Profiling influences of gene overexpression on heterologous resveratrol production in Saccharomyces cerevisiae

Abstract

Metabolic engineering of heterologous resveratrol production in Saccharomyces cerevisiae faces challenges as the precursor L-tyrosine is stringently regulated by a complex biosynthetic system. We overexpressed the main gene targets in the upstream pathways to investigate their influences on the downstream resveratrol production. Single-gene overexpression and DNA assembly-directed multigene overexpression affect the production of resveratrol as well as its precursor p-coumaric acid. Finally, the collaboration of selected gene targets leads to an optimal resveratrol production of 66.14±3.74 mg·L^–1, 2.27 times higher than the initial production in YPD medium (4% glucose). The newly discovered gene targets TRP1 expressing phosphoribosylanthranilate isomerase, ARO3 expressing 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, and 4CL expressing 4-coumaryl-CoA ligase show notable positive impacts on resveratrol production in S. cerevisiae.

Graphical abstract

Keywords

resveratrol / aromatic amino acid / DNA assembly / metabolic engineering / gene overexpression

References

[1] Jeandet P, Delaunois B, Aziz A, Donnez D, Vasserot Y, Cordelier S, Courot E. Metabolic engineering of yeast and plants for the production of the biologically active hydroxystilbene, resveratrol. Journal of Biomedicine & Biotechnology, 2012, 579089

[2] Mei Y Z, Liu R X, Wang D P, Wang X, Dai C C. Biocatalysis and bio-transformation of resveratrol in microorganisms. Biotechnology Letters, 2015, 37(1): 9–18

[3] Borodina I, Nielsen J. Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals. Biotechnology Journal, 2014, 9(5): 609–620

[4] Becker J V, Armstrong G O, vander Merwe M J, Lambrechts M G, Vivier M A, Pretorius I S. Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol. FEMS Yeast Research, 2003, 4(1): 79–85

[5] Beekwilder J, Wolswinkel R, Jonker H, Hall R, deVos C H, Bovy A. Production of resveratrol in recombinant microorganisms. Applied and Environmental Microbiology, 2006, 72(8): 5670–5672

[6] Zhang Y, Li S Z, Li J, Pan X, Cahoon R E, Jaworski J G, Wang X, Jez J M, Chen F, Yu O. Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and Mammalian cells. Journal of the American Chemical Society, 2006, 128(40): 13030–13031

[7] Shin S Y, Jung S M, Kim M D, Han N S, Seo J H. Production of resveratrol from tyrosine in metabolically engineered Saccharomyces cerevisiae. Enzyme and Microbial Technology, 2012, 51(4): 211–216

[8] Trantas E, Panopoulos N, Ververidis F. Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae. Metabolic Engineering, 2009, 11(6): 355–366

[9] Yan Y, Kohli A, Koffas M A. Biosynthesis of natural flavanones in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 2005, 71(9): 5610–5613

[10] Kumar S, Omer S, Chitransh S, Khan B M. Cinnamate 4-hydroxylase downregulation in transgenic tobacco alters transcript level of other phenylpropanoid pathway genes. International Journal of Advanced Biotechnology and Research, 2012, 3(2): 545–557

[11] Wang Y, Halls C, Zhang J, Matsuno M, Zhang Y, Yu O. Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering. Metabolic Engineering, 2011, 13(5): 455–463

[12] Wang Y, Yu O. Synthetic scaffolds increased resveratrol biosynthesis in engineered yeast cells. Journal of Biotechnology, 2012, 157(1): 258–260

[13] Luttik M A, Vuralhan Z, Suir E, Braus G H, Pronk J T, Daran J M. Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: Quantification of metabolic impact. Metabolic Engineering, 2008, 10(3-4): 141–153

[14] Rodriguez A, Kildegaard K R, Li M, Borodina I, Nielsen J. Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis. Metabolic Engineering, 2015, 31: 181–188

[15] Koopman F, Beekwilder J, Crimi B, van Houwelingen A, Hall R D, Bosch D, van Maris A J, Pronk J T, Daran J M. De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae. Microbial Cell Factories, 2012, 11(1): 155

[16] Juminaga D, Baidoo E E K, Redding-Johanson A M, Batth T S, Burd H, Mukhopadhyay A, Petzold C J, Keasling J D. Modular engineering of L-tyrosine production in Escherichia coli. Applied and Environmental Microbiology, 2012, 78(1): 89–98

[17] Reid R J, Sunjevaric I, Kedacche M, Rothstein R. Efficient PCR-based gene disruption in Saccharomyces strains using intergenic primers. Yeast (Chichester, England), 2002, 19(4): 319–328

[18] Gietz R D, Schiestl R H, Willems A R, Woods R A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast (Chichester, England), 1995, 11(4): 355–360

[19] Sun J, Shao Z Y, Zhao H, Nair N, Wen F, Xu J H, Zhao H. Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae. Biotechnology and Bioengineering, 2012, 109(8): 2082–2092

[20] Shao Z, Zhao H, Zhao H. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Research, 2008, 37(2): e16

[21] Sydor T, Schaffer S, Boles E. Considerable increase in resveratrol production by recombinant industrial yeast strains with use of rich medium. Applied and Environmental Microbiology, 2010, 76(10): 3361–3363

[22] Braus G, Paravicini G, Hütter R. A consensus transcription termination sequence in the promoter region is necessary for efficient gene expression of the TRP1 gene of Saccharomyces cerevisiae. Molecular & General Genetics, 1988, 212(3): 495–504

[23] Kim S, Mellor J, Kingsman A J, Kingsman S M. Multiple control element in the TRP1 promoter of Saccharomyces cerevisiae. Molecular and Cellular Biology, 1986, 6(12): 4251–4258

[24] Teshiba S, Furter R, Niederberger P, Braus G, Paravicini G. Cloning of the ARO3 gene of Saccharomyces cerevisiae and its regulation. Molecular & General Genetics, 1986, 205(2): 353–357

[25] Du J, Yuan Y B, Si T, Lian J Z, Zhao H M. Customized optimization of metabolic pathways by combinatorial transcriptional engineering. Nucleic Acids Research, 2012, 40(18): e142

[26] Luo Y, Li B Z, Liu D, Zhang L, Chen Y, Jia B, Zeng B X, Zhao H, Yuan Y J. Engineered biosynthesis of natural products in heterologous hosts. Chemical Society Reviews, 2015, 44(15): 5265–5290

[27] Santos C N, Stephanopoulos G. Melanin-based high-throughput screen for L-tyrosine production in Escherichia coli. Applied and Environmental Microbiology, 2008, 74(4): 1190–1197

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg