intervals of 1 residence time, deviated by less than 3%. The dissolved-oxygen concentration, which was continuously monitored, was kept above 60% of air saturation. Under said conditions the strain consumed all the galactose, and mainly produced biomass and CO 2 , and only minor amounts of ethanol. Moreover, the RQ was close to unity, indicating that metabolism was predominantly in respirative mode. 
     For the determination of stilbenoids, samples were taken at approximately 300 hrs into fermentation corresponding to 15 residence times. Cells were harvested by centrifugation 5000 g for 5 minutes. For the determination of extracellular levels of stilbenoids, an aliquot of 25 ml of supernatant was extracted once with 10 ml ethyl acetate. The ethyl acetate was freeze dried and the dry product redissolved in 0.6 ml methanol. The samples were than 50-fold diluted in water transferred into HPLC vials, and analyzed by HPLC. Furthermore, to evaluate whether the level of stilbenoids that was produced exceeded the solubility of the medium, or were either bound to the cell-membranes 1 ml aliquots of cell culture, thus including both cells and medium, were mixed with 1 ml of 100% ethanol, and mixed vigorously prior to centrifugation. The supernatant was then transferred into HPLC vials and directly analyzed for the content of stilbenoids. For the determination of intracellular levels of stilbenoids, an aliquot of 50 ml culture was sampled, and cells and medium were separated by centrifugation. The pellet was washed with 50 ml of water to remove any stilbenoids that were cell-bound or trapped into the pellet; after re-centrifugation the pellet was then dissolved in 1 ml water. The resulting cell suspension was distributed into extraction tubes and broken with glass beads using a fast-prep machine. The crude extracts were pooled into 10 ml of 100% methanol, and extracted in a rotary chamber for 24 hours in a dark cold room at 4° C. Thereafter, the cell debris was removed via centrifugation for 5 min. at 5000 g and the remaining methanol was removed by freeze-drying overnight. The dry residue was redissolved in 0.4 ml methanol and 0.1 ml water. The samples were than 50-fold diluted in water and then transferred into HPLC vials, and analyzed by HPLC. 
     The following table summarizes the results: 
     
       
         
           
               
            
               
                   
               
               
                 Intracellular and extracellular levels of stilbenoids of PALCPR continuous culture at 300 hrs. 
               
               
                 5 g/l glucose, 35 g/l galactose, D = 0.05, pH = 5.5., 1000 rpm 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Resveratrol 
                 Pinosylvin 
                 Resveratrol 
                 Pinosylvin 
                 Resveratrol 
                 Pinosylvin 
                 Resveratrol 
                 Pinosylvin 
               
               
                 Intracelullar 
                 Intracelullar 
                 Extracelullar 
                 Extracelullar 
                 Extracellular 
                 Extracellular 
                 Total 
                 Total 
               
               
                 (a) 
                 (b) 
                 (c) 
                 (d) 
                 In EtOH (e) 
                 In EtOH (f) 
                 (a + e) 
                 (b + f) 
               
               
                   
               
               
                 2.27 
                 16.45 
                 23.69 
                 12.55 
                 23.65 
                 113.57 
                  25.92 
                 130.02 
               
               
                 8.74 
                 12.65 
                 91.43 
                  9.65 
                 91.26 
                  87.35 
                 100.00 
                 100.00 
               
               
                 0.25 
                  1.83 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                   
               
            
           
         
       
     
     Intracellular levels of stilbenoids were expressed in mg per gram biomass (dry weight), according to the calculation explained in the following section. The concentration of resveratrol and pinosylvin in the extract was determined as 227 mg/l and 1646 mg/l respectively; the volume of the extract was 0.5 ml, hence the absolute amount of resveratrol and pinosylvin extracted was 0.5*227/1000=0.1135 mg and 0.5*1646/1000=0.8230 mg respectively. The stilbenoids were extracted from a 50 ml culture-aliquot and hence the intracellular concentrations of resveratrol and pinosylvin expressed per liter culture were 0.1135*(1000/50)=2.27 mg/l and 0.8230*(1000/50)=16.46 mg/l. The biomass concentration of said culture was 9 g/l. The intracellular resveratrol- and pinosylvin levels expressed per gram dry weight therefore were 2.27/9=0.25 mg/g dry weight and 16.46/9=1.83 mg/g dry weight respectively. 
     REFERENCES 
     
         
         U.S. Pat. No. 6,521,748 
         US-A-2001053847 
         US-A-2004059103 
         US-A-2004023357 
         WO2006089898 
         WO2008/009728 
         Allina, S. M., Pri-Hadash, A., Theilmann, D. A., Ellis, B. E. and Douglas, C. J. (1998) 4-coumarate: Coenzyme A ligase in hybrid poplar. Properties of enzymes, cDNA cloning, and analysis of recombinant clones. Plant Physiol. 116, 743-754. 
         Aoyama, Y., Yoshida, Y., Kubota, S., Kumaoka, H. and Furumichi, A. (1978). NADPH-cytochrome P-450 reductase of yeast microsomes. Arch. Biochem. Biophys. 185, 362-369. 
         Becker J V, Armstrong G O, van der Merwe M J, Lambrechts M G, Vivier M A, Pretorius I S. (2003). Metabolic engineering of  Saccharomyces cerevisiae  for the synthesis of the wine-related antioxidant resveratrol. FEMS Yeast Res. 4, 79-85. 
         Blanquet, S., Meunier, J. P., Minekus, M., Marol-Bonnin, S., and Alric, M. (2003). Recombinant  Saccharomyces cerevisiae  Expressing P450 in Artificial 
         Digestive Systems: a Model for Biodetoxication in the Human Digestive Environment. Appl. Environ. Microbiol. 69, 2884-2892. 
         Celotti E and others. (1996). Resveratrol content of some wines obtained from dried Valpolicella grapes: Recioto and Amarone. Journal of Chromatography A 730, 47-52. 
         Cochrane, F. C., Davin, L. B. and Lewis N. G. (2004). The  Arabidopsis  phenylalanine ammonia lyase gene family: kinetic characterization of the four PAL isoforms. Phytochemistry 65, 1557-1564. 
         Couzin, J. (2004) Aging Research&#39;s Family Feud. Science 303, 1276-1279. 
         Ehlting, J., Büttner, D., Wang, Q., Douglas, C. J., Somssich, I. E. and Kombrink, E. (1999). Three 4-coumarate:coenzyme A ligases in  Arabidopsis thaliana  represents two evolutionary divergent classes in angiosperms. The plant journal. 19, 9-20. 
         Erdeniz N, Mortensen U H, Rothstein R. 
         Cloning-free PCR-based allele replacement methods. 
         Genome Res. 1997:7:1174-83. 
         Filpula, D., Vaslet, C. A., Levy, A., Sykes, A. and Strausberg, R. L. Nucleotide sequence of gene for phenylalanine ammonia-lyase from  Rhodotorula rubra . (1988). Nucleic Acids Res. 16, 11381. 
         Gehm, B. D., McAndrews, J. M., Chien, P. Y. and Jameson, J. L. (1997). Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor. Proc. Natl. Acad. Sci. USA 94, 14138-14143. 
         Gems, D., Johnstone, I. L. and Clutterbuck, A. J. (1991). An autonomously replicating plasmid transforms  Aspergillus nidulans  at high frequency. Gene 98, 61-67. 
         Hain, R., Reif, H. J., Krause, E., Langebartels, R., Kindl, H., Vornam, B., Wiese, W., Schmelzer, E., Schreier, P. H., Stocker, R. H. and Stenzel, K. (1993). Disease resistance results from foreign phytoalexin expression in a novel plant. Nature 361, 153-156. 
         Hwang E I, Kaneko M, Ohnishi Y, Horinouchi S. (2003). Production of plant-specific flavanones by  Escherichia coli  containing an artificial gene cluster. Appl. Environ. Microbiol. 69, 2699-706. 
         Huang, M-T. (1997). Diet for cancer prevention. Food Sci. 24, 713-727 
         Hart, J. H. (1981) Annu. Rev. Phytopathology 19, 437-458. 
         Hart, J. H., Shrimpton, D. M. (1979) Phytopathology 69, 1138-1143. 
         Hall, S. S. (2003) In Vino Vitalis? Compounds Activate Life-Extending Genes. Science 301, 1165. 
         Hamberger, B. and Hahlbrock, K. (2004). The 4-coumarate:CoA ligase gene family in  Arabidopsis thaliana  comprises one rare, sinapate-activating and three commonly occurring isoenzymes. Proc. Natl. Acad. Sci. USA. 101, 2209-2214. 
         Jang, M., Cai, L., Udeani, G O., Slowing, K V., Thomas, C F., Beecher, C W W., Fong, H H S., Farnsworth, N R., Kinghorn, A D., Mehta, R G., Moon, R C., Pezzuto, J M. (1997). Cancer Chemopreventive Activity of Resveratrol, a Natural Product Derived from Grapes. Science 275, 218-220. 
         Jeandet, P. Bessis, R., Maume, B. F., Meunier, P., Peyron, D. and Trollat, P. (1995). Effect of Enological Practices on the Resveratrol Isomer Content of Wine J. Agric. Food Chem. 43, 316-319. 
         Jeandet, P. Bessis, R., Sbaghi, M. and Meunier, P. (1994). Occurrence of a resveratrol β-D-glucoside in wine: Preliminary studies.  Vitis  33, 183-184. 
         Koopmann, E., Logemann, E. and Hahlbrock, K. (1999). Regulation and Functional Expression of Cinnamate 4-Hydroxylase from Parsley. Plant Physiol. 119, 49-55. 
         Kopp, P. (1998). Resveratrol, a phytooestrogen found in red wine. A possible explanation for the conundrum of the “French Paradox”? Eur. J. Endocrinol. 138, 619-620. 
         Kyndt J A, Meyer T E, Cusanovich M A, Van Beeumen J J. (2002). Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein. FEES Lett. 512, 240-244. 
         LaGrange, D. C., Pretorius, I. S. and Van Zyl, W. H. (1997). Cloning of the  Bacillus pumilus  beta-xylosidase gene (xynB) and its expression in  Saccharomyces cerevisiae . Appl. Microbiol. Biotechnol. 47, 262-266. 
         Lin X., Kaul S., Rounsley S. D., Shea T. P., Benito M.-I., Town C. D., Fujii C. Y., Mason T. M., Bowman C. L., Barnstead M. E., Feldblyum T. V., Buell C. R., Ketchum K. A., Lee J. J., Ronning C. M., Koo H. L., Moffat K. S., Cronin L. A., Shen M., Pai G., Van Aken S., Umayam L., Tallon L. J., Gill J. E., Adams M. D., Carrera A. J., Creasy T. H., Goodman H. M., Somerville C. R., Copenhaver G. P., Preuss D., Nierman W. C., White O., Eisen J. A., Salzberg S. L., Fraser C. M., Venter J. C. (1999). Sequence and analysis of chromosome 2 of the plant  Arabidopsis thaliana . Nature 402, 761-768. 
         Lobo, R. A. (1995). Benefits and risks of estrogen replacement therapy. Am. J. Obstet. Gynecol. 173, 982-989. 
         Martin, V. J. J., Pitera, D. J., Withers, S. T., Newman, J. D. and Keasling, J. D. (2003). Engineering a mevalonate pathway in  Escherichia coli  for production of terpenoids. Nature biotechnology 21, 796-802. 
         Mizutani, M., Ohta, D. and Sato, R. (1997). Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from  Arabidopsis  and its expression manner in planta. Plant Physiol. 113, 755-763. 
         Ro, D. K., Mah, N., Ellis, B. E. and Douglas, C. J. (2001). Functional Characterization and Subcellular Localization of Poplar ( Populus trichocarpa×Populus deltoides ) Cinnamate 4-Hydroxylase. Plant Physiol. 126, 317-329. 
         Ro D. K., Douglas C. J. (2004). Reconstitution of the entry point of plant phenylpropanoid metabolism in yeast ( Saccharomyces cerevisiae ): implications for control of metabolic flux into the phenylpropanoid pathway. J. Biol. Chem. 279, 2600-2607. 
         Rosler J, Krekel F, Amrhein N, Schmid J. (1997). Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity. Plant Physiol. 113, 175-179. activity. Plant physiol. 113, 175-179. 
         Samappito, S., Page, J. E., Schmidt, J., De-Eknamkul, W. and Kutchan, T. M. (2003). Aromatic and pyrone polyketides synthesized by a stilbene synthase from  Rheum tataricum . Phytochemistry 62, 313-323. 
         Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y. 
         Schoppner, A.; Kindl, H. (1984) Purification and properties of a stilbene synthase from induced cell suspension cultures of peanut. J. Biol. Chem. 259, 6806-6811. 
         Seshime, Y., Juvvadi, P. R., Fujii, I. and Kitamoto, K. (2005). Genomic evidences for the existence of a phenylpropanoid metabolic pathway in  Aspergillus oryzae . Biochem Biophys Res Commun. 337, 747-51. 
         Urban P., Mignotte, C., Kazmaier M., Delorme F. And Pompon D. (1997). Cloning, Yeast Expression, and Characterization of the Coupling of Two Distantly Related  Arabidopsis thaliana  NADPH-Cytochrome 450 Reductases with P450 CYP73A5. J. Biol. Chem. 272, 19176-19186. 
         Verduyn C, Postma E, Scheffers W A, Van Dijken J P. 
         Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. 
         Yeast. 1992:8:501-17. 
         Watts, K. T., Lee, P. C. and Schmidt-Dannert, C. (2004). Exploring recombinant flavonoid biosynthesis in metabolically engineered  Escherichia coli . Chembiochem 5, 500-507. 
       
    
     The following is a summary of the nucleotide and amino acid sequences appearing herein: 
     SEQ ID NO: 1 is a nucleotide sequence from  Arabidopsis thaliana  encoding a phenylalanine ammonia lyase (PAL2). 
     SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1. 
     SEQ ID NO: 3 is a nucleotide sequence from  Arabidopsis thaliana  encoding a cinnamate 4-hydroxylase (C4H). 
     SEQ ID NO: 4 is the amino acid sequence encoded by SEQ ID NO: 3. 
     SEQ ID NO: 5 is a nucleotide sequence from  Arabidopsis thaliana  encoding a 4-coumarate:CoenzymeA ligase (4CL1). 
     SEQ ID NO: 6 is the amino acid sequence encoded by SEQ ID NO: 5. 
     SEQ ID NO: 7 is a nucleotide sequence from  Rheum tataricum  encoding a resveratrol synthase (VST). 
     SEQ ID NO: 8 is the amino acid sequence encoded by SEQ ID NO: 7. 
     SEQ ID NO: 9 is a nucleotide sequence from  Rheum tataricum  encoding a resveratrol synthase (VST), which is codon-optimized for expression in  S. cerevisiae.    
     SEQ ID NO: 10 is the amino acid sequence encoded by SEQ ID NO: 9. 
     SEQ ID NO: 11 is a nucleotide sequence from  Rhodobacter capsulatus  encoding a tyrosine ammonia lyase (TAL). 
     SEQ ID NO: 12 is the amino acid sequence encoded by SEQ ID NO: 11. 
     SEQ ID NO: 13 is a nucleotide sequence from  Rhodobacter capsulatus  encoding a tyrosine ammonia lyase (TAL), which is codon-optimized for expression in  S. cerevisiae.    
     SEQ ID NO: 14 is the amino acid sequence encoded by SEQ ID NO: 13. 
     SEQ ID NO: 15 is a nucleotide sequence from  S. cerevisiae  encoding a NADPH:cytochrome P450 reductase (CPR1). 
     SEQ ID NO: 16 is the amino acid sequence encoded by SEQ ID NO: 15. 
     SEQ ID NO: 17 is a nucleotide sequence from  Arabidopsis thalianus  encoding a NADPH:cytochrome P450 reductase (AR2). 
     SEQ ID NO: 18 is the amino acid sequence encoded by SEQ ID NO: 17. 
     SEQ ID NOs 19-32 are primer sequences appearing in Table 1, Example 1. 
     SEQ ID NOs 33-34 are primer sequences appearing in Example 16. 
     SEQ ID NOs 35-38 are primer sequences appearing in Example 17. 
     SEQ ID NOs 39-46 are primer sequences appearing in Example 36, Table 1. 
     SEQ ID NOs 47-58 are primer sequences appearing in Example 37, Table 2. 
     SEQ ID NO: 59 is the gene sequence appearing in Example 39. 
     SEQ ID NO: 60 is the first gene sequence appearing in Example 41. 
     SEQ ID NOs 61-62 are primer sequences appearing in Example 41. 
     SEQ ID NO: 63 is the VST1 amino acid sequence in Example 41. 
     SEQ ID NO. 64 is the second gene sequence appearing in Example 41.