Nosarka, Zainab2024-10-282024-10-282023-09Nosarka, Zainab. (2023). Metabolic Engineering of Streptomyces. [Master's dissertation, University of the Witwatersrand, Johannesburg]. https://hdl.handle.net/10539/42002https://hdl.handle.net/10539/42002A dissertation submitted in fulfilment of the requirements for the degree Master of Science in Molecular and Cell Biology, to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023.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.en©2023 University of the Witwatersrand, Johannesburg. All rights reserved. The copyright in this work vests in the University of the Witwatersrand, Johannesburg. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of University of the Witwatersrand, Johannesburg.Metabolic engineeringStreptomycesSecondary metabolitesSynthetic biologyGene editingCRISPR/Cas9Intergeneric conjugationUCTDSDG-17: Partnerships for the goalsDissertationUniversity of the Witwatersrand, Johannesburg