School of Molecular & Cell Biology (ETDs)

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Browsing School of Molecular & Cell Biology (ETDs) by SDG "SDG-17: Partnerships for the goals"

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    Metabolic Engineering of Streptomyces
    (University of the Witwatersrand, Johannesburg, 2023-09) Nosarka, Zainab; Moodley, Sanchia; Rumbold, Karl
    The Streptomyces genus demonstrates remarkable potential as a source and host for the production and discovery of industrially relevant secondary metabolites. The genus natively produces approximately 75% of antibiotics and several other compounds encoded by biosynthetic gene clusters (BCGs). However, most BCGs are poorly expressed or dormant under laboratory conditions and thus require metabolic engineering. Several technologies have been developed for this purpose but the pCRISPomyces-2 plasmid system, which employs Cas9 engineering, exhibits the most promising efficacy. This dissertation outlined foundational research to determine the capacity to introduce pCRISPomyces-2 plasmids into Streptomyces albulus BCRC11814 that produces high quantities of ɛ-poly-L-lysine, an antimicrobial and anti-phage compound. In addition, the strain has several other industrially relevant BCGs that have not been studied but possess engineering potential. To achieve the outlined aim, pCRISPomyces-2 plasmids were Sanger sequenced to ensure structural integrity and related functionality. A ClustalW alignment referenced against the plasmid’s nucleotide sequence verified a sequence identity > 98%. Subsequently, an intergeneric conjugation system was established by transforming pCRISPomyces-2 plasmids into Escherichia coli donor cells with an average transformation efficiency (TE) of 1.49 × 105 cfu/µg. Attempts to optimise TE were hindered by the plasmids’ large size and inherent Cas9 toxicity. Thereafter, the transformed donor cells were conjugated with S. albulus BCRC11814 and a comparative model strain Streptomyces coelicolor A3(2). Successful exconjugants were only obtained with S. coelicolor A3(2). The absence of conjugal mating with S. albulus BCRC11814, despite optimisation attempts, was hypothesised to result from the presence of a methyl-specific restriction modification system. This was confirmed by comparative electro-transformation with methylated and non-methylated DNA that demonstrated specificity to dam and dcm methylated DNA. Furthermore, spontaneous resistance to the selectable marker apramycin was confirmed in both Streptomyces strains. Additional efforts are required to effectively introduce pCRISPomyces-2 plasmids into S. albulus BCRC11814.
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    Parageobacillus thermoglucosidasius and the water-gas shift reaction: Investigating the influences of gas composition and H2- uptake hydrogenases
    (University of the Witwatersrand, Johannesburg, 2024) Mol, Michael; Maayer, De
    H2 gas is an increasingly important commercial reagent in a range of industries, including as a potential fuel and energy carrier. Production of H2 gas is carried out almost exclusively reliant on fossil fuel reformation through a relatively limited set of environmentally harmful processes. Pressing environmental concerns and the shift in climate policies towards greater incorporation of cleaner H2 gas production strategies and the implementation of H2 as a more sustainable and environmentally friendly alternative energy vector have necessitated development of renewable and cleaner H2 production processes. Chapter one discusses available literature on the present applications, predominant production processes and emerging alternative production strategies of H2 gas, with particular focus on the applicability and mechanism of biological H2 gas production by the thermophile, Parageobacillus thermoglucosidasius, via the water-gas shift reaction. The application of P. thermoglucosidasius to produce H2 gas is presently in its formative laboratory-scale stage of development. Although progress has been made at a fundamental level, various aspects of P. thermoglucosidasius H2 gas metabolism, remain uncharacterised or poorly understood. In Chapter two, the potential influences of two putative H2-uptake hydrogenases, encoded on the genome of a P. thermoglucosidasius strain previously identified to conduct the hydrogenogenic biological water-gas shift reaction was explored through knock- out mutagenesis. Furthermore, in Chapter three, to establish the practical implementation of pertinent and inexpensive gas feedstocks for the water-gas shift reaction, we explored the influences of various industrial mimetic gas feedstock compositions on the H2 gas evolution-, metabolic- and growth-profiles of P. thermoglucosidasius. Considering the highly variable compositions of industrial off-gases, which may contain oxygen and the highly sensitive nature of this system to oxygen, we further explored the impacts of varied volumetric and temporal oxygen perturbations on the system. Aside from hydrogen production, P. thermoglucosidasius and closely related thermophilic taxa from the genera Parageobacillus and Saccharococcus represent appealing targets for various other biotechnological developments. In Chapter four, we performed a comprehensive comparative genomic and phylogenomic analysis to further explore the biotechnological potential of Parageobacillus and Saccharococcus spp. for a range of whole cell applications as well as a source of industrially relevant thermostable enzymes