High-yield ectoine production in engineered Corynebacterium glutamicum by fine metabolic regulation via plug-in repressor library

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 CglysCS301Y). 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.