L-homoserine as a nonessential amino acid, can be used as a feed additive due to its significant value as well as in many other applications in agriculture. In this study, Escherichia coli W3110 was engineered for the production of L-homoserine from glucose. The lysA, metA, and thrBC genes were inactivated to block the competing and degradation pathways of L-homoserine. To enhance the carbon flux to homoserine, the effects of the lysC, thrA, and metL genes on L-homoserine production and the impact of L-homoserine on the aspartokinases (AKs) AKI, AKII, and AKIII were studied. The results demonstrated that, the bottleneck of the homoserine biosynthetic pathway was overcome and carbon flux to homoserine was enhanced by overexpressing the metL gene in E. coli W3110. The rhtA gene was also overexpressed under the control of the pN25 promoter to improve the survivability under the stress of high concentration as well as the productivity of L-homoserine. Moreover, we verified that the inactivation of the TdcC transporter further increased L-homoserine production. A high-L-homoserine-producing strain was constructed from wild type E. coli W3110 using metabolic engineering strategies. This improved the production of L-homoserine from 0 to 39.54 g/L in fed-batch fermentation. (C) 2016 Elsevier Ltd. All rights reserved.