Characterising the Two-Component System-Regulated Biosynthesis of Formicamycin in Streptomyces formicae

Noble, Katherine (2024) Characterising the Two-Component System-Regulated Biosynthesis of Formicamycin in Streptomyces formicae. Doctoral thesis, University of East Anglia.

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Abstract

Abstract

A crucial part of overcoming antimicrobial resistance is in the development and
overproduction of novel antibiotics. Isolation and genetic analysis of Streptomyces formicae
identified the formicamycins; novel antibiotics with potent activity against clinically
relevant, drug-resistant pathogens and a high barrier to the development of resistance.
There are three known cluster-situated regulators that work cohesively to control
activation, repression and export of these secondary metabolites. A two-component
system, ForGF, was shown to be the main activator of this pathway and this project aimed
to further elucidate its role and increase production levels of the compounds. Using a
variety of molecular, biochemical and biophysical analyses such as surface plasmon
resonance, gene-reporter fusion assays and qRT-PCR, it has been possible to identify the
binding site of the response regulator and show the impact of binding on promoter activity
and transcription levels. A combination of CRISPR/Cas9 mutagenesis and downstream
analysis of in vivo and in vitro proteins have also been utilised to characterise the interaction
of the two components with one another and their surroundings. It has been shown that
manipulating ForGF and other regulators within the biosynthetic gene cluster leads to
overexpression of the formicamycins, overcoming the problem of low production under
standard laboratory conditions.

By exploiting this mechanism of control, it has also been possible to apply similar initial
processes to other such cluster-situated two-component systems within the same strain.
This genetic manipulation has resulted in changes to the bioactivity of a small library of
strains when challenged with a number of pathogenic organisms. This has significant
potential for further application for the targeted investigation and overproduction of other
novel antimicrobials for clinical development.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Zoe White
Date Deposited:	19 Sep 2024 16:13
Last Modified:	19 Sep 2024 16:13
URI:	https://ueaeprints.uea.ac.uk/id/eprint/96782
DOI:

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