Stepwise Optimization and Transcription Factor Engineering for Efficient Ectoine Production in Escherichia coli

Abstract Ectoine, a high-value compatible solute, is widely used in cosmetics, biopharmaceuticals, and the pharmaceutical industry. Although non-halophilic chassis such as Escherichia coli are preferred for industrial ectoine production, unclear metabolic distributions and physiological constraints have limited titers and productivity. Here, we report a systematic metabolic engineering strategy for high-performance ectoine biosynthesis in E. coli. Introduction of the ectoine gene cluster from Halomonas elongata, coupled with enhanced flux from phosphoenolpyruvate to l-aspartate-β-semialdehyde, established de novo ectoine production. Combinatorial optimization for promoters and expression of an EctBE407D variant collectively increased the ectoine titer by 353% in strain ECT08. Furthermore, the supply of synthetic precursors l-glutamate and oxaloacetic acid was enhanced, elevating the bioproduct to 9.09 g/L in strain ECT18, coupled with a deliberate downregulation of the TCA cycle flux. Transcriptional regulator screening further identified Lrp as a growth-coupled engineering target, whose manipulation enhanced both titer and biomass by 19.83% and 28.62%, respectively. Importantly, metabolic profiles indicated that the Lrp supervised a multi-node regulatory program that alleviated carbon supply, precursor conversion, nitrogen donor availability, and energy efficiency. In fed-batch fermentation, the final strain ECT31 was cultivated in a 7 L bioreactor and induced at 10 h, producing 81.39 g/L ectoine with a yield of 0.31 g/g and a productivity of 0.75 g/L/h. These systematic strategies establish a robust paradigm for the efficient microbial production of aspartate-derived compounds.