Metabolic Engineering Escherichia Coli To Produce P-coumaric Acid

p-Coumaric acid is an important natural phenolic compound with a variety of pharmacological activities,and also a precursor for the biosynthesis of many natural compounds.It is widely used in food,cosmetics and medicine.Compared with the traditional process including chemical synthesis and plant extraction,microbial production of p-coumaric acid has great potential to decrease the production cost and reduce the waste emission.However,the efficiency of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production.On the one hand,the low efficiency of coumaric acid may be due to the low catalytic activity of TAL enzyme.On the other hand,cell growth may be inhibited due to the cytotoxicity to coumaric acid.In order to solve these problem,this study includes two aspects as follows.Firstly,enzyme directed evolution was used to screen high catalytic activity TAL.The random mutant library of Rhodotorula glutinis tal gene encoding tyrosine ammonia lyase(TAL)was constructed through error-prone PCR technology which is one type of directed evolution.A high-throughput screening method was established to screen TAL mutant library.A mutant with a doubled TAL catalytic activity was screened from about 10,000 Escherichia coli colonies which are from above mutant library.There were three mutational amino acid sites in this TAL sequence,namely S9Y,A11N,and E518A.It was further verified by a single point saturation mutation that when S9 site was mutated to Y,I or N,or A11 site was mutated to N,T or Y,the catalytic activity of TAL increased by more than 1 times.Through combinatorial mutation of three types of mutations at the S9 and A11 sites,the TAL catalytic activities of S9Y/A11N and S9N/A11Y mutants were significantly higher than that of other mutants.Then,the plasmid containing S9N/A11Y mutant was transformed into CP032,a tyrosine-producing E.coli strain.The engineered strain produced 394.2 mg/L p-coumaric acid which is 2.2-fold higher than that of the control strain,via shake flask fermentation at 48 h.This work provides a new insight for the biosynthesis study of p-coumaric acid.Secondly,the laboratory adaptive evolution was used to improve the tolerance of the chassis cells to coumaric acid.A previously engineered tyrosine producing E.coli strain,CP032,was cultured in LB medium with different concentrations of coumaric acid to improve tolerance of product inhibition.After 4 rounds of adaptive domestication,the results showed that the evolved strain tolerance to coumaric acid increased from 1.3 g/L to 1.5 g/L,which was 15%higher compared to that of the parent strain CP032.Through the genome resequencing analysis of the domestication strain,a number of genes such as glutardiamine-lysine transporter(cadB)and transporter AraE,that may be related to the tolerance of coumaric acid were screened and mutated.These sites have important value for improving the tolerance mechanism of engineered strains to p-coumaric acid.