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Title: Investigations into the biosynthesis of chloroeremomycin
Author: Charles, E. H.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2001
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This thesis describes work on the biosynthesis of the glycopeptide antibiotic chloroeremomycin. Both gene cloning and isotopically-labelled precursor feeding techniques have been used to study the biosynthetic pathway to this antibiotic. Two genes from the chloroeremomycin biosynthetic gene cluster with homology to non-haem haloperoxidases have been cloned, and the proteins overexpressed and purified. Attempts at demonstrating the involvement of either of these two proteins in chloroeremomycin halogenation have not been successful. However, the second of these two enzymes shows homology to thioesterase enzymes, consistent with the possibility that it could fulfil an editing function in the non-ribosomal biosynthesis of the antibiotic heptapeptide backbone. A system for studying glycopeptide biosynthesis using stable isotopically labelled precursors has been developed. Using this system, the timing of the chlorination of the chloroeremomycin tyrosine residues has been investigated. Putative pathways to the unusual amino acids 4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine have also been studied in this way, together with the methylation of the leucine residue and 4-epi-vancosamine sugar substituent. An enzyme showing homology to glycolate oxidases, and proposed to have a role in the biosynthesis of 4-hydroxyphenylglycine, has been cloned and overexpressed. The enzyme has been shown to be insoluble when expressed in E. coli. Attempts to refold the protein after purification of the inclusion bodies have been successful, but have yielded inactive enzyme. A fourth gene from the chloroeremomycin biosynthetic gene cluster, with homology to a sugar biosynthesis methylation enzyme, has also been cloned and the protein overexpressed and purified. Attempts at proving the involvement of this protein in the biosynthesis of the deoxysugar 4-epi-vancosamine have been unsuccessful. However, the unavailability of the enzyme's predicted substrate has meant that the assays used so far have relied on the success of other enzymes upstream in the biosynthetic pathway.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available