Boosting L-homoserine production by non-auxotrophic E. coli strain via iterative rational design and tolerance...

L-Homoserine is a non-proteinogenic amino acid utilized as a precursor in the biosynthesis of various value-added chemicals. In this study, we developed a new L-homoserine-producing strain by integrating rational re-engineering with tolerance evolution. The non-auxotrophic, plasmid-free chassis, based on previously engineered strain HS15 (Metabolic Engineering, 2021, 67: 321-329), was constructed through dynamic regulation of the biosynthesis of competitive essential amino acids and fine-tuning of the chromosomal expression of key pathway genes via semi-rationally designed 5'-untranslated regions. Subsequently, the helicase-deaminase-based mutator system accelerated adaptive laboratory evolution, significantly enhancing the strain's tolerance to over 100 g/L L-homoserine. The designated strain NS18 achieved a titer of 144.5 g/L with a yield of 0.48 g/g and a productivity of 3.01 g/L/h in a cost-effective, minimal salt fermentation medium, representing the highest titer and productivity reported to date. Multi-omics analyses revealed a redox-correlated mechanism underlying the enhanced L-homoserine production. Furthermore, the upregulation of branched-chain amino acid synthesis suggests a more favorable cellular adaptation in evolved strain NS18 to environmental stresses. In summary, this research effectively tackles inherent metabolic constraints and offers important insights into optimizing microbial cells at the systems level for L-homoserine production.