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Title: Non-ribosomal biosynthesis of calcium-dependent antibiotic : a lipopeptide antibiotic produced by Streptomyces coelicolor A3(2)
Author: Neary, Joanne Marie
Awarding Body: The University of Manchester
Current Institution: University of Manchester
Date of Award: 2003
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Abstract:
The calcium-dependent antibiotic is a lipopeptide antibiotic produced by S. coelicolor A3 (2). Its structure comprises a cyclic undecapeptide containing a number of non-proteinogenic amino acids and an N- terminal 2,3-epoxyhenanoyl side-chain. A number of natural CDA variants have been characterised which arise by modification of specific amino acid residues within the peptide. The CDA biosynthetic gene cluster has been fully sequenced as a consequence of the S. coelicolor Genome Sequencing Project at the Sanger Centre, which was completed in 2002. The CDA biosynthetic gene cluster contains open reading frames encoding three non-ribosomal peptide synthetase (NRPS) genes (cdaPS1-3), fatty acid biosynthesis genes and a number of putative genes encoding enzymes for amino precursor biosynthesis and tailoring of the peptide. cdaPS1-3 appear to have a modular structure typical of linear NRPS genes where each module is responsible for recruitment of a specific amino acid into the peptide. It has been hypothesised that a domain of unknown function at the start of module 1 is responsible for the transfer of the 2,3-epoxyhexanoyl side-chain to the undecapeptide. Module 1 was the focus of the investigation since this module is believed to have an initiating role in CDA biosynthesis. It was concluded from in vitro studies with overproduced module 1 protein from E. cola and S. lividans strains that module 1 adenylation domain is specific for the antibiotic serine, however attachment of serine to the PCP domain of module 1 was not demonstrated. A conserved serine residue in the PCP domain of module 1 was shown to be essential for binding of the 4'-PP cofactor by site directed mutagenesis of the PCP domain DNA: when this sequence was changed to encode alanine CDA production was abolished. A number of substrates were proposed for feeding to PCP domain mutants to re-establish CDA biosynthesis and this work is ongoing. The enzyme responsible for hydroxylation of asparagine at position 9 in CDA was investigated. This enzyme was thought to be a member of the α-ketoglutarate dependent Fe(II) di-oxidase family of enzymes because of the homology shared by the putative asparagine oxygenase (SC03236; asnO) and clavaminic acid synthase (CAS). An inframe deletion of SC03236 was introduced into the chromosome of S. coelicolor strains and analysis of secondary metabolites produced by the mutants suggested that CDA containing asparagine rather than hydroxyasparagine at position 9 was produced. It was concluded therefore that SC03236 was the gene encoding asnO. This was confirmed by complementation of deletion mutants with vector borne SC03236, which indicated a reversion to production of wild type CDA containing hydroxyasparagine by the complemented strains. Over-production of AsnO protein in E. coli was carried out and the protein used in hydroxylation assays with a number of different substrates and the required co-factors for the presumed α-ketoglutarate dependent Fe(II) di-oxidase. However, these experiments proved inconclusive and work in this area is ongoing.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.506447  DOI: Not available
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