K. Eppelmann et al., Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis, J BIOL CHEM, 276(37), 2001, pp. 34824-34831
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.