Abstract

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-OD 600 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. Graphical abstract