De novo production of resveratrol from glucose or ethanol by engineered Saccharomyces cerevisiae

Abstract

Resveratrol is a natural antioxidant compound, used as food supplement and cosmetic ingredient. Microbial production of resveratrol has until now been achieved by supplementation of expensive substrates, p-coumaric acid or aromatic amino acids. Here we engineered the yeast Saccharomyces cerevisiae to produce resveratrol directly from glucose or ethanol via tyrosine intermediate. First we introduced the biosynthetic pathway, consisting of tyrosine ammonia-lyase from Herpetosiphon aurantiacus, 4-coumaryl-CoA ligase from Arabidopsis thaliana and resveratrol synthase from Vitis vinifera, and obtained 2.73 ± 0.05 mg L(-1) resveratrol from glucose. Then we over-expressed feedback-insensitive alleles of ARO4 encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate and ARO7 encoding chorismate mutase, resulting in production of 4.85 ± 0.31 mg L(-1) resveratrol from glucose as the sole carbon source. Next we improved the supply of the precursor malonyl-CoA by over-expressing a post-translational de-regulated version of the acetyl-CoA carboxylase encoding gene ACC1; this strategy further increased resveratrol production to 6.39 ± 0.03 mg L(-1). Subsequently, we improved the strain by performing multiple-integration of pathway genes resulting in resveratrol production of 235.57 ± 7.00 mg L(-1). Finally, fed-batch fermentation of the final strain with glucose or ethanol as carbon source resulted in a resveratrol titer of 415.65 and 531.41 mg L(-1), respectively.

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…For enhanced resveratrol production in S. cerevisiae, authors overexpressed the mutated acetyl-CoA carboxylase which is resistant to the degradation by the sucrose nonfermenting protein 1. [126] In this study, they also increased the tyrosine pool by overexpressing feedback-inhibition resistant 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase (ScAro4p K229L ) and chorismate mutase (ScAro7p G141S ). Consequently, resveratrol production was improved by 234%.…”

Advances in Metabolic Engineering of Saccharomyces cerevisiae for the Production of Industrially and Clinically Important Chemicals

“…In addition, chemical synthesis of these complex structures often requires multiple reaction steps and is not a commercially atractive route due to low product yields [46]. Currently, advances in metabolic engineering allowed commercial-scale microbial production of a number of ine chemicals [47][48][49]. Besides, there is an ongoing academic interest for reconstitution of biosynthetic pathways of several natural products, including complex pathways, in S. cerevisiae.…”

“…Glutathione Overexpression of YAP1 [58] Manipulation of the sulphate assimilation pathway by overexpressing MET14 and MET16 [59] Improved oxidized glutathione production by overexpression of GSH1, GSH2, and ERV1 and the deletion of GLR1 [60] Adaptive laboratory evolution in the presence of increasing levels of acrolein and screening for enhanced glutathione production [61] Whole-genome engineering via genome shuling and screening for enhanced glutathione production [62] Artemisinin/artemisinic acid Reconstruction of the complete biosynthetic pathway of artemisinic acid, including the three-step oxidation of amorphadiene to artemisinic acid by expression of CYP71AV1, CPR1, CYB5, ADH1 and ALDH1 from Artemisia annua [48] Taxol/taxadiene Expression of a truncated version of the endogenous tHMG1 and GGPPS from Taxus chinensis or Sulfolobus acidocaldarius together with TDC1 from T. chinensis [66] Prediction of the eiciency of diferent GGPPS enzymes via computer aided protein modelling [67] Forskolin Expression of a promiscuous cytochrome P450 from Salvia pomifera [68] Polyketides Heterologous expression of 6-MSA synthase gene from Penicillium patulum together with PPTases from either Bacillus subtilis or Aspergillus nidulans [69] Construction of polyketide precursor pathways by expressing prpE from Salmonella typhimurium and PCC pathway from Streptomyces coelicolor [70] Enhanced cofactor supply by expressing 2-PS from Gerbera hybrida [71] Resveratrol Reconstruction of a de novo pathway by expressing TAL from Herpetosiphon aurantiacus, 4-CL1 from Arabidopsis thaliana and VST1 from Vitis vinifera [49] Expression of 4CL1 from A. thaliana and STS from Arachis hypogaea [73] Expression of PAL from Rhodosporidium toruloides, C4H and 4-CL1 from A. thaliana, and STS from A. hypogaea [74] Expression of 4-coumaroyl-coenzyme A ligase (4CL1) from A. thaliana and stilbene synthase (STS) from V. vinifera [75] Overexpression of the resveratrol biosynthesis pathway, enhancement of P450 activity, increasing the precursor supply for resveratrol synthesis via phenylalanine pathway [76] Dihydrochalcones Expression of the heterologous pathway genes in a TSC13-overexpressing S. cerevisiae strain [78] Alkaloids Expression of 14 monoterpene indole alkaloid pathway genes from Catharanthus roseus and enhanced secondary metabolism to produce strictosidine de novo [79] Construction of the complete de novo biosynthetic pathway to norcoclaurine by expressing a mammalian TyrH enzyme and DODC from Pseudomonas putida, along with four genes required for biosynthesis of its electron carrier cosubstrate …”

“…Recently, in order to produce resveratrol from cheaper carbon sources, de novo biosynthesis of resveratrol via tyrosine intermediate in S. cerevisiae has been established by constructing an engineered pathway, involving tyrosine ammonia-lyase from Herpetosiphon aurantiacus, 4-coumaryl-CoA ligase from Arabidopsis thaliana and resveratrol synthase from Vitis vinifera [49]. To direct lux towards tyrosine, feedback-insensitive ARO4 encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and ARO7 encoding a chorismate mutase were overexpressed.…”

Synthetic genome engineering forging new frontiers for wine yeast

“…Nevertheless, earlier work on the development of resveratrol-producing S. cerevisiae strains fed with expensive p-coumaric acid or aromatic amino acids [39] has recently been superseded by the construction of a metabolically engineered strain capable of de novo production of resveratrol from glucose or ethanol via tyrosine as an intermediate (Figure 16). [40] The first step was to introduce the resveratrol biosynthetic pathway, comprising tyrosine ammonia-lyase from the bacterium Herpetosiphon aurantiacus, 4-coumaryl-CoA ligase from A. thaliana and resveratrol synthase from V. vinifera. The yield of resveratrol was significantly improved by the overexpression of feedback-insensitive alleles of the ARO4-encoded 3-deoxy-D-arabino-heptulosonate-7-phosphate and ARO7-encoded chorismate mutase, and the ACC1-encoded acetyl-CoA carboxylase boosting the supply of the precursor malonyl-CoA.…”