Metabolic engineering of Bacillus licheniformis DW2 for ectoine production.

Ectoine is a high-value protective agent with extensive applications in the fields of fine chemicals and biopharmaceuticals, and it is naturally synthesized by Halomonas in extreme environment, however, the current production level cannot meet the growing market demand. In this study, we aimed to develop an efficient and environmentally friendly ectoine production process using Bacillus licheniformis as the host organism. Through introducing ectoine synthetase gene cluster ectABC from Halomonas elongate, as well as optimizing ectABC expression by promoter and 5'-UTR optimization, ectoine titer was increased to 0.55 g/L. Furthermore, subsequent introduction of exogenous phosphoenolpyruvate carboxylase PPC and down-regulated expression of phosphoenolpyruvate carboxykinase PCK optimized the carbon flux through C4 anaplerotic pathway, and further benefited ectoine synthesis. Furthermore, the carbon flux towards aspartic acid accumulation was increased through optimization of glyoxylate and TCA cycles, accompanied with introducing lysC and asd, and blocking by-products pathways, ectoine titer produced by B. licheniformis ECT12 was 2.00 g/L. Moreover, NADPH supply was enhanced by overexpression of exogenous NADH kinase Pos5, and ectoine transportation was improved by introducing compatible solute transporter ProP from Escherichia coli, and the resulting B. licheniformis ECT14 was able to produce 2.60 g/L ectoine. Last but not the least, the ectoine yield of 3.29 g/L was attained in a 5-L fermenter. Taken together, this study not only established B. licheniformis as a framework for sustainable production of ectoine, but also paved the way for achieving the industrial production of ectoine and aspartic acid derivatives in the future.