Profiling influences of gene overexpression on heterologous resveratrol production in <i>Saccharomyces cerevisiae</i>

doi:10.1007/s11705-016-1601-3

Front. Chem. Sci. Eng.

2017, Vol. 11

Issue (1) : 117-125 https://doi.org/10.1007/s11705-016-1601-3

RESEARCH ARTICLE

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

Duo Liu^1,²,Bingzhi Li^1,²,Hong Liu^1,²,Xuejiao Guo^1,²,Yingjin Yuan^1,²(

)

¹. Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
². SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, China

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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.

Keywords resveratrol aromatic amino acid DNA assembly metabolic engineering gene overexpression

Corresponding Author(s): Yingjin Yuan

Online First Date: 15 December 2016 Issue Date: 17 March 2017

Cite this article:

Duo Liu,Bingzhi Li,Hong Liu, et al. Profiling influences of gene overexpression on heterologous resveratrol production in Saccharomyces cerevisiae[J]. Front. Chem. Sci. Eng., 2017, 11(1): 117-125.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-016-1601-3
https://academic.hep.com.cn/fcse/EN/Y2017/V11/I1/117

Fig.1 Schematic illustration of gene overexpression for improving resveratrol production in S. cerevisiae. (a) The upstream biosynthetic pathways of L-PHE, L-TYR and L-TRP are chosen to screen the gene targets that are beneficial to resveratrol production in yeast; (b) metabolic engineering faces the difficulty when multiple highly biosynthetic regulated reactions are operated; (c) each single gene in charge of the related reactions in the upstream pathways is overexpressed; (d) the yeast in vivo DNA assembly process is utilized to combine multigene overexpression. Abbreviations: E4P, erythrose 4-phosphate; PEP, phosphoenol pyruvate; DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydro-shikimate; SHIK, shikimate; SHP, shikimate-3-phosphate; EP3P, 5-enolpyruvyl shikimate-3-phosphate; CHA, chorismate; PPA, prephenate; PPY, phenylpyruvate; HPP, para-hydroxy-phenyl pyruvate; L-PHE, L-phenylalanine; L-TYR, L-tyrosine; ANA, anthranilate; PRA, N-(5-phospho-D-ribosyl) anthranilate; CDRP, 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose-5-phosphate; IGP, indoleglycerol phosphate; L-TRP, L-tryptophan; Aro4p, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase; Aro3p, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase; Aro1p, pentafunctional arom protein which catalyzes steps 2 through 6 in the biosynthesis of chorismate; Aro2p, bifunctional chorismate synthase and flavin reductase; Aro7p, chorismate mutase; Pha2p, prephenate dehydratase; Tyr1p, prephenate dehydrogenase; Aro8p, aromatic aminotransferase I; Aro9p, aromatic aminotransferase II; Trp2p, anthranilate synthase; Trp3p, indole-3-glycerol-phosphate synthase; Trp4p, anthranilate phosphoribosyl transferase; Trp1p, phosphoribosylanthranilate isomerase; Trp5p, tryptophan synthase; Gcn4p, bZIP transcriptional activator of amino acid biosynthetic genes; TAL, tyrosine ammonia lyase; 4CL, 4-coumaryl-CoA ligase; STS, stilbene synthase

Fig.2 Construction and verification of resveratrol synthesis pathway in S. cerevisiae. (a) The resveratrol pathway was constructed by assembly of these DNA segments: “Delta1-URA3-FBA1t”, “FBA1t-TPI1p-STS-PGK1t”, “PGK1t-TEF1p-4CL-CYC1t”, “CYC1t-TDH3p-TAL-TEF1t” and “TEF1t-Delta 2”. The assembled pathway was integrated into the delta site (5’-UTR of Ty1 transposon) on the chromosome; (b) the p-coumaric acid and resveratrol synthesized by yeast were detected by HPLC. The amounts of the two products changed along with the cell’s cultivation period in YPD (4% glucose); (c) the cells cultivated in different culture media produced different amounts of resveratrol and p-coumaric acid. The values represented the production at 96 h. All error bars indicate±SD, n = 3

Fig.3 The effects of single-gene overexpression on production of p-coumaric acid and resveratrol. (a) The overexpression of single gene is achieved by OE-PCR and ligation of single transcriptional unit on pRS425; (b) each single gene overexpression caused different changes of the production of p-coumaric acid and resveratrol. All error bars indicate±SD, n = 3

Fig.4 The effects of multigene overexpression on production of p-coumaric acid and resveratrol. (a) The combinatorial overexpression of two or three gene targets is gained by assembly of ORF library and certain “terminator-promoter” parts through yeast recombination. T0, T1, T2 and T3 represent yeast terminators. P1, P2 and P3 represent yeast promoters. Comparing with the definite ORF used in single gene overexpression, the ORF1, ORF2 and ORF3 represent three groups of mixed ORFs of the gene targets in the upstream pathways. Each group of ORFs owns distinct homologous to be assembled with other parts in a certain order; (b) the results of production in 40 randomly picked strains were presented by changing fold of production from the initial strain contain primary heterologous pathway

Fig.5 Effects of combinatorial overexpression of the selected genes on production of p-coumaric acid and resveratrol. The optimal overexpression of single and multigene genes was selectively co-transformed into the resveratrol-synthesizing cells. The gene targets included both selected endogenous genes in upstream pathways and the certain heterologous gene in the resveratrol synthesis pathway. The effects of combinatorial multigene overexpression were detected and analyzed. All error bars indicate±SD, n = 3

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