Increased Resveratrol Production in Wines Using Engineered Wine Strains Saccharomyces cerevisiae EC1118 and Relaxed Antibiotic or Auxotrophic Selection Ping Sun, Jing-Long Liang, Lin-Zhi Kang, Xiao-Yan Huang, and Jia-Jun Huang Dept. of Bioengineering, College of Food Science, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou 510640, China
Zhi-Wei Ye, Li-Qiong Guo, and Jun-Fang Lin Dept. of Bioengineering, College of Food Science, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou 510640, China Inst. of Food Biotechnology, South China Agricultural University, Guangzhou 510640, China DOI 10.1002/btpr.2057 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com)
Resveratrol is a polyphenolic compound with diverse beneficial effects on human health. Red wine is the major dietary source of resveratrol but the amount that people can obtain from wines is limited. To increase the resveratrol production in wines, two expression vectors carrying 4-coumarate: coenzyme A ligase gene (4CL) from Arabidopsis thaliana and resveratrol synthase gene (RS) from Vitis vinifera were transformed into industrial wine strain Saccharomyces cerevisiae EC1118. When cultured with 1 mM p-coumaric acid, the engineered strains grown with and without the addition of antibiotics produced 8.249 and 3.317 mg/L of trans-resveratrol in the culture broth, respectively. Resveratrol content of the wine fermented with engineered strains was twice higher than that of the control, indicating that our engineered strains could increase the production of resveratrol during wine fermenC 2015 American Institute of Chemical Engineers Biotechnol. Prog., 000:000–000, tation. V 2015 Keywords: resveratrol, Saccharomyces cerevisiae EC1118, wine fermentation, heterogenous expression
Introduction Resveratrol (3,5,40 -trihydroxy-trans-stilbene) is a nonflavonoid, polyphenolic compound mainly found in grapes (Vitis vinifera), peanuts (Arachis hypogaea), and Japanese knotweed (Polygonum cuspidatum) in response to abiotic and biotic stress.1 Red wine is the major dietary source of resveratrol, which is known as an important factor in “French Paradox”.2 Subsequent studies confirmed that resveratrol possesses numerous beneficial effects on human health including cardioprotection, antioxidative, antiinflammatory, antineoplastic, and anti-aging activities, etc.3–7 Because of these health benefits, resveratrol has attracted extensive attention in health food industry, cosmetics industry, and pharmaceuticals industry. Nevertheless, the limited amount and high price of resveratrol hamper its application as a widespread nutraceutical. Resveratrol can be extracted from plant tissues but the degree of purity and product yields obtained by this method is very low, resulting in a large demand of plant materials and solvents, as well as a high cost. Biotechnology using microorganisms is an alternative useful, environmentally and ecologically friendly strategy for the production of resveratrol.8 Resveratrol biosynthesis Correspondence concerning this article should be addressed to J. -F. Lin at [email protected] and L.-Q. Guo at [email protected] C 2015 American Institute of Chemical Engineers V
branches from the phenylpropanoid pathway in certain plant species (Figure 1). As an intermediate, p-coumaric acid is converted into p-coumaroyl-CoA by 4-coumarate: coenzyme A ligase (4CL). Then resveratrol synthase (RS) catalyzes the condensation of resveratrol from one molecule of 4coumaroyl-CoA and three molecules of malonyl-CoA, which is involved in fatty acid biosynthesis.9 Thus, microorganisms are able to produce resveratrol by introducing two specific genes 4CL and RS from the stilbene pathway and using pcoumaric acid as a starting precursor. Saccharomyces cerevisiae has been a good platform for synthesizing resveratrol owing to its food grade status, high growth rate, and well-established use in fermentation reactions for industrial production of biomolecules. Given this, the 4CL216 gene from a hybrid poplar and the STS gene (equal to RS gene) from grapevine were introduced into laboratory strains of S.cerevisiae FY23 which were then grown with p-coumaric acid, producing 1.45 lg/L of glycosylated resveratrol.10 Similarly, S. cerevisiae CEN.PK113-3b harboring the 4CL2 gene of tobacco (Nicotiana tabacum) and the STS gene of V. vinifera was capable of accumulation of resveratrol (5.8 mg/L).11 The S.cerevisiae WAT11 strains, in which the 4CL gene from Arabidopsis thaliana and the STS gene from V. vinifera were co-expressed as a fusion protein, was able to convert p-coumaric acid into resveratrol, in amounts of 5.25 mg/L.12 Transformation of S. cerevisiae 1
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ored to obtain recombinant industrial wine strains of S. cerevisiae EC1118 with the ability to produce resveratrol during fermentation in wines without addition of antibiotics, which can be used as a starting point for further resveratrol industrial production and functional wine production.
Materials and Methods Strains and plasmids
Figure 1. Biosynthetic pathway of resveratrol in specific plants. The names of the key enzymes are abbreviated as follows: PAL, phenylalanine ammonia lyase; TAL, tyrosine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate: coenzyme A ligase; RS, resveratrol synthase.
W303-1A strains with the 4CL gene from A. thaliana and the STS gene from A. hypogaea enabled them to produce 3.1 mg/L resveratrol.13 Although resveratrol is found in wines, the amount that people could consume by this method is limited. Resveratrol is a phytoalexin and its content in grapes depends on the degree of stress exposure. Fungal and bacterial infections, preharvest chemical treatments, and ultra-violet radiation are potent factors that make resveratrol content increase in grapes and consequently in wines too.14 Using transgenic yeast is an alternative way to increase the resveratrol content in wines. To date, a majority of expression vectors used in metabolic engineering for resveratrol production in S. cerevisiae are episomal plasmids with auxotrophic markers. The deficiency of these vectors is that they rely on the presence of appropriate auxotrophic strains like S. cerevisiae FY23, CEN.PK113-3b, and W303-1A strains mentioned above. They are not suitable for wine strains, which are not auxotrophic but have a good performance in fermentation. To take economic and commercial conditions into account, it is not always possible to construct auxotrophic yeast strains derived from industrial strains. Episomal expression vectors with dominant drug resistance markers were first used to produce resveratrol in yeast by Sydor et al.15and they reported a high accumulation of resveratrol with use of antibiotics in rich medium. Nevertheless, the hygromycin adds to the cost and the use of antibiotics limits the practical implementation of resveratrol-producing yeast strains in industrial wine production. S. cerevisiae EC1118 is a commonly used wine yeast with excellent alcohol tolerance and good performance in wine fermentation. Herein, we endeav-
S. cerevisiae LALVIN EC1118 strain is a commercial wine yeast strain that isolated, studied and selected from Champagne fermentations then produced and commercialized by Lallemand (Ontario, Canada). Its ability to ferment at low temperature, good flocculation and excellent alcohol tolerance, make the EC1118 be one of the most widely used yeasts in the world and an excellent strain recommended for all types of wines, including sparkling, and late harvest wines and cider. The strain was grown at 25–30 C and routinely maintained at 4 C on YPD plates containing 2% glucose (w/v), 2% peptone, 1% yeast extract and 2% agar, and in glycerol stocks at 280 C.16 Escherichia coli DH5a was stocked at 80 C in our laboratory and cultivated at 37 C in LB liquid medium containing 0.5% yeast extract, 1% tryptone, and 1% sodium chloride or on LB plates with 2% agar. Plasmids pRS42K-4CL with G418 resistance harboring 4CL gene (Genbank accession number: NM 104046) and pRS42H-RS with hygromycin resistance harboring RS gene (Genbank accession number: KC417318) were constructed and conserved by our laboratory (Figure 2). Transformation of S. cerevisiae EC1118 S. cerevisiae EC1118 were transformed with the two plasmids pRS42K-4CL and pRS42H-RS together by LiAc/SS carrier DNA/PEG method.17 The recombinant strains were screened on the YPD plates containing 200 mg/L G418 and 200 mg/L hygromycin. PCR analysis was performed to confirm that both 4CL and RS genes were introduced in the recombinant strains successfully. Fermentation conditions Fresh yeast colonies containing plasmids pRS42K-4CL and pRS42H-RS were grown overnight in YPD liquid medium. Then cultures were harvested by centrifugation at 4000 rpm for 5 min and re-suspended to an OD600 5 1.0 in a flask with 50 mL fresh YPD medium containing p-coumaric acid, with or without antibiotics (200 mg/L G418 and 200 mg/L hygromycin). Shake flask fermentation were performed at 150 rpm, 28 C for an additional 5 days. As the control, S. cerevisiae EC1118 was cultured without antibiotics under the same condition. When fermentation finished, cultures were extracted with equal volume of ethyl acetate twice and dried by rotary evaporation. The residue was dissolved in 1 mL methanol and then subjected to High Performance Liquid Chromatography (HPLC) analysis. Selection of substrate concentration To determine the optimal substrate concentration for resveratrol production in culture medium, S. cerevisiae EC1118 strains transformed with pRS42K-4CL and pRS42H-RS were inoculated in 10 mL YPD medium and precultured overnight, then the cells were transferred into
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Figure 2. Maps of the expression vectors pRS42H-RS and pRS42K-4CL.
50 mL fresh medium supplemented with 0.5, 1, 3, and 5 mM of p-coumaric acid, respectively. To observe the change of cell density, 1 mL samples were collected at 12, 36, 60, 84, 108, 120, and 132 h to record OD at 600 nm. After 132 h of cultivation, samples were extracted with double volumes of ethyl acetate for p-coumaric acid and resveratrol quantification.
3A-C), indicating that both 4CL and RS genes were successfully expressed to proper functional enzymes that converted p-coumaric acid into resveratrol. Chromatogram A represents the authentic standard of trans-resveratrol; Chromatogram B represents extracts from the culture broth of recombinant; Chromatogram C represents extracts from the culture broth of the negative control.
Wine fermentation
Optimal substrate concentration
Kyoho grapes used for wine production were brought from local supermarket. Grapes were washed, stemmed, drained, and squeezed. Then the must, grape skins and seeds were mixed in a 1-L fermenting container leaving 1/4 space. Fermentation was performed at 28 C after inoculation of 3% yeast cells (107 mL21) in the mixture. Seven days later, fermentation was finished. Wine was obtained by filtering with gauzes and clarified naturally. Samples were collected and extracted with double volumes of ethyl acetate for HPLC analysis.
To determine the optimal substrate concentration, the recombinant strains were grown with different concentrations of p-coumaric acid. A growth inhibition was observed with increasing concentrations of p-coumaric acid but not obvious even if at a high concentration (5 mM). The increasing cell density of recombinant strains coupled with the consumption of p-coumaric acid resulted in the accumulation of resveratrol in the fermentation broth with time. After 132 h fermentation, the remanent p-coumaric acid and resveratrol produced in culture broth was measured by HPLC analysis (Figure 4). The result suggested that a majority of p-coumaric acid were not consumed during fermentation when supplemented with 5 mM p-coumaric acid. The addition of 1 mM p-coumaric acid yielded in the highest accumulation of resveratrol and the more addition of p-coumaric acid failed to increase the resveratrol production.
HPLC analysis of resveratrol and p-coumaric acid The above extracts were analyzed on an HPLC system using a phenomenex ODS reverse phase C-18 column (5 lm, 25034.6 mm) following previous methods12 with some modifications. For resveratrol detection, samples were separated by a 25 min linear gradient from 95% water / 5% acetonitrile to 30% water / 70% acetonitrile at a flow rate of 0.9 mL min21. For p-coumaric acid detection, samples were separated with 65%, 0.1% acetic acid water : 35% acetonitrile. Their characteristic UV absorption spectra were 306 and 308 nm, respectively. The injection volume was 20 lL. The metabolites were confirmed by the retention time compared to that of authentic standards.
Results Resveratrol production in recombinant S. cerevisiae The recombinant strains S. cerevisiae EC 1118 carrying pRS42K-4CL and pRS42H-RS were cultured in liquid YPD medium supplemented with p-coumaric acid. When analyzing the culture broth of recombinant strains by HPLC, a peak was detected at the same retention time as trans-resveratrol standard while there was no peak presented in the corresponding HPLC chromatogram of negative control (Figure
Stability of episomal vectors in resveratrol production The same recombinant strain was grown in liquid culture medium containing 1mM p-coumaric acid with or without addition of G418 and hygromycin. It was found that the addition of antibiotics in culture medium would inhibit the propagation of yeast cells. The cell density of engineered strains was not as high as that of the control strain, the reason for which was that the replication and expression of episomal vectors slowed down the growth of yeast cells. The resveratrol production in the culture broth without antibiotics is 3.317 mg/L while that in the medium with antibiotics reached 8.249 mg/L. The result showed that episomal vectors can autonomously replicate and effectively express target genes in recombinant strains without addition of corresponding antibiotics and keep stably in a certain extent. However, the decrease of resveratrol implied that episomal plasmids might be partially lost during continuous passage culture without selection pressures. A high growth rate was
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Figure 3. HPLC analyses of resveratrol produced by engineered S. cerevisiae EC1118.
not corresponding to a high yield of target product, stating that a nonproducing population accumulating with time when cultured without antibiotics (Figure 5).
Resveratrol production in wine fermentation To test their ability to produce resveratrol during wine production, control strains EC1118 and engineered strains
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Figure 4. Effects of p-coumaric acid concentration (from 0.5 to 5 mM) on resveratrol production. Samples were collected after 132 h fermentation. The added p-coumaric acid represents the amount of p-coumaric acid added in culture medium at the beginning of the fermentation. The remaining p-coumaric acid represents the amount of p-coumaric acid remained in culture medium after 132 h fermentation.
containing 4CL and RS genes were inoculated in grape juice, respectively. After 7 days fermentation at 28 C, resveratrol content of the wine fermented with engineered strains was about 0.4028 mg/L, twice higher than that of the control (0.1997 mg/L). The wine fermentation performed in this study was aimed to test resveratrol production capacity of the transgenic wine industrial yeast strain S. cerevisiae EC1118, therefore the processing technology was simplified and not a complete one that adopted in wine industry. Without a suitable processing technology, the content of resveratrol of the wine fermented with engineered strains was still low when compared to wines that commercially available.14 Nevertheless, the result indicated that our engineered strains could increase the production of resveratrol during wine fermentation.
Discussion In this study, the growth inhibition of yeast cells was not obvious at a high concentration of p-coumaric acid, which was quite different with the previous findings.18 It was probably owing to the overnight preculture. The cells were grown in liquid medium without p-coumaric acid and reached the logarithmic phase with a high viability before being added into fresh culture broth that supplemented with p-coumaric acid. Furthermore, there might be two reasons for no more accumulation of resveratrol in recombinant yeast when added more p-coumaric acid. One was that a PAD1 gene encoding phenylacrylic acid decarboxylase in S. cerevisiae converted part of p-coumaric acid into 4-vinylphenol and further into 4-ethylphenol, which was considered as a taint flavor in wines, by vinylphenol reductase.19–21 Besides, the limited content of malonyl-CoA, another substrate for resveratrol synthesis, imposed restrictions on the production of resveratrol. According to the previous report, the presence of p-coumaric acid can generally reach a concentration of 60 mg/L in grape juice.22 The engineered wine strain could use p-coumaric acid as substrates to yield resveratrol, developing a competitive mechanism of p-coumaric acid in winemaking, which increased the resveratrol content in wines on one hand and probably decreased the 4-ethylphenol on the other hand. Commercially available expression vectors of the pESC series were commonly used to produce resveratrol in
Figure 5.
Growth curves of engineered strain cultured with or without addition of antibiotics. The control strain was grown under the same conditions without addition of antibiotics.
yeast,12,13,18,23,24 which rely on the presence of an appropriate auxotrophic marker and need the induction of galactose. The expression vectors used in our study were constructed based on two shuttle vectors pRS42K and pRS42H with Geneticin resistance and hygromycin B resistance, respectively.25 These vectors allow one to produce resveratrol in industrial wine strains and other natural yeast isolates. Sydor et al.15 used similar expression vectors in a industrial Brazilian sugar cane-fermenting yeast and got a high accumulation of resveratrol. However, it is not sure whether it has a good performance in wine-making without addition of antibiotics. S. cerevisiae EC1118 is a wine strain with high performance in fermentation and brewing. The accumulation of resveratrol in culture broth of the engineered S. cerevisiae EC1118 reached 8.249 mg/L, which is higher than that of previous reports studied in a similar way.10–13,23 Moreover, the target genes were droved by a constitutive promoter (TEF1p) which is no need to be induced. The stability of episomal vectors during fermentation was tested in this study and the result confirmed that engineered strains could produce resveratrol during fermentation with or without selection
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pressures. These properties make it available to increase the levels of resveratrol in wine fermentation and further improve the nutritional value of wines to meet the demands of a rising number of health-conscious wine consumers.26 However, the further essential research on safety evaluation and sensory analyses must be carried out to make sure that new wines are not detrimental for human beings. In conclusion, this work has explored the possibility of resveratrol production in nonauxotrophic industrial yeast strains with or without antibiotics, and further increased the content of resveratrol in wine by using the engineered wine strains. It has also laid a foundation for resveratrol industrial production and broadened the application scope of yeasts in production of bioactive compounds in food industry.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 31272217, 31372116); the Projects of Science and Technology of Guangdong Province (Grant No. 2013B010404041).
Literature Cited 1. Jeandet P, Douillet-Breuil A-C, Bessis R, Debord S, Sbaghi M, Adrian M. Phytoalexins from the Vitaceae: Biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism. J Agric Food Chem. 2002;50:2731–2741. 2. Kopp P. Resveratrol, a phytoestrogen found in red wine. A possible explanation for the conundrum of the “French paradox”? Eur J Endocrinol. 1998;138:619–620. 3. Jang M. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science. 1997;275:218–220. 4. Roberti M, Pizzirani D, Simoni D, Rondanin R, Baruchello R, Bonora C, Buscemi F, Grimaudo S, Tolomeo M. Synthesis and biological evaluation of resveratrol and analogues as apoptosisinducing agents. J Med Chem. 2003;46:3546–3554. 5. Kundu JK, Surh Y-J. Cancer chemopreventive and therapeutic potential of resveratrol: mechanistic perspectives. Cancer Lett. 2008;269:243–261. 6. Csiszar A. Anti-inflammatory effects of resveratrol: possible role in prevention of age-related cardiovascular disease. Ann N Y Acad Sci. 2011;1215:117–122. 7. Das DK, Mukherjee S, Ray D. Erratum to: resveratrol and red wine, healthy heart and longevity. Heart Fail Rev. 2011;16:425–435. 8. Donnez D, Jeandet P, Clement C, Courot E. Bioproduction of resveratrol and stilbene derivatives by plant cells and microorganisms. Trends Biotechnol. 2009;27:706–713. 9. Halls C, Yu O. Potential for metabolic engineering of resveratrol biosynthesis. Trends Biotechnol. 2008;26:77–81. 10. Becker J, Armstrong G, Vandermerwe M, Lambrechts M, Vivier M, Pretorius I. Metabolic engineering of for the synthesis of the wine-related antioxidant resveratrol. FEMS Yeast Res. 2003;4:79–85. 11. Beekwilder J, Wolswinkel R, Jonker H, Hall R, de Vos CHR, Bovy A. Production of resveratrol in recombinant microorganisms. Appl Environ Microbiol. 2006;72:5670–5672.
12. Zhang Y, Li S-Z, Li J, Pan X, Cahoon RE, Jaworski JG, Wang X, Jez JM, Chen F, Yu O. Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and Mammalian cells. J Am Chem Soc. 2006;128:13030–13031. 13. Shin S-Y, Han NS, Park Y-C, Kim M-D, Seo J-H. Production of resveratrol from p-coumaric acid in recombinant Saccharomyces cerevisiae expressing 4-coumarate:coenzyme A ligase and stilbene synthase genes. Enzyme Microb Technol. 2011;48: 48–53. 14. Fernandez-Mar MI, Mateos R, Garcıa-Parrilla MC, Puertas B, Cantos-Villar E. Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review. Food Chem. 2012;130: 797–813. 15. Sydor T, Schaffer S, Boles E. Considerable increase in resveratrol production by recombinant industrial yeast strains with use of rich medium. Appl Environ Microbiol. 2010;76:3361–3363. 16. Quiros M, Martınez-Moreno R, Albiol J, Morales P, VazquezLima F, Barreiro-Vazquez A, Ferrer P, Gonzalez R. Metabolic flux analysis during the exponential growth phase of Saccharomyces cerevisiae in wine fermentations. PLoS One. 2013;8: e71909. 17. Gietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007; 2:31–34. 18. Wang Y, Halls C, Zhang J, Matsuno M, Zhang Y, Yu O. Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering. Metab Eng. 2011;13:455– 463. 19. Salameh D, Brandam C, Medawar W, Lteif R, Strehaiano P. Highlight on the problems generated by p-coumaric acid analysis in wine fermentations. Food Chem. 2008;107:1661–1667. 20. Mukai N, Masaki K, Fujii T, Kawamukai M, Iefuji H. PAD1 and FDC1 are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. J Biosci Bioeng. 2010;109: 564–569. 21. Vanbeneden N, Gils F, Delvaux F, Delvaux FR. Formation of 4-vinyl and 4-ethyl derivatives from hydroxycinnamic acids: Occurrence of volatile phenolic flavour compounds in beer and distribution of Pad1-activity among brewing yeasts. Food Chem. 2008;107:221–230. 22. Chatonnet P, Viala C, Dubourdieu D. Influence of polyphenolic components of red wines on the microbial synthesis of volatile phenols. Am J Enol Vitic. 1997;48:443–448. 23. Trantas E, Panopoulos N, Ververidis F. Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae. Metab Eng. 2009;11:355–66. 24. Wang Y, Yu O. Synthetic scaffolds increased resveratrol biosynthesis in engineered yeast cells. J Biotechnol. 2012;157:258– 260. 25. Taxis C, Knop M. System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae. Biotechniques. 2006;40:73–78. 26. Barreiro-Hurle J, Colombo S, Cantos-Villar E. Is there a market for functional wines? Consumer preferences and willingness to pay for resveratrol-enriched red wine. Food Qual Prefer. 2008; 19:360–371. Manuscript received Feb. 14, 2014, and revision received Feb. 6, 2015.
Zhi-Wei Ye, Li-Qiong Guo, and Jun-Fang Lin Dept. of Bioengineering, College of Food Science, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou 510640, China Inst. of Food Biotechnology, South China Agricultural University, Guangzhou 510640, China DOI 10.1002/btpr.2057 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com)
Resveratrol is a polyphenolic compound with diverse beneficial effects on human health. Red wine is the major dietary source of resveratrol but the amount that people can obtain from wines is limited. To increase the resveratrol production in wines, two expression vectors carrying 4-coumarate: coenzyme A ligase gene (4CL) from Arabidopsis thaliana and resveratrol synthase gene (RS) from Vitis vinifera were transformed into industrial wine strain Saccharomyces cerevisiae EC1118. When cultured with 1 mM p-coumaric acid, the engineered strains grown with and without the addition of antibiotics produced 8.249 and 3.317 mg/L of trans-resveratrol in the culture broth, respectively. Resveratrol content of the wine fermented with engineered strains was twice higher than that of the control, indicating that our engineered strains could increase the production of resveratrol during wine fermenC 2015 American Institute of Chemical Engineers Biotechnol. Prog., 000:000–000, tation. V 2015 Keywords: resveratrol, Saccharomyces cerevisiae EC1118, wine fermentation, heterogenous expression
Introduction Resveratrol (3,5,40 -trihydroxy-trans-stilbene) is a nonflavonoid, polyphenolic compound mainly found in grapes (Vitis vinifera), peanuts (Arachis hypogaea), and Japanese knotweed (Polygonum cuspidatum) in response to abiotic and biotic stress.1 Red wine is the major dietary source of resveratrol, which is known as an important factor in “French Paradox”.2 Subsequent studies confirmed that resveratrol possesses numerous beneficial effects on human health including cardioprotection, antioxidative, antiinflammatory, antineoplastic, and anti-aging activities, etc.3–7 Because of these health benefits, resveratrol has attracted extensive attention in health food industry, cosmetics industry, and pharmaceuticals industry. Nevertheless, the limited amount and high price of resveratrol hamper its application as a widespread nutraceutical. Resveratrol can be extracted from plant tissues but the degree of purity and product yields obtained by this method is very low, resulting in a large demand of plant materials and solvents, as well as a high cost. Biotechnology using microorganisms is an alternative useful, environmentally and ecologically friendly strategy for the production of resveratrol.8 Resveratrol biosynthesis Correspondence concerning this article should be addressed to J. -F. Lin at [email protected] and L.-Q. Guo at [email protected] C 2015 American Institute of Chemical Engineers V
branches from the phenylpropanoid pathway in certain plant species (Figure 1). As an intermediate, p-coumaric acid is converted into p-coumaroyl-CoA by 4-coumarate: coenzyme A ligase (4CL). Then resveratrol synthase (RS) catalyzes the condensation of resveratrol from one molecule of 4coumaroyl-CoA and three molecules of malonyl-CoA, which is involved in fatty acid biosynthesis.9 Thus, microorganisms are able to produce resveratrol by introducing two specific genes 4CL and RS from the stilbene pathway and using pcoumaric acid as a starting precursor. Saccharomyces cerevisiae has been a good platform for synthesizing resveratrol owing to its food grade status, high growth rate, and well-established use in fermentation reactions for industrial production of biomolecules. Given this, the 4CL216 gene from a hybrid poplar and the STS gene (equal to RS gene) from grapevine were introduced into laboratory strains of S.cerevisiae FY23 which were then grown with p-coumaric acid, producing 1.45 lg/L of glycosylated resveratrol.10 Similarly, S. cerevisiae CEN.PK113-3b harboring the 4CL2 gene of tobacco (Nicotiana tabacum) and the STS gene of V. vinifera was capable of accumulation of resveratrol (5.8 mg/L).11 The S.cerevisiae WAT11 strains, in which the 4CL gene from Arabidopsis thaliana and the STS gene from V. vinifera were co-expressed as a fusion protein, was able to convert p-coumaric acid into resveratrol, in amounts of 5.25 mg/L.12 Transformation of S. cerevisiae 1
2
Biotechnol. Prog., 2015, Vol. 00, No. 00
ored to obtain recombinant industrial wine strains of S. cerevisiae EC1118 with the ability to produce resveratrol during fermentation in wines without addition of antibiotics, which can be used as a starting point for further resveratrol industrial production and functional wine production.
Materials and Methods Strains and plasmids
Figure 1. Biosynthetic pathway of resveratrol in specific plants. The names of the key enzymes are abbreviated as follows: PAL, phenylalanine ammonia lyase; TAL, tyrosine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate: coenzyme A ligase; RS, resveratrol synthase.
W303-1A strains with the 4CL gene from A. thaliana and the STS gene from A. hypogaea enabled them to produce 3.1 mg/L resveratrol.13 Although resveratrol is found in wines, the amount that people could consume by this method is limited. Resveratrol is a phytoalexin and its content in grapes depends on the degree of stress exposure. Fungal and bacterial infections, preharvest chemical treatments, and ultra-violet radiation are potent factors that make resveratrol content increase in grapes and consequently in wines too.14 Using transgenic yeast is an alternative way to increase the resveratrol content in wines. To date, a majority of expression vectors used in metabolic engineering for resveratrol production in S. cerevisiae are episomal plasmids with auxotrophic markers. The deficiency of these vectors is that they rely on the presence of appropriate auxotrophic strains like S. cerevisiae FY23, CEN.PK113-3b, and W303-1A strains mentioned above. They are not suitable for wine strains, which are not auxotrophic but have a good performance in fermentation. To take economic and commercial conditions into account, it is not always possible to construct auxotrophic yeast strains derived from industrial strains. Episomal expression vectors with dominant drug resistance markers were first used to produce resveratrol in yeast by Sydor et al.15and they reported a high accumulation of resveratrol with use of antibiotics in rich medium. Nevertheless, the hygromycin adds to the cost and the use of antibiotics limits the practical implementation of resveratrol-producing yeast strains in industrial wine production. S. cerevisiae EC1118 is a commonly used wine yeast with excellent alcohol tolerance and good performance in wine fermentation. Herein, we endeav-
S. cerevisiae LALVIN EC1118 strain is a commercial wine yeast strain that isolated, studied and selected from Champagne fermentations then produced and commercialized by Lallemand (Ontario, Canada). Its ability to ferment at low temperature, good flocculation and excellent alcohol tolerance, make the EC1118 be one of the most widely used yeasts in the world and an excellent strain recommended for all types of wines, including sparkling, and late harvest wines and cider. The strain was grown at 25–30 C and routinely maintained at 4 C on YPD plates containing 2% glucose (w/v), 2% peptone, 1% yeast extract and 2% agar, and in glycerol stocks at 280 C.16 Escherichia coli DH5a was stocked at 80 C in our laboratory and cultivated at 37 C in LB liquid medium containing 0.5% yeast extract, 1% tryptone, and 1% sodium chloride or on LB plates with 2% agar. Plasmids pRS42K-4CL with G418 resistance harboring 4CL gene (Genbank accession number: NM 104046) and pRS42H-RS with hygromycin resistance harboring RS gene (Genbank accession number: KC417318) were constructed and conserved by our laboratory (Figure 2). Transformation of S. cerevisiae EC1118 S. cerevisiae EC1118 were transformed with the two plasmids pRS42K-4CL and pRS42H-RS together by LiAc/SS carrier DNA/PEG method.17 The recombinant strains were screened on the YPD plates containing 200 mg/L G418 and 200 mg/L hygromycin. PCR analysis was performed to confirm that both 4CL and RS genes were introduced in the recombinant strains successfully. Fermentation conditions Fresh yeast colonies containing plasmids pRS42K-4CL and pRS42H-RS were grown overnight in YPD liquid medium. Then cultures were harvested by centrifugation at 4000 rpm for 5 min and re-suspended to an OD600 5 1.0 in a flask with 50 mL fresh YPD medium containing p-coumaric acid, with or without antibiotics (200 mg/L G418 and 200 mg/L hygromycin). Shake flask fermentation were performed at 150 rpm, 28 C for an additional 5 days. As the control, S. cerevisiae EC1118 was cultured without antibiotics under the same condition. When fermentation finished, cultures were extracted with equal volume of ethyl acetate twice and dried by rotary evaporation. The residue was dissolved in 1 mL methanol and then subjected to High Performance Liquid Chromatography (HPLC) analysis. Selection of substrate concentration To determine the optimal substrate concentration for resveratrol production in culture medium, S. cerevisiae EC1118 strains transformed with pRS42K-4CL and pRS42H-RS were inoculated in 10 mL YPD medium and precultured overnight, then the cells were transferred into
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Figure 2. Maps of the expression vectors pRS42H-RS and pRS42K-4CL.
50 mL fresh medium supplemented with 0.5, 1, 3, and 5 mM of p-coumaric acid, respectively. To observe the change of cell density, 1 mL samples were collected at 12, 36, 60, 84, 108, 120, and 132 h to record OD at 600 nm. After 132 h of cultivation, samples were extracted with double volumes of ethyl acetate for p-coumaric acid and resveratrol quantification.
3A-C), indicating that both 4CL and RS genes were successfully expressed to proper functional enzymes that converted p-coumaric acid into resveratrol. Chromatogram A represents the authentic standard of trans-resveratrol; Chromatogram B represents extracts from the culture broth of recombinant; Chromatogram C represents extracts from the culture broth of the negative control.
Wine fermentation
Optimal substrate concentration
Kyoho grapes used for wine production were brought from local supermarket. Grapes were washed, stemmed, drained, and squeezed. Then the must, grape skins and seeds were mixed in a 1-L fermenting container leaving 1/4 space. Fermentation was performed at 28 C after inoculation of 3% yeast cells (107 mL21) in the mixture. Seven days later, fermentation was finished. Wine was obtained by filtering with gauzes and clarified naturally. Samples were collected and extracted with double volumes of ethyl acetate for HPLC analysis.
To determine the optimal substrate concentration, the recombinant strains were grown with different concentrations of p-coumaric acid. A growth inhibition was observed with increasing concentrations of p-coumaric acid but not obvious even if at a high concentration (5 mM). The increasing cell density of recombinant strains coupled with the consumption of p-coumaric acid resulted in the accumulation of resveratrol in the fermentation broth with time. After 132 h fermentation, the remanent p-coumaric acid and resveratrol produced in culture broth was measured by HPLC analysis (Figure 4). The result suggested that a majority of p-coumaric acid were not consumed during fermentation when supplemented with 5 mM p-coumaric acid. The addition of 1 mM p-coumaric acid yielded in the highest accumulation of resveratrol and the more addition of p-coumaric acid failed to increase the resveratrol production.
HPLC analysis of resveratrol and p-coumaric acid The above extracts were analyzed on an HPLC system using a phenomenex ODS reverse phase C-18 column (5 lm, 25034.6 mm) following previous methods12 with some modifications. For resveratrol detection, samples were separated by a 25 min linear gradient from 95% water / 5% acetonitrile to 30% water / 70% acetonitrile at a flow rate of 0.9 mL min21. For p-coumaric acid detection, samples were separated with 65%, 0.1% acetic acid water : 35% acetonitrile. Their characteristic UV absorption spectra were 306 and 308 nm, respectively. The injection volume was 20 lL. The metabolites were confirmed by the retention time compared to that of authentic standards.
Results Resveratrol production in recombinant S. cerevisiae The recombinant strains S. cerevisiae EC 1118 carrying pRS42K-4CL and pRS42H-RS were cultured in liquid YPD medium supplemented with p-coumaric acid. When analyzing the culture broth of recombinant strains by HPLC, a peak was detected at the same retention time as trans-resveratrol standard while there was no peak presented in the corresponding HPLC chromatogram of negative control (Figure
Stability of episomal vectors in resveratrol production The same recombinant strain was grown in liquid culture medium containing 1mM p-coumaric acid with or without addition of G418 and hygromycin. It was found that the addition of antibiotics in culture medium would inhibit the propagation of yeast cells. The cell density of engineered strains was not as high as that of the control strain, the reason for which was that the replication and expression of episomal vectors slowed down the growth of yeast cells. The resveratrol production in the culture broth without antibiotics is 3.317 mg/L while that in the medium with antibiotics reached 8.249 mg/L. The result showed that episomal vectors can autonomously replicate and effectively express target genes in recombinant strains without addition of corresponding antibiotics and keep stably in a certain extent. However, the decrease of resveratrol implied that episomal plasmids might be partially lost during continuous passage culture without selection pressures. A high growth rate was
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Figure 3. HPLC analyses of resveratrol produced by engineered S. cerevisiae EC1118.
not corresponding to a high yield of target product, stating that a nonproducing population accumulating with time when cultured without antibiotics (Figure 5).
Resveratrol production in wine fermentation To test their ability to produce resveratrol during wine production, control strains EC1118 and engineered strains
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Figure 4. Effects of p-coumaric acid concentration (from 0.5 to 5 mM) on resveratrol production. Samples were collected after 132 h fermentation. The added p-coumaric acid represents the amount of p-coumaric acid added in culture medium at the beginning of the fermentation. The remaining p-coumaric acid represents the amount of p-coumaric acid remained in culture medium after 132 h fermentation.
containing 4CL and RS genes were inoculated in grape juice, respectively. After 7 days fermentation at 28 C, resveratrol content of the wine fermented with engineered strains was about 0.4028 mg/L, twice higher than that of the control (0.1997 mg/L). The wine fermentation performed in this study was aimed to test resveratrol production capacity of the transgenic wine industrial yeast strain S. cerevisiae EC1118, therefore the processing technology was simplified and not a complete one that adopted in wine industry. Without a suitable processing technology, the content of resveratrol of the wine fermented with engineered strains was still low when compared to wines that commercially available.14 Nevertheless, the result indicated that our engineered strains could increase the production of resveratrol during wine fermentation.
Discussion In this study, the growth inhibition of yeast cells was not obvious at a high concentration of p-coumaric acid, which was quite different with the previous findings.18 It was probably owing to the overnight preculture. The cells were grown in liquid medium without p-coumaric acid and reached the logarithmic phase with a high viability before being added into fresh culture broth that supplemented with p-coumaric acid. Furthermore, there might be two reasons for no more accumulation of resveratrol in recombinant yeast when added more p-coumaric acid. One was that a PAD1 gene encoding phenylacrylic acid decarboxylase in S. cerevisiae converted part of p-coumaric acid into 4-vinylphenol and further into 4-ethylphenol, which was considered as a taint flavor in wines, by vinylphenol reductase.19–21 Besides, the limited content of malonyl-CoA, another substrate for resveratrol synthesis, imposed restrictions on the production of resveratrol. According to the previous report, the presence of p-coumaric acid can generally reach a concentration of 60 mg/L in grape juice.22 The engineered wine strain could use p-coumaric acid as substrates to yield resveratrol, developing a competitive mechanism of p-coumaric acid in winemaking, which increased the resveratrol content in wines on one hand and probably decreased the 4-ethylphenol on the other hand. Commercially available expression vectors of the pESC series were commonly used to produce resveratrol in
Figure 5.
Growth curves of engineered strain cultured with or without addition of antibiotics. The control strain was grown under the same conditions without addition of antibiotics.
yeast,12,13,18,23,24 which rely on the presence of an appropriate auxotrophic marker and need the induction of galactose. The expression vectors used in our study were constructed based on two shuttle vectors pRS42K and pRS42H with Geneticin resistance and hygromycin B resistance, respectively.25 These vectors allow one to produce resveratrol in industrial wine strains and other natural yeast isolates. Sydor et al.15 used similar expression vectors in a industrial Brazilian sugar cane-fermenting yeast and got a high accumulation of resveratrol. However, it is not sure whether it has a good performance in wine-making without addition of antibiotics. S. cerevisiae EC1118 is a wine strain with high performance in fermentation and brewing. The accumulation of resveratrol in culture broth of the engineered S. cerevisiae EC1118 reached 8.249 mg/L, which is higher than that of previous reports studied in a similar way.10–13,23 Moreover, the target genes were droved by a constitutive promoter (TEF1p) which is no need to be induced. The stability of episomal vectors during fermentation was tested in this study and the result confirmed that engineered strains could produce resveratrol during fermentation with or without selection
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pressures. These properties make it available to increase the levels of resveratrol in wine fermentation and further improve the nutritional value of wines to meet the demands of a rising number of health-conscious wine consumers.26 However, the further essential research on safety evaluation and sensory analyses must be carried out to make sure that new wines are not detrimental for human beings. In conclusion, this work has explored the possibility of resveratrol production in nonauxotrophic industrial yeast strains with or without antibiotics, and further increased the content of resveratrol in wine by using the engineered wine strains. It has also laid a foundation for resveratrol industrial production and broadened the application scope of yeasts in production of bioactive compounds in food industry.
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 31272217, 31372116); the Projects of Science and Technology of Guangdong Province (Grant No. 2013B010404041).
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