L-Homoserine is a valuable amino acid as a platform chemical in the synthesis of various important compounds. Development of microbial strains for high-level L-homoserine production is an attractive research direction in recent years. Herein, we converted a wild-type Escherichia coli to a non-auxotrophic and plasmid-free hyperproducer of L-homoserine using systematically metabolic engineer strategies. First, an initial strain was obtained through regulating L-homoserine degradation pathway and enhancing synthetic flow. To facilitate L-homoserine production, flux-control genes were tuned by optimizing the copy numbers in chromosome, and transport system was modified to promote L-homoserine efflux. Subsequently, a strategy of cofactors synergistic utilization was proposed and successfully applied to achieve L-homoserine hyperproduction. The final engineered strain could efficiently produce 85.29 g/L L-homoserine, which was the highest production level ever reported from a plasmid-free, antibiotic-free, inducer-free and nonauxotrophic strain. These strategies used here can be considered for developing microbial cell factory of other L-aspartate derivatives.

L-高丝氨酸是一种有价值的氨基酸，可作为合成各种重要化合物的平台化学品。开发用于高水平 L-高丝氨酸生产的微生物菌株是近年来一个有吸引力的研究方向。在这里，我们转化了野生型大肠杆菌使用系统的代谢工程策略，对 L-高丝氨酸的非营养缺陷型和无质粒的超生产者。首先，通过调节L-高丝氨酸降解途径和增强合成流动获得初始菌株。为了促进 L-高丝氨酸的产生，通过优化染色体中的拷贝数来调整通量控制基因，并修改转运系统以促进 L-高丝氨酸流出。随后，提出了一种辅助因子协同利用的策略，并成功应用于实现L-高丝氨酸的过度生产。最终的工程菌株可以有效地生产 85.29 g/L L-高丝氨酸，这是迄今为止报道的无质粒、无抗生素、无诱导剂和非营养缺陷型菌株的最高生产水平。