Surfactin–Bacillaene Copathway Engineering Strategy Boosts Fengycin Production and Antifungal Activity in Bacillus velezensis HN-Q-8

Previous studies have demonstrated that <i>Bacillus velezensis</i> HN-Q-8 shows significant inhibitory effects against various plant pathogenic fungi causing potato diseases, primarily attributed to the production of fengycin. However, the low yield of fengycin in wild-type strains limits its practical application, and the influence of its biosynthesis pathway on volatile organic compound production remains unclear. In this study, to enhance fengycin production in <i>Bacillus velezensis</i> HN-Q-8, we applied metabolic engineering by targeting competitive pathways. Specifically, a double mutant (<i>ΔsrfAAΔbaeBE</i>) was constructed by knocking out the surfactin synthase gene <i>srfAA</i> and the bacillaene synthesis gene <i>baeBE</i>. The fengycin yield of the <i>ΔsrfAAΔbaeBE</i> mutant in the basal (sodium glutamate) fermentation medium reached 98.83 mg/L, representing a 2.39-fold increase over the wild-type strain. Subsequent medium optimization by supplementing peptone further boosted production to 155.61 mg/L, which was 3.77-fold higher than the wild-type level. The lipopeptide extract from the double mutant strain <i>ΔsrfAAΔbaeBE</i> demonstrated potentiated antifungal activity against four major potato fungal pathogens: <i>Alternaria solani</i> (early blight), <i>Rhizoctonia solani</i> (black scurf), <i>Fusarium oxysporum</i> (wilt), and <i>Botrytis cinerea</i> (gray mold). The active volatile compounds released by <i>ΔsrfAAΔbaeBE</i>, such as benzaldehyde and 2,5-dimethylpyrazine were significantly increased. The knockout of <i>srfAA</i> and <i>baeBE</i> also distinctly altered the physiology of the strain: the double mutant exhibited enhanced biofilm formation, an accelerated early growth rate followed by early decline, and a severely reduced sporulation capacity. These results confirmed the feasibility of molecularly modifying <i>Bacillus velezensis</i> HN-Q-8 to improve fengycin production and antifungal activity for further agricultural application.