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Synthetic scaffolds increased resveratrol biosynthesis in engineered yeast cells
Journal of Biotechnology 157 (2012) 258–260
Contents lists available at SciVerse ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
Synthetic scaffolds increased resveratrol biosynthesis in engineered yeast cells Yechun Wang, Oliver Yu ∗ Donald Danforth Plant Science Center, 975 North Warson Road, Saint Louis, MO 63132, United States
a r t i c l e
i n f o
Article history: Received 27 August 2011 Received in revised form 28 October 2011 Accepted 2 November 2011 Available online 9 November 2011 Keywords: Resveratrol Metabolic engineering Synthetic scaffold
a b s t r a c t Resveratrol is a polyphenolic compound produced by a few higher plants when under attack by pathogens such as bacteria or fungi. Besides antioxidant benefits to humans, this health-promoting compound has been reported to extend longevity in yeasts, flies, worms, fishes and obesity mice. Here we utilized the synthetic scaffolds strategy to improve resveratrol production in Saccharomyces cerevisiae. We observed a 5.0-fold improvement over the non-scaffolded control, and a 2.7-fold increase over the previous reported with fusion protein. This work demonstrated the synthetic scaffolds can be used for the optimization of engineered metabolic pathway. © 2011 Elsevier B.V. All rights reserved.
Resveratrol is a naturally occurring defense compound produced by a few plant species in response to pathogen attacks. Resveratrol has significant physiological effects on human and other animals, which may be related to its antioxidant activities. Most importantly, when large quantities of resveratrol were fed to laboratory animals, the compound significantly increased the longevity of the subjects, including roundworm, fruit fly, fish, and mouse (Bauer et al., 2004; Howitz et al., 2003; Valenzano et al., 2006). Although the mechanism of this effect is still under investigation, the specific effects of resveratrol on longevity attracted extensive attentions in aging research. The health-promoting properties of resveratrol have stimulated the development of heterologous expression system based on Saccharomyces cerevisiae and Escherichia coli. Detail progresses have been described in previous review papers (Halls and Yu, 2008; Wang et al., 2010, 2011a). In recent years, a new scaffolding strategy has been successfully applied on increasing glucaric acid and mevalonate biosynthetic flux (Dueber et al., 2009; Moon et al., 2010). The technology is based on engineered synthetic protein scaffolds. These scaffolds interact with the enzymes of natural compound biosynthesis pathway via small peptide ligands in a programmable manner that improve production of end-product. By taking advantage of these scaffolds, a 77-fold improvement of mevalonate production was achieved (Dueber et al., 2009) and 5-fold increase in glucaric acid titers over the non-scaffolded control (Moon et al., 2010). Here we constructed the scaffolds to recruit the enzymes (4-coumarate:CoA ligase (4CL1) and stilbene synthase (STS)) of resveratrol biosynthesis pathway to improve resveratrol production in yeast cells.
∗ Corresponding author. Tel.: +1 314 587 1441; fax: +1 314 587 1541. E-mail address: [email protected] (O. Yu). 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.11.003
Nine scaffold plasmids consisting of GBD, SH3 and PDZ protein interaction domains were kindly provided by Dr. John Dueber from Department of Bioengineering, University of California, Berkeley, CA. The scaffold plasmids were cut by BglII/XhoI, and the small scaffold fragments (from 771 bp to 2319 bp) were subcloned into the yeast expression vector pESC-TRP carrying galatose-inducible (Gal1) promoter, downstream of a BamHI/XhoI multi-cloning site. The information of nine scaffolds yeast expression vectors were showed in Table 1 (Dueber et al., 2009). For building ligand-fusion proteins, the sequences of SH3 and PDZ, were synthesized by Genewiz Inc. (South Plainfield, NJ), the ligands SH3 and PDZ were cloned into other yeast expression vector pESC-HIS and pESC-URA (Stratagene), respectively, under the control of a Gal1 promoter at the multi-cloning sites. The resultant plasmids were designated as pESC-HIS-SH3 and pESC-URA-PDZ. The plasmid encoding the resveratrol pathway enzymes 4CL1 and STS were amplified from our previous plasmid (Zhang et al., 2006) by PCR using primers with BamHI/XhoI restriction enzyme sites. The 4CL1 gene was inserted into the pESC-HIS-SH3 vector, and STS into the pESC-URA-PDZ vector (as pESC-HIS-4CL1-SH3 and pESC-URA-STS-PDZ, respectively). pESC-HIS-4CL1-SH3, pESC-URA-STS-PDZ and pESC-scaffolds, were transformed into WAT11 cells (Pompon et al., 1996; Urban et al., 1997) made competent cells with the Frozen-EZ Yeast kit (Zymo Research, Orange, CA). pESC-4CL1-SH3, pESC-STS-PDZ, and pESC-TRP empty vector without the ligands were also transformed into WAT11 cells as controls. Yeast cultures (20 mL) were grown overnight at 30 ◦ C in appropriate SD drop-out liquid media (US Biological, MA) containing 2% (w/v) glucose. Cultures were then collected by centrifugation and washed three times in sterile deionized water. Cells were resuspended in induction medium containing 2% galatose to an OD600 of 1.0. The substrate p-coumaric acid was added to a final concentration of 100 M, and re-supplied
Y. Wang, O. Yu / Journal of Biotechnology 157 (2012) 258–260
Fig. 1. Effects of nine synthetic scaffolds on resveratrol production in yeast cells. The maximum production of resveratrol was observed in G1S2P4 construct, in which the resveratrol level is 5.0-fold higher than the non-scaffolded control (G0S0P0). Data are the averages of three biological repeats.
every 24 h (100 M). Approximately 36 h after induction, aliquots (400 L) were extracted with 800 L ethyl acetate. Extracts were dried with an Eppendorf VacufugeTM (Eppendorf Scientific, MA) at room temperature and re-dissolved in 80% methanol for HPLC analysis (Wang et al., 2011b). To determine whether these scaffolds work on resveratrol biosynthesis in yeast cells, we cloned the nine scaffold configurations into nine yeast expression vectors. Scaffolds were constructed
259
by assembling three protein–protein interaction domains linked together by nine residue Gly-Ser linkers. The binding domains SH3 and PDZ were used to target 4CL and STS, respectively. As expected, yeast cells transformed with pESC-HIS-4CL-SH3, pESCURA-STS-PDZ and pESC-scaffold vectors can successfully produce resveratrol in large quantities when fed with the substrate pcoumaric acid, based on HPLC analysis (Figs. 1 and 2). Compared to the controls yeast cells transformed with pESC-HIS-4CL-SH3, pESCURA-STS-PDZ and the pESC-TRP vector (without the scaffolds), a more than 5.0-fold increase in the resveratrol production (6.7 mg/L) was observed for the optimal scaffold (GBD1 SH32 PDZ4 ) at 36 h after galactose induction (Fig. 1). After longer incubation, the scaffolding construct still have more than 2-fold increase in resveratrol production (14.4 mg/L) at 96 h after induction (Supplementary Table 1). More importantly, this yield is 2.7-fold higher than the previously reported with fusion proteins (Zhang et al., 2006), suggesting the more flexible joining of the two enzymes by scaffolding appear to be more productive than the more rigid fusion protein constructs. Production improvements depended on the number of domain repeats in the scaffolds. For example, scaffolds GBD1 SH32 PDZ4 and GBD1 SH34 PDZ4 differ by two SH3 domains resulted in producing different resveratrol titers: 5.17-fold versus 1.4-fold, respectively. Compared to the non-scaffold GBD0 SH30 PDZ0 , scaffolds GBD1 SH34 PDZ1 and GBD1 SH34 PDZ4 showed low increase in resveratrol production (1.23-fold and 1.4-fold). Apparently, the number of binding domains had dramatic impacts on increasing pathway flux. The enzymes, 4CL1 and STS, are monomer and
Fig. 2. HPLC analysis and UV absorption spectra of extracts from yeast cells with vectors pESC-HIS-4CL-SH3 and pESC-URA-STS-PDZ expression pESC-scaffold vectors. Partial HPLC chromatograms showed the almost identical retention time at 14.5 min from resveratrol in yeast cells (A) and authentic resveratrol standard (B). Both of them show similar UV spectrum at 306 nm.
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Y. Wang, O. Yu / Journal of Biotechnology 157 (2012) 258–260
Table 1 Yeast expression vectors carrying scaffold domains of GBD, SH3 and PDZ. Scaffold plasmid
Number of SH3 domain
Number of SH3 domain for 4CL1
Number of pDZ domain for STS
pESC-TRP-757 pESC-TRP-758 pESC-TRP-759 pESC-TRP-760 pESC-TRP-761 pESC-TRP-762 pESC-TRP-763 pESC-TRP-764 pESC-TRP-765 pESC-TRP
1 1 1 1 1 1 1 1 1 0
1 1 1 2 2 2 4 4 4 0
1 2 4 1 2 4 1 2 4 0
homodimeric proteins (Austin et al., 2004; Hu et al., 2010; Shomura et al., 2005), respectively. As expected, scaffolds GBD1 SH32 PDZ4 and GBD1 SH31 PDZ2 , with a 1:2 ratio of SH3 domain:PDZ domain, showed higher resveratrol titer than other scaffold control. However, we still do not know the detail mechanisms of how scaffolding metabolic enzymes increased metabolic efficiency. Further biochemical and structural studies on the interactions between scaffold protein domains and enzymes of pathway will elucidate more details in the future. The entire set of vectors generated in this work will help people to investigate these mechanisms of protein scaffolding in yeast. Although the amount of resveratrol produced by the scaffolds in Saccharomyces cerevisiae is relatively low when compared to several recent reports on resveratrol engineering, these results demonstrated that the synthetic scaffolds could be applied on resveratrol metabolic engineering to significantly increase the yield of final products. When combined with other metabolic engineering method, the resveratrol production can be further increased in the future. Acknowledgements This work is supported in part by grants from DOE (DESC0001295), NSF (MCB-0923779) and USDA (2010-65116-20514). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jbiotec.2011.11.003. References Austin, M.B., Bowman, M.E., Ferrer, J.L., Schroder, J., Noel, J.P., 2004. An aldol switch discovered in stilbene synthases mediates cyclization specificity of type III polyketide synthases. Chem. Biol. 11, 1179–1194.
Bauer, J.H., Goupil, S., Garber, G.B., Helfand, S.L., 2004. An accelerated assay for the identification of lifespan-extending interventions in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 101, 12980–12985. Dueber, J.E., Wu, G.C., Malmirchegini, G.R., Moon, T.S., Petzold, C.J., Ullal, A.V., Prather, K.L., Keasling, J.D., 2009. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759. Halls, C., Yu, O., 2008. Potential for metabolic engineering of resveratrol biosynthesis. Trends Biotechnol. 26, 77–81. Howitz, K.T., Bitterman, K.J., Cohen, H.Y., Lamming, D.W., Lavu, S., Wood, J.G., Zipkin, R.E., Chung, P., Kisielewski, A., Zhang, L.L., Scherer, B., Sinclair, D.A., 2003. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425, 191–196. Hu, Y., Gai, Y., Yin, L., Wang, X., Feng, C., Feng, L., Li, D., Jiang, X.N., Wang, D.C., 2010. Crystal structures of a Populus tomentosa 4-coumarate:CoA ligase shed light on its enzymatic mechanisms. Plant Cell 22, 3093–3104. Moon, T.S., Dueber, J.E., Shiue, E., Prather, K.L., 2010. Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli. Metab. Eng. 12, 298–305. Pompon, D., Louerat, B., Bronine, A., Urban, P., 1996. Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol. 272, 51–64. Shomura, Y., Torayama, I., Suh, D.Y., Xiang, T., Kita, A., Sankawa, U., Miki, K., 2005. Crystal structure of stilbene synthase from Arachis hypogaea. Proteins 60, 803–806. Urban, P., Mignotte, C., Kazmaier, M., Delorme, F., Pompon, D., 1997. Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J. Biol. Chem. 272, 19176–19186. Valenzano, D.R., Terzibasi, E., Genade, T., Cattaneo, A., Domenici, L., Cellerino, A., 2006. Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr. Biol. 16, 296–300. Wang, Y., Chen, H., Yu, O., 2010. Metabolic engineering of resveratrol and other longevity boosting compounds. Biofactors 36, 394–400. Wang, Y., Chen, S., Yu, O., 2011a. Metabolic engineering of flavonoids in plants and microorganisms. Appl. Microbiol. Biotechnol. 91, 949–956. Wang, Y., Halls, C., Zhang, J., Matsuno, M., Zhang, Y., Yu, O., 2011b. Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering. Metab. Eng. 13, 455–463. Zhang, Y., Li, S.Z., Li, J., Pan, X., Cahoon, R.E., Jaworski, J.G., Wang, X., Jez, J.M., Chen, F., Yu, O., 2006. Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and Mammalian cells. J. Am. Chem. Soc. 128, 13030–13031.
Contents lists available at SciVerse ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
Synthetic scaffolds increased resveratrol biosynthesis in engineered yeast cells Yechun Wang, Oliver Yu ∗ Donald Danforth Plant Science Center, 975 North Warson Road, Saint Louis, MO 63132, United States
a r t i c l e
i n f o
Article history: Received 27 August 2011 Received in revised form 28 October 2011 Accepted 2 November 2011 Available online 9 November 2011 Keywords: Resveratrol Metabolic engineering Synthetic scaffold
a b s t r a c t Resveratrol is a polyphenolic compound produced by a few higher plants when under attack by pathogens such as bacteria or fungi. Besides antioxidant benefits to humans, this health-promoting compound has been reported to extend longevity in yeasts, flies, worms, fishes and obesity mice. Here we utilized the synthetic scaffolds strategy to improve resveratrol production in Saccharomyces cerevisiae. We observed a 5.0-fold improvement over the non-scaffolded control, and a 2.7-fold increase over the previous reported with fusion protein. This work demonstrated the synthetic scaffolds can be used for the optimization of engineered metabolic pathway. © 2011 Elsevier B.V. All rights reserved.
Resveratrol is a naturally occurring defense compound produced by a few plant species in response to pathogen attacks. Resveratrol has significant physiological effects on human and other animals, which may be related to its antioxidant activities. Most importantly, when large quantities of resveratrol were fed to laboratory animals, the compound significantly increased the longevity of the subjects, including roundworm, fruit fly, fish, and mouse (Bauer et al., 2004; Howitz et al., 2003; Valenzano et al., 2006). Although the mechanism of this effect is still under investigation, the specific effects of resveratrol on longevity attracted extensive attentions in aging research. The health-promoting properties of resveratrol have stimulated the development of heterologous expression system based on Saccharomyces cerevisiae and Escherichia coli. Detail progresses have been described in previous review papers (Halls and Yu, 2008; Wang et al., 2010, 2011a). In recent years, a new scaffolding strategy has been successfully applied on increasing glucaric acid and mevalonate biosynthetic flux (Dueber et al., 2009; Moon et al., 2010). The technology is based on engineered synthetic protein scaffolds. These scaffolds interact with the enzymes of natural compound biosynthesis pathway via small peptide ligands in a programmable manner that improve production of end-product. By taking advantage of these scaffolds, a 77-fold improvement of mevalonate production was achieved (Dueber et al., 2009) and 5-fold increase in glucaric acid titers over the non-scaffolded control (Moon et al., 2010). Here we constructed the scaffolds to recruit the enzymes (4-coumarate:CoA ligase (4CL1) and stilbene synthase (STS)) of resveratrol biosynthesis pathway to improve resveratrol production in yeast cells.
∗ Corresponding author. Tel.: +1 314 587 1441; fax: +1 314 587 1541. E-mail address: [email protected] (O. Yu). 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.11.003
Nine scaffold plasmids consisting of GBD, SH3 and PDZ protein interaction domains were kindly provided by Dr. John Dueber from Department of Bioengineering, University of California, Berkeley, CA. The scaffold plasmids were cut by BglII/XhoI, and the small scaffold fragments (from 771 bp to 2319 bp) were subcloned into the yeast expression vector pESC-TRP carrying galatose-inducible (Gal1) promoter, downstream of a BamHI/XhoI multi-cloning site. The information of nine scaffolds yeast expression vectors were showed in Table 1 (Dueber et al., 2009). For building ligand-fusion proteins, the sequences of SH3 and PDZ, were synthesized by Genewiz Inc. (South Plainfield, NJ), the ligands SH3 and PDZ were cloned into other yeast expression vector pESC-HIS and pESC-URA (Stratagene), respectively, under the control of a Gal1 promoter at the multi-cloning sites. The resultant plasmids were designated as pESC-HIS-SH3 and pESC-URA-PDZ. The plasmid encoding the resveratrol pathway enzymes 4CL1 and STS were amplified from our previous plasmid (Zhang et al., 2006) by PCR using primers with BamHI/XhoI restriction enzyme sites. The 4CL1 gene was inserted into the pESC-HIS-SH3 vector, and STS into the pESC-URA-PDZ vector (as pESC-HIS-4CL1-SH3 and pESC-URA-STS-PDZ, respectively). pESC-HIS-4CL1-SH3, pESC-URA-STS-PDZ and pESC-scaffolds, were transformed into WAT11 cells (Pompon et al., 1996; Urban et al., 1997) made competent cells with the Frozen-EZ Yeast kit (Zymo Research, Orange, CA). pESC-4CL1-SH3, pESC-STS-PDZ, and pESC-TRP empty vector without the ligands were also transformed into WAT11 cells as controls. Yeast cultures (20 mL) were grown overnight at 30 ◦ C in appropriate SD drop-out liquid media (US Biological, MA) containing 2% (w/v) glucose. Cultures were then collected by centrifugation and washed three times in sterile deionized water. Cells were resuspended in induction medium containing 2% galatose to an OD600 of 1.0. The substrate p-coumaric acid was added to a final concentration of 100 M, and re-supplied
Y. Wang, O. Yu / Journal of Biotechnology 157 (2012) 258–260
Fig. 1. Effects of nine synthetic scaffolds on resveratrol production in yeast cells. The maximum production of resveratrol was observed in G1S2P4 construct, in which the resveratrol level is 5.0-fold higher than the non-scaffolded control (G0S0P0). Data are the averages of three biological repeats.
every 24 h (100 M). Approximately 36 h after induction, aliquots (400 L) were extracted with 800 L ethyl acetate. Extracts were dried with an Eppendorf VacufugeTM (Eppendorf Scientific, MA) at room temperature and re-dissolved in 80% methanol for HPLC analysis (Wang et al., 2011b). To determine whether these scaffolds work on resveratrol biosynthesis in yeast cells, we cloned the nine scaffold configurations into nine yeast expression vectors. Scaffolds were constructed
259
by assembling three protein–protein interaction domains linked together by nine residue Gly-Ser linkers. The binding domains SH3 and PDZ were used to target 4CL and STS, respectively. As expected, yeast cells transformed with pESC-HIS-4CL-SH3, pESCURA-STS-PDZ and pESC-scaffold vectors can successfully produce resveratrol in large quantities when fed with the substrate pcoumaric acid, based on HPLC analysis (Figs. 1 and 2). Compared to the controls yeast cells transformed with pESC-HIS-4CL-SH3, pESCURA-STS-PDZ and the pESC-TRP vector (without the scaffolds), a more than 5.0-fold increase in the resveratrol production (6.7 mg/L) was observed for the optimal scaffold (GBD1 SH32 PDZ4 ) at 36 h after galactose induction (Fig. 1). After longer incubation, the scaffolding construct still have more than 2-fold increase in resveratrol production (14.4 mg/L) at 96 h after induction (Supplementary Table 1). More importantly, this yield is 2.7-fold higher than the previously reported with fusion proteins (Zhang et al., 2006), suggesting the more flexible joining of the two enzymes by scaffolding appear to be more productive than the more rigid fusion protein constructs. Production improvements depended on the number of domain repeats in the scaffolds. For example, scaffolds GBD1 SH32 PDZ4 and GBD1 SH34 PDZ4 differ by two SH3 domains resulted in producing different resveratrol titers: 5.17-fold versus 1.4-fold, respectively. Compared to the non-scaffold GBD0 SH30 PDZ0 , scaffolds GBD1 SH34 PDZ1 and GBD1 SH34 PDZ4 showed low increase in resveratrol production (1.23-fold and 1.4-fold). Apparently, the number of binding domains had dramatic impacts on increasing pathway flux. The enzymes, 4CL1 and STS, are monomer and
Fig. 2. HPLC analysis and UV absorption spectra of extracts from yeast cells with vectors pESC-HIS-4CL-SH3 and pESC-URA-STS-PDZ expression pESC-scaffold vectors. Partial HPLC chromatograms showed the almost identical retention time at 14.5 min from resveratrol in yeast cells (A) and authentic resveratrol standard (B). Both of them show similar UV spectrum at 306 nm.
260
Y. Wang, O. Yu / Journal of Biotechnology 157 (2012) 258–260
Table 1 Yeast expression vectors carrying scaffold domains of GBD, SH3 and PDZ. Scaffold plasmid
Number of SH3 domain
Number of SH3 domain for 4CL1
Number of pDZ domain for STS
pESC-TRP-757 pESC-TRP-758 pESC-TRP-759 pESC-TRP-760 pESC-TRP-761 pESC-TRP-762 pESC-TRP-763 pESC-TRP-764 pESC-TRP-765 pESC-TRP
1 1 1 1 1 1 1 1 1 0
1 1 1 2 2 2 4 4 4 0
1 2 4 1 2 4 1 2 4 0
homodimeric proteins (Austin et al., 2004; Hu et al., 2010; Shomura et al., 2005), respectively. As expected, scaffolds GBD1 SH32 PDZ4 and GBD1 SH31 PDZ2 , with a 1:2 ratio of SH3 domain:PDZ domain, showed higher resveratrol titer than other scaffold control. However, we still do not know the detail mechanisms of how scaffolding metabolic enzymes increased metabolic efficiency. Further biochemical and structural studies on the interactions between scaffold protein domains and enzymes of pathway will elucidate more details in the future. The entire set of vectors generated in this work will help people to investigate these mechanisms of protein scaffolding in yeast. Although the amount of resveratrol produced by the scaffolds in Saccharomyces cerevisiae is relatively low when compared to several recent reports on resveratrol engineering, these results demonstrated that the synthetic scaffolds could be applied on resveratrol metabolic engineering to significantly increase the yield of final products. When combined with other metabolic engineering method, the resveratrol production can be further increased in the future. Acknowledgements This work is supported in part by grants from DOE (DESC0001295), NSF (MCB-0923779) and USDA (2010-65116-20514). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jbiotec.2011.11.003. References Austin, M.B., Bowman, M.E., Ferrer, J.L., Schroder, J., Noel, J.P., 2004. An aldol switch discovered in stilbene synthases mediates cyclization specificity of type III polyketide synthases. Chem. Biol. 11, 1179–1194.
Bauer, J.H., Goupil, S., Garber, G.B., Helfand, S.L., 2004. An accelerated assay for the identification of lifespan-extending interventions in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 101, 12980–12985. Dueber, J.E., Wu, G.C., Malmirchegini, G.R., Moon, T.S., Petzold, C.J., Ullal, A.V., Prather, K.L., Keasling, J.D., 2009. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759. Halls, C., Yu, O., 2008. Potential for metabolic engineering of resveratrol biosynthesis. Trends Biotechnol. 26, 77–81. Howitz, K.T., Bitterman, K.J., Cohen, H.Y., Lamming, D.W., Lavu, S., Wood, J.G., Zipkin, R.E., Chung, P., Kisielewski, A., Zhang, L.L., Scherer, B., Sinclair, D.A., 2003. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425, 191–196. Hu, Y., Gai, Y., Yin, L., Wang, X., Feng, C., Feng, L., Li, D., Jiang, X.N., Wang, D.C., 2010. Crystal structures of a Populus tomentosa 4-coumarate:CoA ligase shed light on its enzymatic mechanisms. Plant Cell 22, 3093–3104. Moon, T.S., Dueber, J.E., Shiue, E., Prather, K.L., 2010. Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli. Metab. Eng. 12, 298–305. Pompon, D., Louerat, B., Bronine, A., Urban, P., 1996. Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol. 272, 51–64. Shomura, Y., Torayama, I., Suh, D.Y., Xiang, T., Kita, A., Sankawa, U., Miki, K., 2005. Crystal structure of stilbene synthase from Arachis hypogaea. Proteins 60, 803–806. Urban, P., Mignotte, C., Kazmaier, M., Delorme, F., Pompon, D., 1997. Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J. Biol. Chem. 272, 19176–19186. Valenzano, D.R., Terzibasi, E., Genade, T., Cattaneo, A., Domenici, L., Cellerino, A., 2006. Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr. Biol. 16, 296–300. Wang, Y., Chen, H., Yu, O., 2010. Metabolic engineering of resveratrol and other longevity boosting compounds. Biofactors 36, 394–400. Wang, Y., Chen, S., Yu, O., 2011a. Metabolic engineering of flavonoids in plants and microorganisms. Appl. Microbiol. Biotechnol. 91, 949–956. Wang, Y., Halls, C., Zhang, J., Matsuno, M., Zhang, Y., Yu, O., 2011b. Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering. Metab. Eng. 13, 455–463. Zhang, Y., Li, S.Z., Li, J., Pan, X., Cahoon, R.E., Jaworski, J.G., Wang, X., Jez, J.M., Chen, F., Yu, O., 2006. Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and Mammalian cells. J. Am. Chem. Soc. 128, 13030–13031.