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O-succinyl-l-homoserine (OSH) is a promising platform chemical for the production of C4 chemicals with huge market potential which can be produced by fermentation from glucose. To construct a strain capable of producing OSH with high yield, the metJ(encodes transcriptional repressor) and metI(encodes a subunit of dl-methionine transporter) were deleted in Escherichia coliW3110 to obtain a strain E. coli∆JI. Then, overexpression of metL(encodes bifunctional aspartate kinase/homoserine dehydrogenase II) and inactivation of metB(encodes cystathionine γ-synthase) were implemented in one step, and the OSH titer of the resulting strain E. coli∆JIB*TrcmetLwas dramatically increased to 7.30 g/L. The feedback regulation was further relieved by progressively overexpressing metAfbr(encodes homoserine O-succinyltransferase), yjeH(encodes l-methionine exporter), and thrAfbr(encodes bifunctional aspartate kinase/homoserine dehydrogenase I) to increase the metabolic flux from aspartate to OSH. The 100% rationally designed strain E. coli∆JIB*TrcmetL/pTrc-metAfbr-Trc-thrAfbr-yjeHproduced 9.31 g/L OSH from 20 g/L glucose (0.466 g/g glucose) in batch fermentation, which represents the highest OSH yield from glucose reported to date. The culture profiles of the newly constructed strains were recorded to investigate their productive properties. The effects of l-methionine addition on the fermentation process of the optimal strain were also studied. Our results demonstrate that tuning the expression level of metL, inactivation of metB, and attenuation of feedback resistance of the crucial enzymes in the biosynthetic pathway are the key factors that impact the OSH production in E. coli.