Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.782596
Title: Mechanisms for the generation of structural diversity in polyketide biosynthesis
Author: Walker, Paul
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2019
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Abstract:
Kalimantacin β-Branching Pathway Polyketide synthases encode a remarkable number of enzymes to catalyse chain extension, reductive processing and tailoring reactions leading to structural diversity in the polyketide products. Interaction of a modular ACP-bound substrate with a transacting HCS cassette leads to β-branching, as exemplified by kalimantacin A which contains four β-branches with an endo or exo double bond or a saturated methyl group (green dots). This diversity of β-branches, incorporated by a single HCS cassette, provides a fascinating case study to probe the mechanism and selectivity for consecutive β-branch incorporations. The HCS cassette, modular ACPs and tailoring enzymes from the kalimantacin gene cluster were cloned, expressed and purified. In vitro reconstitution of the β-branching pathway demonstrated the formation of an endo-β-methyl branch by the HCS cassette. The α,β-unsaturation was rigorously assigned by NMR experiments using pantetheineand ACP-bound intermediates. Subsequent reduction (BatK) of this substrate resulted in the formation of a saturated-β-methyl branch. A previously unassigned modular ECH domain was identified, cloned and expressed as a single domain (mECH) or ACP4- mECH di-domain (4M). An NMR assay utilising a single carbon-13 label incorporated into key biosynthetic mimics was developed and definitively showed the formation of an exo-β-methyl branch via mECH-catalysed decarboxylation. Mupirocin/Thiomarinol Starter Unit Generation Previous stable isotope labelling studies carried out on the mupirocin (P. fluorescens) and thiomarinol (Pseudoalteromonas sp.) producing strains was the basis for a hypothesis for the formation of a 3- or 4-carbon starter unit in the biosynthesis of the fatty acid moiety of both compounds. The cassette of enzymes hypothesised to be responsible for the generation of a 3-hydroxypropionate (3-HP) and 4-hydroxybutyrate (4-HB) starter unit consists of an ACP (M/TacpD), an adenylation domain (MupQ/TmlQ) and a reductase (MupS/TmlS). Each of these proteins were expressed and purified, and the proposed pathway was then reconstituted in vitro which conclusively demonstrated 3-HP and 4-HB formation.
Supervisor: Willis, Chris ; Race, Paul ; Crump, Matthew Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.782596  DOI: Not available
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