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Title: Studies on the biosynthesis of gladiolin
Author: Costa, Marianne
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2018
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Gladiolin is a macrolide antibiotic with potent activity against Mycobacterium tuberculosis. It is biosynthesised by a trans-AT polyketide synthase (PKS) with many unique features including an O-methylation module, with a dedicated Omethyltransferase (OMT) domain. In this work, the OMT-ACP didomains from the gladiolin PKS, as well as three others from trans-AT PKSs, were overproduced and purified. Their activity and substrate tolerance were determined, providing the first biochemical proof of O-methylation on polyketide assembly lines and revealing useful substrate promiscuity. The OMT domains were shown to fall into two clades, which appear to correspond their ability to interact with specific ACP domains. The gene cluster also contained two putative auxiliary genes of unknown function; gbnC that encodes for an asparagine synthetase, and gbnM which encodes for an amidase. Inactivation of gbnM generated a mutant producing a novel amide derivative of gladiolin, which maintained antimicrobial activity. Purified GbnM was shown to catalyse hydrolysis of the amide derivative to produce gladiolin. Although it was established, by in vivo gene deletion, that gbnC, proposed to be responsible for the initial amidation, plays an essential biosynthetic role, in vitro studies were not possible due to insolubility of the protein.
Supervisor: Not available Sponsor: Midlands Integrative Biosciences Training Partnership ; Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry ; QP Physiology ; RM Therapeutics. Pharmacology