L-Homoserine is a nonessential chiral amino acid and the precursor of L-threonine and L-methionine. It has great potential to be used in the pharmaceutical, agricultural, cosmetic, and fragrance industries. However, the current low efficiency in the fermentation process of L-homoserine drives up the cost and therefore limits applications. Here, we systematically analyzed the L-homoserine production network in Escherichia coli to design a redox balance route for L-homoserine fermentation from glucose. Production of L-homoserine from L-aspartate via reduction of the tricarboxylic acid cycle intermediate oxaloacetate lacks reducing power. This deficiency could be corrected by activating the glyoxylate shunt and driving the flux from fumarate to L-aspartate with excess reducing power. This redox balance route decreases cell growth pressure and the theoretical yield of L-homoserine is 1.5 mol/mol of glucose without carbon loss. We fine-tuned the flux from fumarate to L-aspartate, deleted competitive and degradative pathways, enhanced L-homoserine efflux, and generated 84.1 g/L L-homoserine with 1.96 g/L/h productivity and 0.50 g/g glucose yield in a fed-batch fermentation. This study proposes a novel balanced redox metabolic network strategy for highly efficient production of L-homoserine and its derivative amino acids.

L-高丝氨酸是一种非必需的手性氨基酸，是 L-苏氨酸和 L-蛋氨酸的前体。它具有巨大的潜力，可用于制药、农业、化妆品和香水行业。然而，目前L-高丝氨酸发酵过程的低效率推高了成本，从而限制了应用。在这里，我们系统地分析了大肠杆菌中的 L-高丝氨酸生产网络设计从葡萄糖发酵 L-高丝氨酸的氧化还原平衡路线。通过还原三羧酸循环中间体草酰乙酸从 L-天冬氨酸生产 L-高丝氨酸缺乏还原能力。这种缺陷可以通过激活乙醛酸支路并以过量的还原能力驱动从富马酸盐到 L-天冬氨酸的通量来纠正。这种氧化还原平衡路线降低了细胞生长压力，L-高丝氨酸的理论产量为 1.5 mol/mol 葡萄糖，没有碳损失。我们微调了从富马酸盐到 L-天冬氨酸的通量，删除了竞争性和降解途径，增强了 L-高丝氨酸流出，并以 1.96 g/L/h 的生产率和 0.50 g/g 的葡萄糖产量生成了 84.1 g/L L-高丝氨酸补料分批发酵。