L-methionine has attracted a great deal of attention for its nutritional, pharmaceutical, and clinical applications. In this study, Escherichia coli W3110 was engineered via deletion of a negative transcriptional regulator MetJ and over-expression of homoserine O-succinyltransferase MetA together with efflux transporter YjeH, resulting in L-methionine overproduction which is up to 413.16mg/L. The partial inactivation of the L-methionine import system MetD via disruption of metI made the engineered E. coli metJ metI/pTrcA*H more tolerant to high L-ethionine concentration and accumulated L-methionine to a level 43.65% higher than that of E. coli W3110 metJ/pTrcA*H. Furthermore, deletion of lysA, which blocks the lysine biosynthesis pathway, led to a further 8.5-fold increase in L-methionine titer of E. coli metJ metI lysA/pTrcA*H. Finally, addition of Na2S2O3 to the media led to an increase of fermentation titer of 11.45%. After optimization, constructed E. coli metJ metI lysA/pTrcA*H was able to produce 9.75g/L L-methionine with productivity of 0.20g/L/h in a 5L bioreactor. This novel metabolically tailored strain of E. coli provides an efficient platform for microbial production of L-methionine. Biotechnol. Bioeng. 2017;114: 843-851. (c) 2016 Wiley Periodicals, Inc.