Engineering of a microbial coculture of Escherichia coli strains for the biosynthesis of resveratrol

Abstract

BackgroundResveratrol is a plant natural product with many health-protecting effects which makes it an attractive chemical both for academic studies and industrial purposes. However, the low quantities naturally produced by plants as well as the unsustainable procedures of extraction, purification and concentration have prompted many biotechnological approaches to produce this chemical in large quantities from renewable sources. None of these approaches have considered a microbial coculture strategy to produce this compound. The aim of this study was to prove the functionality of a microbial coculture for the biosynthesis of resveratrol.ResultsIn this work, we have successfully applied a coculture system strategy comprised of two populations of Escherichia coli strains, each with a partial and complementary section of the pathway leading to the biosynthesis of the stilbene resveratrol. The first strain is a pheA knockout mutant previously engineered to excrete p-coumaric acid into the medium through the overexpression of genes encoding a tyrosine ammonia lyase from Rhodothorula glutinis, a feedback resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and a transketolase. The second strain in the coculture was engineered to express the second part of the resveratrol biosynthetic pathway through the introduction of synthetic genes encoding the 4-coumaroyl-CoA ligase from Streptomyces coelicolor A2 and the stilbene synthase either from the peanut Arachis hypogaea or the grapevine Vitis vinifera, the latter synthesized employing a gene harmonization strategy and showing better resveratrol production performance. Batch cultures were performed in mineral medium with glycerol as the sole carbon source, where a final titer of 22.6 mg/L of resveratrol was produced in 30 h.ConclusionsTo our knowledge, this is the first time that a coculture of bacterial strains is used for the biosynthesis of resveratrol from glycerol, having the potential for a greater improvement in the product yield and avoiding the use of precursors such as p-coumaric acid, yeast extract or an expensive inhibitor such as cerulenin.

An adsorptive bioprocess for production and recovery of resveratrol with Corynebacterium glutamicum

“…In fact, the titers of resveratrol produced by C. glutamicum are lower in direct comparison with other already published microbial production strains, but the use of this strain as a platform for polyphenol production is highly appealing for the reasons explained in the Introduction. Further research and development of this strain, aiming at increasing the resveratrol titers, may result in a good platform for industrial resveratrol production.…”

“…The concentration of resveratrol that can be obtained from plant cell cultures is at most equal to that reported as naturally occurring in the plant, being the grape cell suspensions ( V. vinifera ) the most promising one, with a resveratrol yield ranging from 2–5 mg L ‐1 . The first studies for microbial resveratrol production reported the use of Saccharomyces cerevisiae and Escherichia coli as cell factories, with a production of around 0.5 g L ‐1 . More recently, other organisms, such as C. glutamicum , which is a workhorse in industrial biotechnology, especially for the production of several aminoacids, proved to be a promising host organisms for resveratrol production .…”

“…3,4 The first studies for microbial resveratrol production reported the use of Saccharomyces cerevisiae and Escherichia coli as cell factories, with a production of around 0.5 g L -1 . 2,[5][6][7][8][9][10] More recently, other organisms, such as C. glutamicum, which is a workhorse in industrial biotechnology, especially for the production of several aminoacids, 11 proved to be a promising host organisms for resveratrol production. 12,13 It is a generally regarded as a safe (GRAS status) microorganism able to achieve high cell densities, 14 with high resistance to the presence of small aromatic compounds, 15 able to grow on p-coumaric acid, ferulic acid, caffeic acid and 3-(4-hydroxyphenyl)-propionic acid as sole carbon and energy sources.…”

Expanding the Chemical Palette of Industrial Microbes: Metabolic Engineering for Type III PKS‐Derived Polyketides

“…[30] This background strain can be further engineered to produce the desired phenylpropanoid CoA esters (such as 4-coumaroyl-CoA) to convert into polyketides. [24][25][26][27][28][29][30][31][32][33][34][35][36][37] A graphical summary of the major pathways targeted in all of these engineering strategies is provided in Figure 2.…”

“…[72] This exciting result suggests that further investigation into THN synthases could reveal interesting chemistry, thus further expanding the applications of polyketide production. [95] Escherichia coli Stilbeniod At4CL1, VvSTS Minimal, cerulenin, acid Flask 2.3 g L À1 resveratrol [18] Escherichia coli Stilbeniod Pc4CL, VvSTS matB, matC, RgTAL Minimal, tyr Flask 35 mg L À1 resveratrol [33] Escherichia coli Stilbeniod Pc4CL, VvSTS RgTAL, ΔtyrR ΔtrpED Complex Flask 4.612 mg L À1 resveratrol [30] Escherichia coli Stilbeniod strain 1: Pc4CL, VvSTS strain 2: RgTAL, aroG(fbr), tktA,…”