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.

中文翻译：

---

通过代谢工程在大肠杆菌中生长耦合生产 L-异亮氨酸


L-异亮氨酸是一种必需氨基酸，广泛用于制药和食品工业。然而，目前的生产效率不足以满足日益增长的需求。在这项研究中，我们旨在开发一种高效的 L-异亮氨酸产大肠杆菌菌株。首先，通过使用反馈抗性酶实现 L-异亮氨酸的积累。接下来，通过引入基于 metA-metB 的 α-酮丁酸生成旁路，建立了生长偶联 L-异亮氨酸合成途径，该旁路将 L-异亮氨酸的产生显着提高至 7.4 g/L。采用从适应性实验室进化中获得的活性改进的胱硫醚 γ-合酶突变体后，L-异亮氨酸产量进一步增加到 8.5 g/L。随后，通过绕过 NADPH 依赖性天冬氨酸氨基转移酶途径并采用 NADH 依赖性途径和转氢酶来改善氧化还原通量。最后，通过改变运输系统来增强 L-异亮氨酸外排。补料分批发酵 48 h 后，所得菌株 ISO-12 的 L-异亮氨酸生产滴度达到 51.5 g/L，葡萄糖产量为 0.29 g/g。本研究开发的菌株实现了比以前报道的更高的 L-异亮氨酸生产效率。这些策略将有助于生产 L-异亮氨酸和相关产品的细胞工厂的发展。