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Cloning, expression and characterisation of the starter module from indanomycin biosynthesis

Derrington, Sasha Rebecca (2014) Cloning, expression and characterisation of the starter module from indanomycin biosynthesis. PhD thesis, University of Leeds.

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Abstract

Nonribosomal peptides and polyketides form important classes of pharmaceutical agents. Several architectures of biosynthetic machinery exist for the construction of these structurally diverse molecules. Many attempts to engineer these proteins have concentrated on the multimodular type I polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). Here, work was carried out to express the NRPS module responsible for starter unit formation from indanomycin biosynthesis in E. coli. Three synthetic genes, for IdmI, IdmJ and IdmK, were cloned and subsequently used in expression trials. Despite multiple attempts, IdmI was always expressed as an insoluble protein. IdmJ was expressed as a soluble protein, and an unexpected post-translational modification was found and investigated. Mutagenesis studies suggested that the unknown post-translational modification was occurring at a cysteine 127. IdmK, the carrier protein, was expressed in a soluble form with good yield. Analysis by mass spectrometry showed that, surprisingly, E. coli was able to phosphopantetheinylate IdmK, which is required for a functional module. Preliminary structure determination was carried out by X-ray crystallography. A complete 3D structure was obtained using NMR spectroscopy of the [15N] and [13C, 15N] labelled protein. Structure determination was performed using CS-ROSETTA, which only uses chemical shift data, and ARIA, which assigns ambiguous NOE distance restraints. Both structure calculations produced comparable structures for IdmK. The structure showed a 3 α-helix bundle, with the same topology, lengths and locations of helices as other carrier proteins. Initial investigations into holo-IdmK suggest that the phosphopantetheine co-factor is directed into the hydrophobic core of the protein. This research has set up the system for future studies to engineer the pathway for novel product biosynthesis.

Item Type: Thesis (PhD)
Keywords: Nonribosomal peptide synthetases Polyketide synthases Recombinant protein expression
Academic Units: The University of Leeds > University of Leeds Research Centres and Institutes > Astbury Centre for Structural Molecular Biology (Leeds)
The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Identification Number/EthosID: uk.bl.ethos.638892
Depositing User: Ms Sasha Rebecca Derrington
Date Deposited: 23 Feb 2015 11:31
Last Modified: 06 Oct 2016 14:42
URI: http://etheses.whiterose.ac.uk/id/eprint/7720

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