Metabolic Engineering Of Escherichia Coli For Resveratrol Production

Resveratrol is a kind of plant-derived natural product.It has been found that resveratrol has antioxidant activity,anti-inflammatory activity,protecting cardiovascular activity and can prolong life span of mice and nematodes.Now,resveratrol,an emerging natural active compound,is widely used in food additives,health products and cosmetics.At present,resveratrol is mainly extracted from grape or Polygonum cuspidatum,which is limited by time and region.With the development of metabolic engineering and synthetic biology,producing resveratrol with engineered microoragnisms has achieved great progresses.However,previous studies mainly focused on engineering metabolic pathways related to resveratrol biosynthesis.Herein,combinatorial optimization of resveratrol biosynthetic pathway and the intracellular environment of E.coli,and dynamic resveratrol biosynthesis were carried out.The main results were as follows.Initially,p-coumaric acid was used as precursor and malonyl-CoA metabolic pathways were modified.The consumption of malonyl-CoA to fatty acids was reduced by inhibition of fabD expression with sRNA anti-fabD and the biosynthesis of malonyl-CoA from acetyl-CoA was enhanced with overexpression of Cgacc.Thus,the concentration of malonyl-CoA was 0.35 nmol/mg DCW,representing 4.3 fold increase.At the same time,the resveratrol production was increased by 1.7 fold(202.92 mg/L).Using L-tyrosine as precursor,modular engineering of PcTAL,At4CL and VvSTS using plasmids with different copy numbers suggested that high-copy-number expression of PcTAL-At4CL and low-copy-number expression of VvSTS were contributed to resveratrol production.Ten transport genes(mdfA,emrD,emrE,acrAB,tolC,marA,yddG,arE,ompW and ompF)were screened to further increase resveratrol production.The results showed that overexpression of acrAB,marA,yddG,araE,ompW and ompF could improve resveratrol production,in which overexpression of ompF signigicantly enhanced resveratrol production by 99.8%.Combining optimization of intracellular environment for resveratrol production(overexpressing chaperones and omp F)and optimization of resveratrol biosynthetic pathway(modular engineering PcTAL,At4CL,VvSTS and improving malonyl-CoA supplement)achieved 238.71 mg/L high-yield resveratrol production from L-tyrosine,3.1 fold increase.Introducing optimized module into L-tyrosine producing strain and using glycerol as carbon source,de novo biosynthesis of resveratrol was achieved and resveratrol titer was up to 332.24 mg/L.In the study of Wu et al.,304.5 mg/L of resveratrol was produced with glucose as carbon source.Compared to Wu et al's study,resveratrol titer in this study was increased by 9.1%.Using Fap R as transcription factor and designing different promoters,dynamic regulation system was constructed and made the concentration of malonyl-CoA a mode of oscillation.At last,compared to the strating strain,resveratrol titer by the strain harboring dynamic regulation system was increased by 1.6 fold to 113.91mg/L.Then,the normalized metabolic flux analysis was conducted,indicating that both static and dynamic system could efficiently improve malonyl-CoA supply and resveratrol production.However,compared to static system,resveratrol biosynthetic metabolic flux in dynamic system increased by 51.4%,suggesting dynamic regulation was more efficient for resveratrol production.