Metabolic Engineering for Resveratrol Derivative Biosynthesis in Escherichia coli

Abstract

We previously reported that the SbROMT3syn recombinant protein catalyzes the production of the methylated resveratrol derivatives pinostilbene and pterostilbene by methylating substrate resveratrol in recombinant E. coli. To further study the production of stilbene compounds in E. coli by the expression of enzymes involved in stilbene biosynthesis, we isolated three stilbene synthase (STS) genes from rhubarb, peanut, and grape as well as two resveratrol O-methyltransferase (ROMT) genes from grape and sorghum. The ability of RpSTS to produce resveratrol in recombinant E. coli was compared with other AhSTS and VrSTS genes. Out of three STS, only AhSTS was able to produce resveratrol from p-coumaric acid. Thus, to improve the solubility of RpSTS, VrROMT, and SbROMT3 in E. coli, we synthesized the RpSTS, VrROMT and SbROMT3 genes following codon-optimization and expressed one or both genes together with the cinnamate/4-coumarate:coenzyme A ligase (CCL) gene from Streptomyces coelicolor. Our HPLC and LC-MS analyses showed that recombinant E. coli expressing both ScCCL and RpSTSsyn led to the production of resveratrol when p-coumaric acid was used as the precursor. In addition, incorporation of SbROMT3syn in recombinant E. coli cells produced resveratrol and its mono-methylated derivative, pinostilbene, as the major products from p-coumaric acid. However, very small amounts of pterostilbene were only detectable in the recombinant E. coli cells expressing the ScCCL, RpSTSsyn and SbROMT3syn genes. These results suggest that RpSTSsyn exhibits an enhanced enzyme activity to produce resveratrol and SbROMT3syn catalyzes the methylation of resveratrol to produce pinostilbene in E. coli cells.

Resveratrol and Related Stilbenoids, Nutraceutical/Dietary Complements with Health‐Promoting Actions: Industrial Production, Safety, and the Search for Mode of Action

“…The final Saccharomyces cerevisiae strain combined with an optimized fermentation procedure gave 531 or 415 mg STB/L when the sole source of carbon was ethanol or glucose, respectively (Li et al., ). Genetic engineering combined with optimized biotechnology tools also may afford production of RSV and of specific derivatives, such as pinostilbene whose health‐promoting potential is not known yet (Jeong et al., ). Food/supplement industry must calculate the cost‐benefit of using this method, with either glucose or ethanol, for STBDC production.…”

Engineering yeast for high-level production of stilbenoid antioxidants

“…Two resveratrol O-methyltransferases from Sorghum bicolor ( SbROMT ) and Vitis vinifera ( VvROMT ) were shown to methylate resveratrol to pinostilbene and pterostilbene, respectively (Fig. 6a)1113. We expressed the two enzymes in the resveratrol producing strain ST4990 to generate strains ST4993 and ST4994.…”

“…Several groups subsequently improved the production of resveratrol and its derivatives in yeast and Escherichia coli . However, all of these studies applied feeding of expensive precursors, such as p -coumaric acid101112131415, tyrosine151617 or phenylalanine18. The highest reported titer of resveratrol was 2.3 g l −1 , when 2.5 g l −1 p -coumaric acid was fed to engineered E. coli 19.…”

Enhanced production of resveratrol derivatives in tobacco plants by improving the metabolic flux of intermediates in the phenylpropanoid pathway

“…Full-length cDNAs for RpSTS and VrROMT were amplified from rhubarb (Rheum palmatum) and grape (Vitis riparia) by homology-based polymerase chain reaction (PCR) using PCR primers based on the RtSTS1 (AF508150) and VvROMT (FM178870) genes, as previously described by Jeong et al (2014Jeong et al ( , 2015. For the heterologous expression of RpSTS and VrROMT in tobacco plants, the resulting RpSTS and VrROMT cDNAs were amplified with Pfu DNA polymerase (Solgent, Korea) using gene-specific primers that introduced 5′ NdeI and 3′ SpeI sites (underlined) ( Table S1).…”