Thesis

Investigating streptomyces clavuligerus linear replicons for improved clavulanic acid production

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Awarding institution
  • University of Strathclyde
Date of award
  • 2024
Thesis identifier
  • T16837
Person Identifier (Local)
  • 201972935
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Abstract
  • Increasing antimicrobial resistance against ß-lactam antibiotics through bacterially produced ß-lactamases has prompted research into effective enzyme inhibitors, such as clavulanic acid. Streptomyces clavuligerus is the primary producer of clavulanic acid, which is used in various drugs such as Augmentin®. Streptomyces clavuligerus contains a dynamic genome and is composed of four giant linear plasmids (GLPs), pSCL1, pSCL2, pSCL3 and pSCL4 and its chromosome. Various genes essential for the maintenance of linear replicons, such as tap and tpg which encode telomeric terminal proteins (TPs), are found on three out of four GLPs (pSCL2, pSCL3, pSCL4). Previous work demonstrated a circularised chromosome and loss of plasmid after Cas9 mediated cleavage of the largest GLP, pSCL4, potentially due to the absence of tap-tpg. To determine the role of tap-tpg in chromosomal and plasmid linearity, we optimised and tested their inactivation using CRISPR-dCas9 multiplexing, targeting tap-tpg₄ on pSCL4, tap tpg₃ on pSCL3 and tap-tpg₂ on pSCL2. We used Illumina short-read sequencing to genotypically analyse our strains, which highlighted that the knockdown of multiple taptpg genes resulted in mutant strains with various combinations of lost plasmids and terminal ends. Noticeably, only the mutants with silenced tap-tpg4 demonstrated the loss of multiple plasmids, and pSCL2 loss was suggested to be a direct consequence of the CRISPR-dCas9 system, as all mutants and vector controls lost the plasmid. Additionally, we demonstrated chromosome circularisation and the loss of the 13-nucleotide binding site of Tap for our tap-tpg₄, tap-tpg₃ and tap-tpg₂ silenced mutant EM10, suggesting that overall tap-tpg expression levels in S. clavuligerus affect end-patching. The mutant strains were phenotypically characterised and mutants which had lost pSCL3, pSCL2 and/or pSCL1 had a significantly higher specific growth rate, therefore we confirmed that these replicons were essential for strain fitness. We also confirmed that S. clavuligerus does not require pSCL3, pSCL2 and pSCL1 for clavulanic acid production, as strains that had lost more replicons did not produce more clavulanic acid. Therefore, biotechnologically, the loss of replicons is of importance in terms of strain fitness rather than clavulanic acid production in S. clavuligerus. Moreover, to study the process of endpatching and determine the binding activity of TPs, overexpression of Tap4, Tap3 and Tap2 proteins was optimised and Tap4 was shown to potentially bind to the chromosomal ssDNA telomeres. With this study we have elucidated the importance of tap-tpg in the plasmid maintenance of S. clavuligerus and confirmed the essentiality of replicons, highlighting that whilst most replicons were dispensable, they are necessary for maintaining strain fitness and optimum growth.
Advisor / supervisor
  • Herron, Paul
Resource Type
DOI
Date Created
  • 2023
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