Reconstruction and metabolic regulation of O-succinyl-L-homoserine biosynthesis pathway in Escherichia coli W3110.
O-Succinyl-L-homoserine (OSH) plays a pivotal role in L-methionine biosynthesis. Microbial cell factories for high-yield OSH production have been progressively optimized, achieving substantial improvements in fermentation titers. In this study, a multi-step progressive optimization strategy was adopted to construct a high-yield OSH-producing strain. First, the feedback inhibition of the key enzyme HST was relieved, and the key genes involved in the byproduct metabolic pathways were knocked out. This modification enabled engineered strain to produce 9.77 ± 0.27g/L OSH in shake-flask fermentation. Second, ribosome binding site (RBS) engineering, promoter engineering, and dynamic metabolic regulation were integrated to strengthen and balance the intracellular supply of the two core precursors, L-homoserine and succinyl-CoA. These strategies greatly increased the OSH titer to 18.54 ± 0.03g/L. Finally, global optimization of cofactor and energy optimization was carried out to further enhance strain performance, and the engineered strain OSHM40 achieved the OSH titer of 20.15 ± 0.21g/L via shake-flask cultivation, and 104.09 ± 2.06g/L in a 5-L bioreactor under fed-batch fermentation, with a sugar-acid conversion rate of 64.99% and a volumetric productivity of 1.43g/L/h. Notably, the OSH titer and sugar-acid conversion rate of this strain represent the highest levels reported to date among all plasmid-free OSH-producing strains. The plasmid-free system constructed in this study effectively avoids the plasmid-induced metabolic burden and genetic instability. This work demonstrates the prominent advantages and great application potential of plasmid-free modular engineering for the efficient biosynthesis of OSH and other high-value amino acids.