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dc.contributor.authorPinkett, Kaitlyn
dc.contributor.otherQueen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.))en
dc.description.abstractThe formation of carbon-fluorine (C-F) bonds represents a very rare form of enzyme catalysis relative to other organohalogens. Instead of an oxidative mechanism, desolvation and nucleophilic incorporation of fluoride is required. Nucleocidin, produced by Streptomyces calvus, is a novel fluorinated analog of adenosine. The fluorine at the 4’-carbon of nucleocidin demands an unusual sequence of cleavage of the 4’C-H bond of the ribose ring and stereospecific glycosylation of fluoride. The nucleocidin biosynthetic gene cluster has recently been identified in Streptomyces calvus. This provides an opportunity to elucidate the functions of the encoded enzymes. To determine the minimal set of genes required to synthesize nucleocidin, a strategy was developed to clone the gene cluster using transformation associated recombination (TAR) in yeast, followed by heterologous expression in another Streptomyces strain. In parallel, CRISPR/Cas9 was used to inactivate selected biosynthetic genes. Plasmids encoding CRISPR/Cas9 that were designed to inactivate orf171, nucG, and nucK were conjugated into S. calvus. Additionally, the biosynthesis of a chlorinated derivative of nucleocidin was attempted by incorporating the putative ascamycin halogenase genes acmX and acmY into S. calvus Spore 4. Integration of acmX into the chromosome of S. calvus Spore 4 was successful as confirmed by PCR, while integration of acmY could not be confirmed.The presence of nucleocidin in the extracts of S. calvus Spore 4 att::[acmX, aac(3)IV] and S. calvus Spore 4 was confirmed by 19FNMR spectroscopy. A new signal was observed in the S. calvus Spore 4 att::[acmY,aac(3)IV] extract that was not observed in the S. calvus Spore 4 extract. This could correspond to a chlorinated derivative, or a new nucleocidin biosynthetic or shunt intermediate. AcmX was successfully heterologously expressed in E. coli and purified. Further studies can focus on assaying AcmX to confirm its function as a variant B FADH2-dependent chlorinase. A putative purine nucleoside phosphorylase (orf206) present in the nucleocidin cluster was heterologously expressed as inclusion bodies in E. coli, setting the stage for purification under denaturing conditions. Finally, enhanced production of nucleocidin was not observed after orf191 encoding a predicted streptomycin-like regulatory gene was integrated onto the chromosome of S. calvus Spore 4. Overall, this work sets the stage for investigating the functions of nucleocidin biosynthetic genes and generating new nucleocidin derivatives.en_US
dc.relation.ispartofseriesCanadian thesesen
dc.rightsAttribution-NonCommercial-NoDerivs 3.0 United States*
dc.rightsQueen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canadaen
dc.rightsProQuest PhD and Master's Theses International Dissemination Agreementen
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dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.titleElucidating the Biosynthesis of Nucleocidin, a Fluorinated Natural Product Produced by Streptomyces Calvusen_US
dc.description.degreeMaster of Scienceen_US
dc.contributor.supervisorZechel, L. Daviden

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