Metabolic Engineering Of Escherichia Coli For L-methionine Production

L-Methionine is one of the eight essential amino acids for human beings. It is the firstlimiting amino acid in diets for poultry, which leads to a bulk demand for methionine. Theproduction of methionine by chemical synthesis is expensive and causes seriousenvironmental pollution. Therefore, great interest rises in the production of L-methionine bysubmerged fermentation. In this study, we knocked out metJ gene of E. coli BL21(DE3) andobtained a derivative strain that was resistant to DL-ethionine(ET) by UV mutagenesis. Thenthe plasmid pACYCDuet-AES containing metA, cysE, yeaS genes was constructed to improvethe methionine production. The medium composition and fermentation conditions at shakeflask level were also investigated in this study. The main contents and results of this study areas follows:The methionine repressor encoded by metJ gene was knocked out by Red recombinationsystem in E. coli BL21(DE3). The production of L-methionine was increased from0to22mg·L-1.A mutant strain (YB12) with DL-Et resistant was obtained by UV mutagenesis of E. coliBL21(DE3) ΔmetJ followed by shake flask fermentation. Strain YB12could accumulate61mg·L-1L-methionine which was about three times of that produced by the origin strain E. coliBL21(DE3) ΔmetJ. The production of L-methionine by YB12was segregational stable.Plasmid pACYCDuet-AES was constructed for expression of key genes (metA, cysE,yeaS) of the L-methionine synthetic pathway. Production of L-methionine was improved to182mg·L-1when these genes were over expressed in YB12.The optimum fermentation medium and conditions for L-methionine production wasconfirmed using single-factor experiments and response surface methods. The optimalfermentation conditions were15mL broth of250mL flask, culturing temperature37℃,inoculum rate10，IPTG concentration0.1mmol·L-1. The accumulation of L-methioninewas added up to395mg·L-1at48h after optimization.