<p>Pheromone ComX is a critical element of the quorum-sensing (QS) system in <i>Bacillus subtilis</i>. 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 <i>B. subtilis</i> 1A751 WR-<i>itu</i>, 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 <i>B. subtilis</i> 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.</p> Graphical Abstract <p></p>