Mechanisms for the Generation of Structural Diversity in Polyketide Biosynthesis

  • Paul Walker

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)


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.
Date of Award7 May 2019
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorChris L Willis (Supervisor), Paul R Race (Supervisor) & Matthew P Crump (Supervisor)

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