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Title: Investigating the programming of Type I iterative polyketide synthase enzymes
Author: Ivison, David
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2013
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Type I iterative polyketide synthase (PKS) enzymes are a family of large, complex proteins with multiple functional domains. They produce a diverse range of polyketide natural products through the selective use and reuse of these domains; the "programming" of a PKS, inherent within the protein itself, refers to the mechanism by which the order of domains' actions is determined. Two approaches were used to investigate the nature of the programming of type I iterative PKS: both intact PKS genes and portions thereof corresponding to individual functional domains were cloned using high-throughput techniques and expressed in heterologous hosts. Recombinant proteins were purified and studied in vitro. In addition, some in vivo studies on PKS genes expressed in heterologous hosts were carried out. Crystallisation of several isolated PKS domains was attempted, but was ultimately unsuccessful. Work in this area is ongoing and could provide useful complimentary information to that obtained through biochemical characterisation. The squalestatin tetraketide synthase (SQTKS) enoylreductase (ER) domain was purified and studied in detail using a variety of substrate mimics, including a range of "unnatural" synthetic small-molecule substrate analogues designed to test the enzyme's selectivity and specificity. These identified polyketide chain length or methylation pattern as being crucial for recognition of a key intermediate. The SQTKS dehydratase (DH) domain was investigated in a similar manner, and was found to produce only the expected product when presented with a smallmolecule substrate mimic. A starter unit:acyl carrier protein transacylase (SAT) domain and a producttemplate/ acyl carrier protein (PT-ACP) didomain from 3-methylorcinaldehyde synthase (MOS) were also successfully cloned, expressed heterologously and partially characterised. Efforts were made to express the full-length gene encoding SQTKS in E. coli and S. cerevisiae; while expression of the expected protein was observed in E. coli, it could not be purified.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available