Metabolic engineering of Bacillus subtilis for high-yield surfactin production

The industrial application of surfactin has been constrained by the low production capacity of Bacillus subtilis. This study developed a high-yield, high-tolerance surfactin-producing strain through a multi-level metabolic engineering strategy. First, we constructed an enhanced P43 promoter (P43LN) by extending its upstream region from 260 to 520 bp and pairing it with a cognate 5' UTR, which exhibited more than threefold higher activity than the commonly used core P43 promoter, providing a versatile tool for stable and high-level expression of key genes. Based on this, the native srfA promoter in the oilfield-derived B. subtilis TD7 was replaced with the P43 promoter, yielding the engineered strain TP1. Subsequently, systematic knockout of competing antimicrobial peptide synthesis gene clusters (pps, bac, and pks) increased surfactin titer to 2.78 g/L. Further synergistic enhancement of NADPH regeneration (via zwf overexpression) and cellular tolerance (via efflux pump gene yerP overexpression) resulted in the final strain TS8, which achieved a shake-flask surfactin titer of 3.51 g/L, representing a 102.7% increase in per-OD₆₀₀ productivity. This work successfully integrates promoter engineering, metabolic flux redistribution, and tolerance modules, not only significantly improving surfactin production but also providing a powerful expression and systematic optimization toolkit for B. subtilis, laying a technical foundation for the efficient biomanufacturing of complex natural products.