Thesis

Understanding the development of industrial strains of Streptomyces

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Awarding institution
  • University of Strathclyde
Date of award
  • 2025
Thesis identifier
  • T17432
Person Identifier (Local)
  • 201975109
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Abstract
  • Due to the current AMR crisis there is a need to produce more antimicrobials, however discovering new antibiotics is a long and expensive process. As a result, there is also a need to increase the production of current antimicrobials and specialised metabolites. Previous work has shown that in Streptomyces coelicolor the primary metabolic enzyme Pyruvate phosphate dikinase (PPDK), which catalyses the reversible reaction of pyruvate to phosphenolpyruvate (PEP), is up-regulated almost 30 fold during antibiotic production on oil based media. This thesis further investigated this observation by studying pyruvate PPDK and its role in facilitating specialised metabolite production. PPDK like Pyruvate kinase (Pyk) can convert PEP to Pyruvate, however the reaction is reversible and the PEP forming reaction is favoured. Actinobacteria are relatively unique having this enzyme, as it is uncommon among bacteria and it importantly enables the organism to grow gluconeogenically as well as glycolytically. Several genes encoding primary metabolic enzymes show multiplicity in Streptomyces and there are commonly two PPDK proteins. However, the two versions of this enzyme are in separate clades when mapped onto a phylogenetic tree, supporting the hypothesis that the two may have different physiological roles. Using CRISPRi technology a number of ppdk knockdown and over expression mutants were produced and their carbon utilisation profiles were analysed. These preliminary data were the basis for analysing growth rate and antibiotic titre on single carbon sources, which showed that by manipulating these central carbon metabolic genes in Streptomyces it is possible to alter antibiotic titre. Further bioinformatic and biochemical analysis of these enzymes also shed light on their physiological roles in Streptomyces. These results also help to further understand carbon flux around the PEP-PYR-OXA (PEP-pyruvate-oxalacetate) node of central carbon metabolism under a range of conditions and to understand how this impacts the availability of precursor molecules for specialised metabolite production.
Advisor / supervisor
  • Hoskisson, Paul A.
  • Hunter, Iain S.
Resource Type
DOI

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