L-valine is an essential amino acid and an important amino acid in the food and feed industry. The relatively low titer and low fermentation yield currently limit the large-scale application of L-valine. Here, we constructed a chromosomally engineered Escherichia coli to efficiently produce L-valine. First, the synthetic pathway of L-valine was enhanced by heterologous introduction of a feedback-resistant acetolactate acid synthase from Bacillus subtilis and overexpression of other two enzymes in the L-valine synthetic pathway. For efficient efflux of L-valine, an exporter from Corynebacterium glutamicum was subsequently introduced. Next, the precursor pyruvate pool was increased by knockout of GTP pyrophosphokinase and introduction of a ppGpp 3′-pyrophosphohydrolase mutant to facilitate the glucose uptake process. Finally, in order to improve the redox cofactor balance, acetohydroxy acid isomeroreductase was replaced by a NADH-preferring mutant, and branched-chain amino acid aminotransferase was replaced by leucine dehydrogenase from Bacillus subtilis. Redox cofactor balance enabled the strain to synthesize L-valine under oxygen-limiting condition, significantly increasing the yield in the presence of glucose. Two-stage fed-batch fermentation of the final strain in a 5 L bioreactor produced 84 g/L L-valine with a yield and productivity of 0.41 g/g glucose and 2.33 g/L/h, respectively. To the best of our knowledge, this is the highest L-valine titer and yield ever reported in E. coli. The systems metabolic engineering strategy described here will be useful for future engineering of E. coli strains for the industrial production of L-valine and related products.

L-缬氨酸是一种必需氨基酸，是食品和饲料工业中的重要氨基酸。相对较低的滴度和较低的发酵产率目前限制了L-缬氨酸的大规模应用。在这里，我们构建了一种染色体工程大肠杆菌来有效地生产 L-缬氨酸。首先，L-缬氨酸的合成途径通过异源引入来自枯草芽孢杆菌的抗反馈乙酰乳酸合酶和L-缬氨酸合成途径中其他两种酶的过表达而得到增强。为了有效流出 L-缬氨酸，谷氨酸棒状杆菌的输出者随后被介绍。接下来，通过敲除 GTP 焦磷酸激酶和引入 ppGpp 3'-焦磷酸水解酶突变体来增加前体丙酮酸库，以促进葡萄糖摄取过程。最后，为了改善氧化还原辅因子平衡，乙酰羟酸异构还原酶被 NADH 优先突变体取代，支链氨基酸转氨酶被枯草芽孢杆菌的亮氨酸脱氢酶取代. 氧化还原辅因子平衡使该菌株能够在限氧条件下合成 L-缬氨酸，在葡萄糖存在下显着提高产量。最终菌株在 5 L 生物反应器中的两阶段补料分批发酵产生 84 g/L L-缬氨酸，产量和生产率分别为 0.41 g/g 葡萄糖和 2.33 g/L/h。据我们所知，这是有史以来在大肠杆菌中报道的最高 L-缬氨酸滴度和产量。这里描述的系统代谢工程策略将有助于大肠杆菌菌株的未来工程，用于 L-缬氨酸和相关产品的工业生产。