Abstract
Ectoine, a compatible solute and valuable cosmetic additive, is synthesized from L-aspartate-β-semialdehyde (ASA) via a three-step pathway catalyzed by EctABC. To investigate the metabolic basis of ectoine overproduction in an engineered E. coli strain E2, we employed an integrated analysis of metabolomics and quantitative PCR (qPCR). The results suggest that E2 exhibits a coordinated upregulation of central metabolism: metabolomic profiles showed elevated levels of PPP intermediates and key precursors (aspartate and oxaloacetate), consistent with increased precursor and NADPH supply. qPCR analysis confirmed the upregulated expression of genes involved in the TCA cycle, aspartate biosynthesis, and the Ectabc operon. Based on this multi-omics insight, the competing pathway of ASA toward methionine biosynthesis was selected as a potential target for metabolic intervention via knockout of the metA gene. Based on these findings, a precise metabolic intervention was implemented: knockout of the metA gene to block this diversion, which, supported by an optimized feeding strategy, resulted in a final ectoine titer of 115.52 g/L in the derived strain E2-1 after 72 hours of fermentation. This study provides insights into the metabolic basis of high-yield ectoine production and presents a paradigm of systems-level analysis for targeted engineering, providing a robust framework and a clear theoretical foundation for the rational design of advanced microbial cell factories.