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Title: Biosynthetic engineering and characterisation of non-natural nonribosomal peptide antibiotics
Author: Nunns, Laura
ISNI:       0000 0004 7655 1554
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2014
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Over the past decades, bacterial resistance has emerged to virtually all classes of antibiotics, highlighting the need for new antibiotics, ideally with novel mechanisms of action.1 In this thesis, the secondary metabolite natural products of interest are the nonribosomal peptide antibiotics. Initially by site-directed mutagenesis, the active site Phe108 residue of the predicted fatty acid chain length determinant, β-ketoacyl synthase-II (KAS-II), encoded by fabF3 from the calcium dependant antibiotic (CDA) biosynthetic gene cluster of S. coelicolor was altered resulting in the production of novel analogues of CDA with altered fatty acid tails. The molecular formula and structure characterisation of the novel CDAs was confirmed by HR-MS, HPLC, LC-MS and MS-MS. In a parallel study, a methyltransferase gene deletion strain (MT1110DeltaglmT) was used to alter the substrate used by a module of the CdaPS3 NRPS. Through this strategy, a novel glutamine (Gln) containing variant of CDA was produced, in place of the natural glutamate (Glu) or methylglutamate (mGlu) residue at the equivalent position. Using a mutasynthetic approach with the genetically engineered mutant strain MT1110DeltaglmT-CdaPS3(Lys278Gln), the synthetic non-natural amino acid (2S,3R)-3-methylglutamine (mGln) was incorporated into CDA at the equivalent position to Glu/mGlu and Gln. The novel Gln and mGln containing CDAs were characterised by HR-MS, HPLC, LC-MS and MS-MS.Expanding on these combinatorial biosynthesis studies, heterologous gene expression identified the specific gene functions of ram20 (chlorinase) and ram29 (mannosyltransferase) genes, over-expressed from the Actinomycetes ATCC 33076 gene cluster of ramoplanin, into the biosynthetic gene cluster of the enduracidin producer S. fungicidicus (ATCC 31731). Novel chlorinated and mannosylated analogues of enduracidin were successfully isolated and characterised by LC-MS and MS-MS. The combination of the biosynthetic engineering techniques developed in this thesis has been demonstrated to be a powerful tool for expanding the structural diversity of the nonribosomal antibiotics, which are desperately needed in the battle to treat and prevent the spread of multiple-drug resistant bacteria.
Supervisor: Micklefield, Jason Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available