Accumulation of the angucycline antibiotic rabelomycin after disruption of an oxygenase gene in the jadomycin B biosynthetic gene cluster of Streptomyces venezuelae.
DNA from a region downstream of and overlapping the polyketide synthase (PKS) gene cluster for jadomycin B biosynthesis in Streptomyces venezuelae was cloned and sequenced. Analysis of the nucleotide sequence located one complete ORF (ORF6), an incomplete one representing the 3' region of ORF4 in the PKS cluster, and a second incomplete one (ORF7). The deduced amino acid sequences for ORFs 6 and 7 resemble those of oxygenases. Since a plausible biosynthetic pathway for jadomycin B includes an angular polyketide intermediate that undergoes oxidative ring fission before condensation with an amino acid, we subcloned one of the presumptive oxygenase genes (ORF6) in a segregationally unstable shuttle vector (pHJL400) and disrupted it by inserting the gene for apramycin resistance. Transformation of S. venezuelae with the disruption vector and selection for apramycin resistance gave mutants blocked in jadomycin biosynthesis. Southern hybridization confirmed that gene replacement had occurred. Cultures of the mutants accumulated a metabolite identified by comparison with an authentic sample as rabelomycin, a non-nitrogenous polyketide-derived antibiotic originally isolated from Streptomyces olivaceus.
Yang
Keqian
K
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1.
Han
Lei
L
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1.
Ayer
Stephen W
SW
Institute for Marine Biosciences, National Research Council of Canada, Halifax, Nova Scotia, Canada B3H 3Z1.
Vining
Leo C
LC
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1.
D000595:Amino Acid Sequence; D000880:Anthraquinones / Q000145:classification / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000145:classification / Q000378:metabolism*; D001483:Base Sequence; D005798:Genes, Bacterial; D007546:Isoquinolines / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics; D005810:Multigene Family; D016296:Mutagenesis; D016366:Open Reading Frames; D010105:Oxygenases / Q000172:deficiency / Q000235:genetics*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D000095602:Angucyclines and Angucyclinones
D000880:Anthraquinones; D000900:Anti-Bacterial Agents; D007546:Isoquinolines; D009097:Multienzyme Complexes; D010105:Oxygenases; C081750:jadomycin B; C003289:rabelomycin; D000095602:Angucyclines and Angucyclinones