Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.781884
Title: Investigating the role of MmyB-like pathway-specific transcriptional activators in antibiotic production
Author: Aldali, Jehad
ISNI:       0000 0004 7967 4971
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2018
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Abstract:
Streptomyces bacteria are known to produce a large number of bioactive secondary metabolites. The genome sequence of the model organism Streptomyces coelicolor A3(2) revealed the presence of multiple previously unknown antibiotic-like gene clusters (Bentley et al., 2002). A transcriptional activator named MmyB was proposed to specifically trigger the production of the methylenomycin antibiotics in S. coelicolor A3(2). S. coelicolor A3(2) itself, which encodes 16 proteins highly similar to MmyB (Xu et al., 2012), each of them proposed to control the expression of a distinct biosynthetic gene cluster or to control the expression of other uncharacterised genes was our main target micro-organism. (O'Rourke et al., 2009). The aim of this project was to clone a series of mmyB-like putative pathway-specific transcriptional activators under the control of a Streptomyces constitutive promoter in integrative vectors. These vectors were then introduced in Streptomyces bacteria, using intergenic conjugation. The effect of mmyB-like gene overexpression was then investigated by: i) analysing the expression of specific sets of antibiotic biosynthetic genes using RT-PCR, ii) assaying the antibiotic activity of metabolic extracts where mmyB-like genes were overexpressed versus negative controls and iii) To identify, isolate and elucidate the structure of antimicrobial using analytical chemistry (LC-MS, HPLC, NMR) in collaboration with analytical chemists. S. coelicolor A3(2) itself which encodes 16 proteins highly similar to MmyB (Xu et al., 2012), each of them proposed to control the expression of a distinct biosynthetic gene cluster or to control the expression of other uncharacterised genes was our main target micro-organism. This project successfully revealed that overexpression of transcriptional activators was a powerful strategy to discover new natural products by switching on silent biosynthetic gene clusters. Our understanding of the molecular mechanisms by which particular families of transcriptional activators function was essential in order to determine which specific genes within a cluster would be overexpressed and would result in overproduction of cryptic metabolites. The antibiotic activity of culture extracts and purified metabolites from engineered S. coelicolor strains overexpressing mmyB-like transcriptional activators was then assessed against Bacillus subtilis. In addition to confirming methylenomycin overproduction, these essays also revealed the production of new natural products (lantibiotic-derived and poly unsaturated fatty acid-derived).
Supervisor: Not available Sponsor: Safārah ; Saudi Arabia
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.781884  DOI: Not available
Keywords: QH426 Genetics
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