Promoter Engineering of the Surfactin Operon Enhances Surfactin Production in the Environmental Strain Bacillus subtilis RI4914

Abstract

This study aimed to enhance surfactin production in Bacillus subtilis by replacing the native promoter of the surfactin synthase operon (P srfA ) with the strong IPTG-inducible promoter Pgrac to address challenges in large-scale biosurfactant production. The engineered strain was developed through homologous recombination, creating an integration cassette fused via overlap extension PCR and transformed into B. subtilis RI4914. Surfactin yields were assessed via oil spreading and UPLC-MS. Results showed that surfactin synthesis in the modified strain was nearly absent without IPTG. With IPTG, however, production surged 8.4-fold compared to the wild type, reaching 4.8 g/L. Productivity and carbon source yield also improved 8-fold and 11-fold, respectively. This promoter engineering strategy significantly enhanced surfactin production, with the observed increase being consistent with the expected behavior of IPTG-inducible Pgrac systems in Bacillus subtilis, suggesting an effective regulatory response associated with the tested expression system.

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