Growth-coupled production of L-isoleucine in Escherichia coli via metabolic engineering

• Published in: Metabolic engineering Vol. 86; pp. 181 - 193
• Main Authors: Lu, Nan, Wei, Minhua, Yang, Xuejing, Li, Yingzi, Sun, Hao, Yan, Qianyu, Zhang, Haibin, He, Jilong, Ma, Jie, Xia, Menglei, Zhang, Chenglin
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.11.2024
• Subjects: Efflux; Escherichia coli; Growth-coupled production; L-isoleucine; Metabolic engineering; Redox flux; Redox flux; Efflux; Metabolic engineering; Growth-coupled production; Escherichia coli; L-isoleucine
• ISSN: 1096-7176; 1096-7184; 1096-7184
• DOI: 10.1016/j.ymben.2024.10.004

L-isoleucine, an essential amino acid, is widely used in the pharmaceutical and food industries. However, the current production efficiency is insufficient to meet the increasing demands. In this study, we aimed to develop an efficient L-isoleucine-producing strain of Escherichia coli. First, accumulation of L-isoleucine was achieved by employing feedback-resistant enzymes. Next, a growth-coupled L-isoleucine synthetic pathway was established by introducing the metA-metB-based α-ketobutyrate-generating bypass, which significantly increased L-isoleucine production to 7.4 g/L. Upon employing an activity-improved cystathionine γ-synthase mutant obtained from adaptive laboratory evolution, L-isoleucine production further increased to 8.5 g/L. Subsequently, the redox flux was improved by bypassing the NADPH-dependent aspartate aminotransferase pathway and employing the NADH-dependent pathway and transhydrogenase. Finally, L-isoleucine efflux was enhanced by modifying the transport system. After fed-batch fermentation for 48 h, the resultant strain, ISO-12, reached an L-isoleucine production titer of 51.5 g/L and yield of 0.29 g/g glucose. The strains developed in this study achieved a higher L-isoleucine production efficiency than those reported previously. These strategies will aid in the development of cell factories that produce L-isoleucine and related products. [Display omitted] •Growth-coupled production of L-isoleucine was achieved in Escherichia coli.•Feedback regulation of key enzymes in L-isoleucine synthesis was abrogated.•A growth-coupled L-isoleucine synthetic pathway was established.•The redox flux was improved via NADH dependent pathways and transhydrogenase.•High production titer and yield of L-isoleucine was fulfilled.