谷氨酸棒杆菌
亮氨酸
生物化学
代谢工程
大肠杆菌
柠檬酸合酶
氨基酸
酶
产量(工程)
柠檬酸循环
转氨酶
化学
生物
基因
冶金
材料科学
作者
Xiaohu Ding,Wenjun Yang,Xiaobin Du,Ning Chen,Qingyang Xu,Minhua Wei,C. H. Zhang
标识
DOI:10.1016/j.ymben.2023.06.003
摘要
L-leucine is an essential amino acid widely used in food and pharmaceutical industries. However, the relatively low production efficiency limits its large-scale application. In this study, we rationally developed an efficient L-leucine-producing Escherichia coli strain. Initially, the L-leucine synthesis pathway was enhanced by overexpressing feedback-resistant 2-isopropylmalate synthase and acetohydroxy acid synthase both derived from Corynebacterium glutamicum, along with two other native enzymes. Next, the pyruvate and acetyl-CoA pools were enriched by deleting competitive pathways, employing the nonoxidative glycolysis pathway, and dynamically modulating the citrate synthase activity, which significantly promoted the L-leucine production and yield to 40.69 g/L and 0.30 g/g glucose, respectively. Then, the redox flux was improved by substituting the native NADPH-dependent acetohydroxy acid isomeroreductase, branched chain amino acid transaminase, and glutamate dehydrogenase with their NADH-dependent equivalents. Finally, L-leucine efflux was accelerated by precise overexpression of the exporter and deletion of the transporter. Under fed-batch conditions, the final strain LXH-21 produced 63.29 g/L of L-leucine, with a yield and productivity of 0.37 g/g glucose and 2.64 g/(L h), respectively. To our knowledge, this study achieved the highest production efficiency of L-leucine to date. The strategies presented here will be useful for engineering E. coli strains for producing L-leucine and related products on an industrial scale.
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