Genes for jadomycin B biosynthesis and regulation in Streptomyces venezuelae ISP5230.
Date
2002
Authors
Wang, Liru.
Journal Title
Journal ISSN
Volume Title
Publisher
Dalhousie University
Abstract
Description
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 gamma-butyrolactone autoregulators controlling morphogenesis and secondary metabolism in streptomycetes. jadW2 is a homologue of 3-beta-keto steroid dehydrogenase, and jadW 3 is a homologue of 3-beta-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.)
Thesis (Ph.D.)--Dalhousie University (Canada), 2002.
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 gamma-butyrolactone autoregulators controlling morphogenesis and secondary metabolism in streptomycetes. jadW2 is a homologue of 3-beta-keto steroid dehydrogenase, and jadW 3 is a homologue of 3-beta-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.)
Thesis (Ph.D.)--Dalhousie University (Canada), 2002.
Keywords
Biology, Molecular., Biology, Microbiology.