The emergence of antibiotic resistance poses a threat to humankind in combination with the significant drop in discovering new antibiotics since the 1980s. Although Streptomyces are prolific producers of bioactive metabolites, genome analysis shows more biosynthetic clusters than compounds produced under laboratory conditions. Besides identification of a new potential compound, its biosynthesis needs to be understood to exploit it for industrial production. This thesis focussed on understanding the interaction of primary metabolism, in particular the Phosphoenolpyruvate-Pyruvate-Oxaloacetate (PEP-PYR-OAA) node, with specialised metabolism to identify targets for metabolic engineering. Streptomyces coelicolor and its polyketide actinorhodin formed a proof of concept. Many central carbon metabolic enzymes are annotated by isofunctional genes. Such expansions were analysed by creating a database of 614 actinobacterial species: more expansions were observed in genera rich in specialised metabolism. This, in combination with screening mutants for changes in polyketide production, identified phosphofructokinase, pyruvate kinase, pyruvate phosphate dikinase, pyruvate dehydrogenase complex and malic enzyme as potential targets. These are expanded in Streptomyces and showed changes in actinorhodin production for at least one of each gene-set. Phosphofructokinase and malic enzymes have been previously studied. Differential gene expression analysis by RNA-Seq for glycolytic and gluconeogeneic conditions showed that most genes of expanded enzymes were differentially-expressed. Analysis of metabolite pools of central carbon metabolism supported this further. The two pyruvate kinases did not show differential expression and were analysed in more detail. A pyk2 mutant exhibited altered growth on glucose, whereas pyk1 mutants showed altered actinorhodin production.Biochemical characterisation suggested Pyk2 as the housekeeping enzyme with low S₀.₅ for both substrates, whereas Pyk1 was activated under low energy state conditions. Overall, the thesis provides a better understanding of the interaction of primary metabolism and expansion in respect to specialised metabolism to be used in strain development for the production of antibacterial compounds.
|Date of Award||23 Feb 2016|
- University Of Strathclyde
|Sponsors||University of Strathclyde & Scottish Funding Council SFC|
|Supervisor||Paul Hoskisson (Supervisor) & Iain Hunter (Supervisor)|