Dynamic regulation of iturin production via reconstructing the quorum-sensing system ComQXPA in Bacillus subtilis

Pheromone ComX is a critical element of the quorum-sensing (QS) system in Bacillus subtilis. It activates the surfactin promoter (P_srf) to initiate surfactin production in a cell-density-dependent manner. In this study, the natural promoter P_itu of B. subtilis 1A751 WR-itu, a lipopeptide iturin mono-producing parent strain, was replaced by the constitutive promoter P₄₃, QS P_srf, and the mutated QS P_M-srf, generating dynamic regulation systems to improve iturin production. HPLC analysis revealed that the P_M-srf promoter-harbouring strain significantly enhanced iturin production to 409.33 ± 16.77 mg·L^- 1, 2.15 times higher than the parent strain. Further identification by UPLC-HRESI-MS/MS and GC-MS indicated that the strain could produce four new C₁₀-C₁₃ iturins. Moreover, the ComX degrading enzymes AprE or NprE were down-regulated by the CRISPR interference (CRISPRi) system, increasing iturin production to 526.46 ± 18.43 mg·L^- 1 and 416.99 ± 17.02 mg·L^- 1, respectively. Interestingly, iturin production was further increased to 579.85 ± 19.83 mg·L^- 1 under simultaneous down-regulation of AprE and NprE, 3.05 times higher than the parent strain. The reconstructed ComQXPA QS system in B. subtilis combines the inhibitory of AprE and NprE to dynamically up and down-regulate the expression of iturin operon, providing an effective pipeline for regulating other bioactive molecules.