Metabolic Engineering Of Escherichia Coli For Resveratrol Production

Resveratrol is a natural polyphenolic compound that belongs to the stilbene class. Resveratrol have a lot of health benefits. Numerous studies have found that its biological activities include antioxidant, anti-tumor, anti-aging, prevention of cardiovascular diseases, anti-inflammatory and regulation of estrogen. The number of articles devoted to trans-resveratrol has been increasing these years. Therefore, it is important to develop an effective method to produce this compound commercially. Engineered microorganisms is generally regarded as the most promising strategies for resveratrol bioproduction. Microbial production of resveratrol has been generally achieved by supplementation of expensive substrates, such as p-coumaric acid or aromatic amino acids. In this study, key enzymes for resveratrol production were expressed in Escherichia coli. The supply of precursors involved in the resveratrol biosynthesis were also enhanced for higher yield of resveratrol production. The main conclusions of this thesis were as follows:(1) Metabolic engineering of Escherichia coli for resveratrol production. In order to achieve the heterologous synthesis of resveratrol, TAL from Rhodotorula glutinis, 4CL from Petroselinum crispum and STS from Vitis vinifera were expressed in E. coli BL21(DE3). Two plasmids were constructed, i.e., pACYC-TAL-4Cl and pRSF-STS. 0.23 mg L-1 of resveratrol were detected after fermentation with glucose as substrate, showed that all three genes were successfully expressed in E. coli. In order to enhance the metabolic pathway from glucose to aromatic aminod acids, DAHP synthase(aroF) and CM/PDT(pheAfbr) were overexpressed, plasmid pCDF-aroF-pheAfbr were constructed. Plasmid pET-matB-matC carrying matB and matC from Rhizobium trifolii was introduced into E. coli to increase the supply of malonyl-CoA. Resveratrol content was detected by LCMS-IT-TOF after fermentation. 3.2 mg·L-1 of resveratrol was obtained after metabolic pathway optimization, increased by 11.9-fold compared with the former strain.(2) A phenylalanine production strain E. coli WSH-Z06 was used to construct a resveratol production strain. The plasmid pAP-B03 carrying aroF and pheAfbr were absent during passaging. pheA in E. coli WSH-Z06 was knockout with CRISPR-Cas9 system to weaken the phenylalanine pathway. A feed-back resistant tyrAfbr was overexpressed to enhance the tyrosine pathway. TAL, 4CL and STS was integrated into E. coli WSH-Z06 genome with CRISPR-Cas9 system for resveratrol production. 71.6 mg·L-1 of resveratrol was obtained according to the LCMS-IT-TOF analysis result. The resveratrol content was increased by 20.3-fold compared with the strain carrying 4 plasmids. IPTG was unnecessary during fermentation with E. coli RES-03, which decreased the cost for resveratrol production.(3) The degradation processes of resveratrol under different temperature(25 °C, 30 °C, 37 °C and 42 °C), pH(3.0, 5.0, 7.0, 9.0 and 11.0) conditions, and other conditions(water, tryptone, yeast extract, LB broth, M9 broth) were studied. It is noticed that higher temperature, alkaline pH condition and the existence of tryptone would increase the degradation rate of resveratrol. The existence of E. coli would not increase the degradation rate of resveratrol. The results can serve as the references for decrease of resveratrol degradation during fermentation. The kinetics of resveratrol degradation at pH 7.0 and the temperatures investigated followed first-order kinetics. The effect of temperature on the degradation rate of resveratrol was expressed by a linear plot of Arrhenius equation logkobs = 24.896- 9360.8(1/T)(r2=0.99). The LCMS-IT-TOF result showed two major metabolites after resveratrol degradation, viniferin and oxyresveratrol.