Metabolic Control for High-Efficiency Ectoine Synthesis in Engineered Escherichia coli.

Abstract

Ectoine is a pivotal natural osmoprotectant that functions as a compatible solute through osmoregulation, enabling microorganisms to thrive in extreme environments such as high salinity. To meet market demands, this study focuses on optimizing its production process. We initially engineered the ectABC gene cluster from Halomonas venusta via 5'-UTR modification, establishing a functional ectoine biosynthesis pathway in E. coli. Subsequent introduction of a rate-limiting enzyme EctB mutant (E407D) and aspartokinase mutant increased titer by 140%. To address lysine byproduct accumulation, an innovative molecular switch was employed to regulate lysA gene expression, achieving dynamic balance between cell growth and product synthesis. Further optimization through cofactor engineering yielded the final strain ECT31, which produced 164.6 g/L ectoine in a 100 L bioreactor within 117 h, the highest reported titer for E. coli-based ectoine production to date. The metabolic engineering strategy presented herein establishes a new pdigm for efficient biosynthesis of amino acid derivatives.