Antimicrobial resistance is a global challenge that requires the discovery of new antimicrobial compounds and a deeper understanding of the underlying production of current ones. Streptomyces clavuligerus is an industrially important bacterium used for the large-scale fermentation of the β-lactamase inhibitor clavulanic acid (CA). However, variability during fermentation undermines productivity, with limited knowledge of how population dynamics and evolutionary change contribute to this instability.
This thesis established and applied barcode sequencing (Bar-seq) to track strain-level population dynamics in industrially relevant S. clavuligerus fermentations. A library of 20 isogenic barcoded strains was constructed based on the vector pSET152. Validation experiments confirmed stable chromosomal integration and accurate quantification of relative abundance of barcoded strains. Bar-seq revealed that population structure in small-scale (50 mL) fermentations was stable over 48–72 hours, but scale-up to 1 L in rich media (TSB) introduced instability and batch-to-batch variation. In contrast, multistage fermentations in industrial media (S2A -CM5) were stable across scales up to 7.5 L, indicating that process-relevant conditions do not result in competitive divergence.
To assess the evolution of S. clavuligerus in fermentations, barcoded populations were passaged for 70 days (152 generations) in CM5. Replicate cultures differed in population dynamics, with distinct strains becoming dominant, while others declined or persisted at lower relative abundance. Whole-genome sequencing and transcriptomics linked these shifts to specific mutations and altered gene expression, though declines in CA titres were observed across all populations. Phenotypic assays showed variable colony morphology and sporulation, but these traits were unreliable indicators of any underlying population change.
Overall, this work demonstrates that population stability depends strongly on media and scale. Long-term adaptation can reduce CA production despite stable short-term population dynamics. Bar-seq provides a powerful tool for linking genotype, phenotype, and productivity in Streptomyces, with direct implications for industrial strain development and process monitoring.
| Date of Award | 30 Jan 2026 |
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| Original language | English |
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| Awarding Institution | - University Of Strathclyde
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| Sponsors | University of Strathclyde |
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| Supervisor | Paul Hoskisson (Supervisor) & Iain Hunter (Supervisor) |
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