Metabolic engineering of Escherichia coli for efficient production of l-homoserine from sugarcane molasses.

l-homoserine is a crucial platform chemical serving as a precursor for high-value compounds, with steadily growing global market demand. However, its industrial bioproduction remains constrained by high feedstock costs, reliance on plasmid-based systems, and suboptimal carbon utilization efficiency. Here we report the systematic engineering of Escherichia coli for efficient l-homoserine production from low-cost sugarcane molasses. Through introducing the sucrose assimilation gene cluster (scrKYAB) from Salmonella typhimurium, the engineered strain acquired the capacity to utilize sucrose, achieving a titer of 3.80 g/L. Subsequent chromosomal integration of the l-homoserine efflux transporters RhtB and Cg0701, combined with deletion of pykF and overexpression of pycP458Scg from Corynebacterium glutamicum, yielded a plasmid-free, non-auxotrophic strain with minimized byproduct accumulation, increasing the titer to 4.73 g/L. Transcriptomic analysis further identified key metabolic targets, among which deletion of the global regulator arcA rewired central carbon flux and improved l-homoserine production by 32 %. Finally, the glucose and fructose assimilation pathways were modified to enable the co-utilization of glucose, fructose, and sucrose. Using untreated sugarcane molasses as the carbon source in a 5 L bioreactor, the engineered strain HS19 achieved an l-homoserine titer of 81.88 g/L. This represents the first successful production of the high‑value chemical L‑homoserine from this low‑cost feedstock in E. coli. When using pretreated sugarcane molasses as the carbon source, the titer reached 97.91 g/L, which represents the highest L‑homoserine titer from non‑grain carbon sources. This work provides a feasible strategy for low‑cost manufacturing of L‑homoserine and its derivatives.