Tailoring Escherichia coli for high-yield production of O-acetyl-L-homoserine through multi-node metabolic regulation.

O-acetyl-L-homoserine (OAH) is a key precursor for the biosynthesis of L-methionine and various C4 compounds, with significant industrial potential. However, efficient microbial production of OAH remains challenging due to complex metabolic regulation and precursor limitations. In this study, we rationally developed a plasmid-free, non-auxotrophic Escherichia coli strain to produce OAH. We modularized the OAH synthetic pathway into L-homoserine and acetyl-CoA modules, enhanced each module individually, and identified a highly efficient L-homoserine O-acetyltransferase (MetX) from Cyclobacterium marinum. Using small RNA screening, we pinpointed critical metabolic nodes and fine-tuned the pathway flux through promoter engineering and regulatory elements. Notably, we balanced the acetyl-CoA and L-homoserine synthesis with moderate expression of pyruvate carboxylase, weakened the TCA cycle by modulating citrate synthase and the branched-chain amino acid pathway by attenuating BCAA aminotransferase, thereby redirecting carbon flux towards OAH production. Additionally, we optimized the threonine attenuator for dynamic regulation of the threonine pathway and enhanced intracellular ATP turnover. Under a two-stage pH control fermentation strategy, the final plasmid-free and non-auxotrophic strain OAH37 achieved a titer of 94.1 g/L OAH, with a yield of 0.42 g/g glucose and a productivity of 1.37 g/L/h. Our work demonstrates the potential of metabolic engineering strategies for efficient microbial synthesis of OAH, providing a foundation for industrial-scale production of this important precursor.