Assembly and heterologous expression of the coumermycin A1 gene cluster and production of new derivatives by genetic engineering

Many secondary metabolites of clinical importance have been isolated from different Streptomyces species. As most of the natural producers remain difficult to handle genetically, heterologous expression of an entire biosynthetic gene cluster in a well characterised host allows improved possibilities for modifications of the desired compound by manipulation of the biosynthetic genes. However, the large size of a functional gene cluster often prevents its direct cloning into a single cosmid clone. Here we describe a successful strategy to assemble the entire coumermycin A1 biosynthetic gene cluster (38.6 kb) into a single cosmid clone by lambda RED recombination technology. Heterologous expression of the reconstituted gene cluster in Streptomyces coelicolor M512 resulted in the heterologous production of coumermycin A1. Inactivation of the methyltransferase gene couO--responsible for the C-methylation at the 8-positions of the aminocoumarin moieties in coumermycin A1--and heterologous expression of the modified cluster resulted in an accumulation of a C-8-unsubstituted coumermycin A1 derivative. Subsequent expression of the halogenase gene clo-hal from the clorobiocin gene cluster in the heterologous producer strain led to the formation of two new hybrid antibiotics, containing either one or two chlorine atoms. The identities of the new compounds were verified by LC-MS, and their antibacterial activities were tested against Bacillus subtilis in an agar diffusion assay.