Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.582366
Title: Elucidation and exploitation of a crotonyl-CoA carboxylase/reductase-independent pathway for unusual polyketide extender unit biosynthesis
Author: Ray, Lauren
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2013
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Abstract:
Polyketides comprise the largest class of natural products and exhibit vast structural diversity, which can be attributed to many factors.1 In recent years it has become clear that a wealth of unusual polyketide synthase extender units contributes significantly to this diversity. To date, the majority of characterised unusual extender units have been found to be biosynthesised via a common pathway involving reductive carboxylation reactions catalysed by crotonyl-CoA carboxylase/reductases (CCRs). Stambomycins, a novel class of macrolides produced by Streptomyces ambofaciens, have unusual branched alkyl side chains present within their carbon skeletons. These are derived from unusual hexyl- and heptylmalony-CoA polyketide synthase extender units that are incorporated into the stambomycin polyketide chain. Bioinformatics analysis of genes within the stambomycin biosynthetic gene cluster suggests the involvement of a CCR-independent mechanism in the biosynthesis of these extender units. Efforts to understand the biosynthetic pathway for the unusual extender units through the use of isotope-labelled precursors and a mechanistic probe have provided strong evidence for a CCR-independent mechanism and demonstrated that they are derived from the fatty acid biosynthetic pathway. The pathway for the provision of the unusual extender units is proposed to involve an acyl-CoA carboxylase, the β-subunit of which is encoded within the stambomycin biosynthetic gene cluster. This is hypothesised to be responsible for the carboxylation of acyl-CoA thioester precursors to yield the corresponding malonyl-CoA derivatives. Biochemical and genetic approaches failed to conclusively prove the role of the acyl-CoA carboxylase in the biosynthesis of the extender units. However, the retention of both deuterium labels in a stambomycin analogue derived from the feeding of [3,3-2H]heptanoic acid to S. ambofaciens is consistent with an acyl-CoA carboxylase dependent pathway for the biosynthesis of the extender unit. The promiscuity of the enzymes involved in the biosynthesis and incorporation of the extender units was investigated and exploited, to furnish a variety of novel stambomycin analogues via precursor directed biosynthesis. These included compounds with acetylene and azide tags. Further derivatisation of the acetylene-tagged stambomycin analogue enabled the addition of a biotin prosthetic group to the macrolides. The polyketide synthase responsible for the biosynthesis of the spiroketal-containing macrolide, ossamycin, produced by Streptomyces hygroscopicus subsp. ossamyceticus, is also proposed to utilise an unusual 2-methylpropylmalonyl-CoA extender unit. However attempts to identify the ossamycin biosynthetic gene cluster, and thus the genes responsible for assembly of 2-methylpropylmalonyl-CoA, were unsuccessful.
Supervisor: Not available Sponsor: Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC) ; Biotica Technology Ltd
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.582366  DOI: Not available
Keywords: QP Physiology
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