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Plipastatin is a cyclic lipopeptide synthesized by non-ribosomal peptide synthetases (NRPS), which has a diverse range of applications in postharvest preservation of fruits and vegetables, biological control, and feed processing. Whereas the yield of plipastatin in wild Bacillus sp. is low, its chemical structure is complex and challenging to synthesize, significantly limiting its production and application. ComQXPA-PsrfA, a quorum-sensing (QS) circuit from Bacillus amyloliquefaciens, was constructed in this study. Two QS promoters MuPsrfA and MtPsrfA, with 35 and 100% increased activity, respectively, were obtained by mutating the original promoter PsrfA. Thus, the natural promoter of plipastatin was replaced by a QS promoter to achieve the dynamic regulation of plipastatin, which increased the yield of plipastatin by 3.5 times. Integrating ComQXPA into plipastatin mono-producing M-24:MtPsrfA increased the yield of plipastatin to 3850 mg/L, representing the highest yield reported to date. Four new plipastatins were identified via UPLC-ESI-MS/MS and GC–MS analysis of fermentation products of mono-producing engineered strains. Among them, three plipastatins contained two double bonds in the fatty acid side chain, representing the first example of a new type of plipastatin. Our results indicate that the QS system ComQXPA-PsrfA of Bacillus can dynamically regulate plipastatin production, and the pipeline could be extended to the other strains to regulate target products dynamically.

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