Metabolic Remodelingof the NOG Pathway and Glyoxylate Cycle for High‐Yield L‐Homoserine Biosynthesis

L‑homoserine is a valuable C4 amino acid with broad applications in agriculture, pharmaceuticals, and daily chemicals. However, existing high‑yielding strains have mainly been evaluated at laboratory scale, and their scale‑up feasibility has not yet been sufficiently verified, leaving industrial production without a direct engineering basis. In this study, a non-auxotrophic, scalable L‑homoserine high‑producer was constructed by targeting carbon loss, rewiring the metabolic network, and maintaining cofactor balance. Specifically, the threonine branch was dynamically attenuated to reduce product degradation, while the non‑oxidative glycolysis (NOG) pathway was introduced and coupled with the glyoxylate shunt to conserve carbon and redirect flux toward the aspartate pathway, thereby increasing precursor availability. The main biosynthetic pathway and cofactor regeneration were further strengthened to accelerate L‑homoserine synthesis. Ultimately, strain HS25 achieved a titer of 126.5 g/L L‑homoserine,a yield of 0.58 g/g glucose, and a productivity of 1.77 g/L/hin 50 L fermentation. These metabolic engineering strategies are scalable and may serve as a generalizable framework for the industrial production of other aspartate‑derived amino acids and their derivatives.