dc.contributor.advisor |
Ackerley, David |
|
dc.contributor.author |
Owen, Jeremy George |
|
dc.date.accessioned |
2010-11-16T20:06:50Z |
|
dc.date.accessioned |
2022-10-20T20:07:34Z |
|
dc.date.available |
2010-11-16T20:06:50Z |
|
dc.date.available |
2022-10-20T20:07:34Z |
|
dc.date.copyright |
2010 |
|
dc.date.issued |
2010 |
|
dc.identifier.uri |
https://ir.wgtn.ac.nz/handle/123456789/22544 |
|
dc.description.abstract |
Non-ribosomal peptide synthetases (NRPS) are large, modular enzymes that synthesise
biologically active secondary metabolites from amino acid precursors without the need for a
nucleic acid template. NRPS play an integral role in microbial physiology and also have
potential applications in the synthesis of novel peptide molecules. Both of these aspects are
examined in this thesis.
Under conditions of iron starvation Pseudomonas syringae synthesises siderophores for active
uptake of iron. The primary siderophore of P. syringae is pyoverdine, a fluorescent molecule
that is assembled from amino acid (aa) precursors by NRPS. Five putative pyoverdine NRPS
genes in P. syringae pv. phaseolicola 1448a (Ps1448a) were identified and characterised in
silico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Creation of
pyoverdine null Ps1448a enabled identification of a previously uncharacterised temperatureregulated
secondary siderophore, achromobactin, which is NRPS independent and has lower
affinity for iron. Pyoverdine and achromobactin null mutants were characterised in regard to
iron uptake, virulence and growth in iron-limited conditions. Determination of the substrate
specificity for the seven adenylation (A) domains of the Ps1448a pyoverdine sidechain NRPS
was also attempted. Although ultimately unsuccessful, these attempts provided a rigorous
assessment of methods for the expression, purification and biochemical characterisation of Adomains.
The Ps1448a NRPS were subsequently employed in domain swapping experiments to test
condensation (C) domain specificity for aa substrates during peptide formation in vivo.
Experiments in which the terminal C- and/or A-domain of the Pseudomonas aeruginosa
(PAO1) pyoverdine NRPS system were replaced with alternative domains from Ps1448a and
PAO1 were consistent with previous in vitro observations that C-domains exhibit strong
sidechain and stereo-selectivity at the downstream aa position, but only stereo-selectivity at
the upstream aa position.
These results prompted investigation into the role of inter-domain communication in NRPS
function, to test the hypothesis that the thiolation (T) domain enters into specific interactions
with other domains, which might provide an alternative explanation for the diminished activity
of recombinant NRPS enzymes. A recently characterised single-module NRPS, bpsA, was
chosen as a reporter gene for these experiments based on its ability to generate blue pigment in
Escherichia coli. Substitution of the native bpsA T-domain consistently impaired function,
consistent with the hypothesis. It was shown that directed evolution could be applied to restore
function in substituted T-domains. Mutations that restored function were mapped in silico, and
a structural model for interaction between the thioester (TE) and T-domain of BpsA was
derived.
The utility of bpsA for discovery and characterisation of phosphopantetheinyl transferase
(PPTase) enzymes was also investigated. In vivo and in vitro assays for determination of
PPTase activity were developed and a high-throughput screen for discovery of new PPTases in
environmental DNA libraries was successfully implemented. |
en_NZ |
dc.language.iso |
en_NZ |
|
dc.publisher |
Te Herenga Waka—Victoria University of Wellington |
en_NZ |
dc.subject |
NRPS |
en_NZ |
dc.subject |
Enzyme engineering |
en_NZ |
dc.subject |
Siderophores |
en_NZ |
dc.title |
Characterisation, Manipulation and Directed Evolution of Non-Ribosomal Peptide Synthetase Enzymes |
en_NZ |
dc.type |
Text |
en_NZ |
vuwschema.contributor.unit |
School of Biological Sciences |
en_NZ |
vuwschema.subject.marsden |
270805 Genetic Engineering and Enzyme Technology |
en_NZ |
vuwschema.type.vuw |
Awarded Doctoral Thesis |
en_NZ |
thesis.degree.discipline |
Biotechnology |
en_NZ |
thesis.degree.grantor |
Te Herenga Waka—Victoria University of Wellington |
en_NZ |
thesis.degree.level |
Doctoral |
en_NZ |
thesis.degree.name |
Doctor of Philosophy |
en_NZ |