Ectoine is a high-value protective and stabilizing agent with different applications in biopharmaceuticals, biotechnology, and fine chemicals. Here, efficient production of ectoine in Corynebacterium glutamicum was achieved by combination of metabolic engineering and plug-in repressor library strategy. First, the ectBAC cluster from Pseudomonas stutzeri was introduced into strain K02, and the titer of the obtained strain was 2.12 g/L. Metabolic engineering was then performed for further optimization, including removal of competing pathways (pck and ldh knockout), deletion of glycolysis repressor (sugR knockout), and enhancement of precursor supply (overexpression of Ecasd and CglysC^S301Y). Next, two repressor libraries were designed for targeted flux control to improve ectoine production. Finally, strain CB5L6 produced 45.52 g/L ectoine and had the highest yield in C. glutamicum. For the first time, plug-in repressor library was employed to engineer C. glutamicum for metabolites production, which will provide a guideline for the construction of microbial cell factories.

Ectoine 是一种高价值的保护剂和稳定剂，在生物制药、生物技术和精细化学品中具有不同的应用。在这里，通过代谢工程和插入式阻遏物库策略的结合，实现了谷氨酸棒杆菌中四氢嘧啶的高效生产。首先，将来自施氏假单胞菌的ectBAC簇导入菌株K02，得到的菌株滴度为2.12g/L。然后进行代谢工程以进一步优化，包括去除竞争途径（pck和ldh敲除）、删除糖酵解阻遏物（sugR敲除）和增强前体供应（Ecasd和CglysC ^S301Y）。接下来，设计了两个阻遏物库用于有针对性的通量控制，以提高四氢嘧啶的产量。最后，菌株CB5L6产生45.52 g/L的四氢嘧啶，在谷氨酸棒杆菌中产量最高。首次采用插件阻遏库对谷氨酸棒杆菌进行代谢物生产工程，为微生物细胞工厂的建设提供指导。