Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599319
Title: Biosynthetic studies on lankacidins from Streptomyces rochei var. volubilis
Author: Garner, S.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
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Abstract:
This dissertation describes work performed to investigate lankacidins biosynthesis in Streptomyces rochei var. volubilis along with the plasmid and extracellular enzyme profile of the strain. The strain was confirmed to produce lankacidins, as well as the 14-membered macrolide lankamycin, as judged by mass spectroscopic analysis of culture extracts. It also appeared to contain a giant plasmid as judged by pulsed-field gel electrophoresis. A genomic library of S. rochei var. volubilis was prepared and screened for genes potentially involved in lankacidins biosynthesis. Two regions, covering a total of 130 kbp, were identified and sequenced. Open reading frame analysis of the sequences putatively identified the hybrid nonribosomal peptide synthetase/polyketide synthase responsible for lankacidins core biosynthesis along with a number of post-synthetase genes for production of mature lankacidins. This hybrid NRPS-PKS is predicted to have several unusual features including iterative use of modules, modules split between multienzymes and discrete PKS domains acting in trans. A number of putative lankacidins biosynthetic genes were transferred by conjugation to a non-producer, Streptomyces lividans TK24, and the presence of the DNA in this host was confirmed by Southern blotting. However, no novel metabolites attributable to the lankacidins genes could be detected under any conditions of growth tested. Addition of N-acetylglycine-SNAC, an analogue of a predicted early intermediate, did not stimulate production of lankacidins-like products from the transformed non-producer. Several carrier protein domains from the synthetase were expressed and purified as His6-tagged fusion proteins from E. coli. These were converted to their respective holo-form by the phosphopantetheinyl transferase Svp (in vivo) and Sfp (in vitro). PKS together with one holo-acyl carrier protein (ACP) led to partial acetylation of that ACP, consistent with the predicted function of the AT as a malonyl-CoA:ACP-acyltranferase. The discrete AT was expressed in E. coli and purified from inclusion bodies but in vitro activity assays were inconclusive.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.599319  DOI: Not available
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