| dc.contributor.author | Wang, Liru. | en_US |
| dc.date.accessioned | 2014-10-21T12:37:13Z | |
| dc.date.available | 2002 | |
| dc.date.issued | 2002 | en_US |
| dc.identifier.other | AAINQ67663 | en_US |
| dc.identifier.uri | http://hdl.handle.net/10222/55839 | |
| dc.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. | en_US |
| dc.description | 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.) | en_US |
| dc.description | Thesis (Ph.D.)--Dalhousie University (Canada), 2002. | en_US |
| dc.language | eng | en_US |
| dc.publisher | Dalhousie University | en_US |
| dc.publisher | | en_US |
| dc.subject | Biology, Molecular. | en_US |
| dc.subject | Biology, Microbiology. | en_US |
| dc.title | Genes for jadomycin B biosynthesis and regulation in Streptomyces venezuelae ISP5230. | en_US |
| dc.type | text | en_US |
| dc.contributor.degree | Ph.D. | en_US |
