Molecular characterization and analysis of the biosynthetic gene cluster for the antitumor antibiotic mitomycin C from Streptomyces lavendulae NRRL 2564

Background: The mitomycins are natural products that contain a variety of f unctional groups, including aminobenzoquinone- and aziridine-ring systems. Mitomycin C (MC) was the first recognized bioreductive alkylating agent,and has been widely used clinically for antitumor therapy. Precursor-feeding s tudies showed that MC is derived from 3-amino-5-hydroxybenzoic acid (AHBA), D-glucosamine, L-methionine and carbamoyl phosphate. A genetically linked AHBA biosynthetic gene and MC resistance genes were identified previously i n the MC producer Streptomyces lavendulae NRRL 2564, We set out to identify other genes involved in MC biosynthesis. Results: A cluster of 47 genes spanning 55 kilobases of S. lavendulae DNA g overns MC biosynthesis. Fourteen of 22 disruption mutants did not express o r overexpressed MC, Seven gene products probably assemble the AHBA intermed iate through a variant of the shikimate pathway, The gene encoding the firs t presumed enzyme in AHBA biosynthesis is not, however, linked within the M C cluster. Candidate genes for mitosane nucleus formation and functionaliza tion were identified. A putative MC translocase was identified that compris es a novel drug-binding and export system, which confers cellular self-prot ection on S. lavendulae. Two regulatory genes were also identified. Conclusions: The overall architecture of the MC biosynthetic gene cluster i n S. lavendulae has been determined. Targeted manipulation of a putative MC pathway regulator led to a substantial increase in drug production. The cl oned genes should help elucidate the molecular basis for creation of the mi tosane ring system, as well efforts to engineer the biosynthesis of novel n atural products.