Sun, Bing‐Yao, Wang, Feng‐Qing, Zhao, Jian, Tao, Xin‐Yi, Liu, Min, and Wei, Dong‐Zhi
l‐homoserine, a nonprotein amino acid, is used to synthesize many active substances in the industry. Here, to develop a robust l‐homoserine‐producing strain, Escherichia coli W3110 was used as a chassis to be engineered. Based on a previous construct with blocked competing routes for l‐homoserine synthesis, five genes were overexpressed by promoter replacement strategy to increase the l‐homoserine production, including enhancement of precursors for l‐homoserine synthesis (ppc, thrA, and asd), reinforcement of the NADPH supply (pntAB) and efflux transporters (rhtA) to improve the l‐homoserine production. However, the plasmid losing was to blame for the wildly fluctuating fermentation performance of engineered strains, ranging between 2.1 and 6.2 g/L. Then, a hok/sok toxin/antitoxin system was introduced into the free plasmid expression cassette to maintain the genetic stability of the episomal plasmid; consequently, the plasmid‐losing rate sharply decreased, resulting in the engineered strain SHL17, which exhibited excellent stability in l‐homoserine production, with 6.3 g/L in shake flasks and 44.4 g/L in a 5‐L fermenter without antibiotic addition. This work verified the effective use of the hok/sok toxin/antitoxin system combined with promoter engineering to improve the genetic stability of E. coli episomal plasmids without antibiotics. [ABSTRACT FROM AUTHOR]