Functional Characterization and Metabolic Engineering of Key Genes in L-Cysteine Biosynthesis in Bacillus licheniformis

This study systematically characterized the L-cysteine biosynthetic pathway in Bacillus licheniformis and demonstrated that exogenous serine supplementation significantly upregulated the expression of pathway-associated genes, confirming serine as the primary precursor driving L-cysteine synthesis. Through targeted gene deletions, we generated knockout strains BL2ΔglyA, BL2ΔsdaAA, BL2ΔmetC, BL2Δ2, and BL2Δ3 to minimize precursor diversion and product degradation. Combinatorial overexpression of the feedback-resistant mutant cysEf and the transporter eamA yielded an engineered strain achieving 1.075 g/L L-cysteine in shake-flask fermentation with an 18.69% molar conversion yield. These findings highlight the potential of B. licheniformis as a platform for sulfur metabolic engineering and provide a sustainable fermentation strategy to replace traditional high-pollution hydrolysis-based L-cysteine production. Additionally, this work reveals fundamental differences in sulfur metabolism networks between Gram-positive and Gram-negative bacteria, elucidating microbial metabolic diversity and the cross-regulatory mechanisms linking sulfur, carbon, and nitrogen metabolism.