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Bio-engineering of Antibiotic Enduracidin Biosynthetic Pathways and PreQ1 Riboswitch
[Thesis]. Manchester, UK: The University of Manchester; 2011.
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Abstract
Non-ribosomally synthesised natural products derived mainly from bacteria and fungi act as important therapeutic agents. Due to their complex structures it is difficult to chemically synthesise such compounds, therefore the engineering of biosynthetic and biocatalytic pathways that are vital for their production are the aims of this thesis. The first project involved the study of the biosynthesis of 3-O-methyl aspartic acid (OmAsp) in the antibiotic A54145. We demonstrated that LptL functions as an asparagine hydroxylase. We also predicted that LptJ and LptK are involved in the biosynthesis of OmAsp, although we did not find evidence of this non-standard amino acid in the antibiotic CDA when we over-expressed both proteins in Streptomyces coelicolor. The second project involved the study of the chlorination and mannosylation of hydroxyphenyl glycine (Hpg) residues in ramoplanin. We over-expressed the putative chlorinase and mannosyl transferase separately in the enduracidin-producer, in which the production of enduracidin has been characterised. We then found that trichlorinated and mannosylated enduracidin analogues were produced.The final project involved the re-engineering of a preQ1 riboswitch. We successfully created ten preQ1 riboswitch mutants fused to the reporter gene lacZ in Bacillus subtilis, all of which showed different levels of β-galactosidase activity. Subsequently, we found that the preQ1 C17U mutant riboswitch can respond specifically to the synthetic ligands 5-(aminomethyl)furo[2,3-d]pyrimidine-2,4-diamine (D6) and 2,4-diamino-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (D9) to control gene expression in a dose dependent manner. The results described here show the successful production of variant antibiotics by bio-engineering and the use of an engineered preQ1 riboswitch as a tool for regulating gene expression.