Bleomycin biosynthesis, insights into a hybrid nonribosomal peptide synthetase/polyketide synthase system

Bleomycin (BLM), an extensively used anticancer antibiotic produced by Streptomyces verticillus, is an example of a peptide-polyketide metabolite. Compounds derived from amino acids and short fatty acid units are produced by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), respectively. Structural and catalytic similarities between modular NRPSs and PKSs prompted us to propose a hybrid NRPS/PKS/NRPS system for BLM assembly. To confirm the hybrid NRPS/PKS/NRPS model for BLM assembly the gene cluster encoding for the biosynthesis of BLM in Streptomyces verticillus was cloned and completely sequenced. The blm gene cluster contained 30 genes clustered with the previously characterized blmAB resistance genes. The sequence of the blm gene cluster supported our NRPS/PKS/NRPS model, consisting of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module (BlmVIII), and other genes encoding for biosynthesis, regulatory, and resistance proteins. One of the most striking features of the Blm system was BlmVIII with a unique modular organization consisting of an embedded methyltransferase domain. To characterize BlmVIII and to test the possibility that BlmVIII could be used for combinatorial biosynthesis of polyketides, a domain swapping strategy was adopted in which the respective domains from BlmVIII were interchanged for the respective PKS domains from a 6-deoxyerthyonlide B synthase (DEBS) model system. In addition, to substantiate the NRPS/PKS/NRPS model for BLM assembly, efforts were made to produce individual NRPS and PKS proteins from the Blm synthetase in E. coli. Although it proved difficult to produce full-length NRPS proteins in E. coli, several domains and NRPS proteins could be produced and phosphopantetheinylated by Svp, a phosphopantetheinyl transferase from Streptomyces verticillus . BlmVIII was also produced in E. coli and could be phosphopantetheinylated by Svp then loaded with the predicted substrate, malonyl-CoA. The in vitro studies on the NRPS proteins and BlmVIII have set the stage for future biochemical investigation of NRPS/PKS and PKS/NRPS interfaces in the biosynthesis of BLM. The genetic and biochemical information gathered here regarding BLM biosynthesis will facilitate future pathway engineering efforts to generate new analogs of BLM.