Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis

Nonribosomal peptides are processed on multifunctional enzymes called nonri bosomal peptide synthetases (NRPSs), whose modular multidomain arrangement allowed the rational design of new peptide products. However, the lack of n atural competence and efficient transformation methods for most of nonribos omal peptide producer strains prevented the in vivo manipulation of these b iosynthetic gene clusters. In this study, we present methods for the constr uction of a genetically engineered Bacillus subtilis surrogate host for the integration and heterologous expression of foreign NRPS genes. In the B. s ubtilis surrogate host, we deleted the resident 26-kilobase srfA gene clust er encoding the surfactin synthetases and subsequently used the same chromo somal location for integration of the entire 49-kilobase bacitracin biosynt hetic gene cluster from Bacillus licheniformis by a stepwise homologous rec ombination method. Synthesis of the branched cyclic peptide antibiotic baci tracin in the engineered B. subtilis strain was achieved at high level, ind icating a functional production and proper posttranslational modification o f the bacitracin synthetases BacABC, as well as the expression of the assoc iated bacitracin self-resistance genes. This engineered and genetically ame nable B. subtilis strain will facilitate the rational design of new bacitra cin derivatives.