| The sequenced region of the Streptomyces venezuelae ISP5230 chromosome, containing a gene cluster for biosynthesis of the antibiotic jadomycin B, was extended in both directions by chromosome walking. At the right-hand end, 13 new genes were added: these began with jadM, which encoded a phosphopantetheinyl transferase and partially overlapped jadL. Expression of jadM in E. coli and examination of the product by SDS-PAGE confirmed formation of a 29 KDa protein predicted from the jadM sequence. Northern hybridization indicated that biosynthesis of jadomycin B correlated with jadM expression. Since cultures of S. venezuelae disrupted in jadM were defective in jadomycin B production, but grew well and produced chloramphenicol normally, jadM was presumed to encode a holo-ACP synthase dedicated to jadomycin B biosynthesis. Downstream of jadM was a gene (jadN) encoding an acyl-CoA synthase/decarboxylase. This enzyme probably condenses acyl-coenzyme A precursors to synthesize the core linear polyketide. The adjacent genes jadX, O, P, Q, S, T, U, and V formed a sub-cluster involved in biosynthesis of the L-digitoxose moiety of jadomycin B. When the sub-cluster was cloned in E. coli and the genes were individually disrupted, transfer of the DNA into S. venezuelae by intergeneric conjugation furnished mutants altered in jadomycin B biosynthesis. HPLC and NMR analysis of intermediates accumulated in cultures of the insertionally inactivated mutants indicated that each gene mediates either formation of L-digitoxose or its attachment to jadomycin aglycone.; Chromosome walking to extend the left-hand end of the jad cluster added three new genes. Of these jadW1 is a homologue of barX and afsA, which are associated with γ-butyrolactone autoregulators controlling morphogenesis and secondary metabolism in streptomycetes. jadW2 is a homologue of 3-β-keto steroid dehydrogenase, and jadW 3 is a homologue of 3-β-keto ACP/CoA reductase. Disrupting jadW1 not only stopped production of jadomycin B and chloramphenicol, but also prevented differentiation of the mycelium. Reintroducing jadW1 into jadW1-disrupted mutants restored jadomycin B production above the wild-type titre, and allowed chloramphenicol production, implying that jadW1 positively regulated synthesis of both antibiotics. Introducing jadW 1 into the wild type had a similar effect, and resulted in accumulation of acetyl-chloramphenicol. In contrast to the negative effect of inactivating jadW1, disrupting jadW 2 increased jadomycin B production 5–10 fold and allowed jadomycin B to be produced without the need for ethanol toxicity stress. (Abstract shortened by UMI.)... |
