Award details

Investigating the Biosynthesis of Unusual Peptide Natural Products

Reference	BBS/E/J/000CA538
Principal Investigator / Supervisor	Dr Andrew Truman
Co-Investigators / Co-Supervisors
Institution	John Innes Centre
Department	John Innes Centre Department
Funding type	Research
Value (£)	97,415
Status	Current
Type	Institute Project
Start date	01/10/2013
End date	30/09/2018
Duration	59 months

Abstract

The overall goal of this research project is to employ complementary genetic and biochemical methods to probe the biosynthesis of unusual peptide-derived compounds. An example is bottromycin, which has potent antibacterial activity and possesses beta-methylated amino acids and a macrocyclic amidine. The bottromycin gene cluster represents a starting point for the discovery of other unusual peptide pathways. The objectives of this research programme are: 1. In vitro analysis of key bottromycin enzymes Radical SAM methyltransferases (RSMTs) are a poorly characterised class of enzyme that requires a highly oxygen sensitive iron-sulfur cluster. A variety of methods will be tested for optimal protein expression, purification and reconstitution. This will be carried out in anaerobic conditions and biophysical techniques (EPR, CD, UV-Vis) will be used to assess iron-sulfur cluster reconstitution. Activity with peptide substrates will be analysed using liquid chromatography-mass spectrometry (LC-MS). Candidate amidine-forming cyclases will be co-expressed with their putative peptide substrate and modifications will be assessed by LC-MS. Confirmed cyclase(s) will be purified and further analysed by crystallography and mutagenesis. 2. Reconstitution of bottromycin biosynthesis A synthetic biology approach will be used to generate partial bottromycin gene clusters, where different sets of genes will generate different intermediates or shunt metabolites. Reporter genes with colorimetric outputs will be encoded in synthetic clusters to quantify transcription. 3. Genome mining for peptide natural products Genome mining has identified a number of putative gene clusters for ribosomal peptides that contain radical SAM proteins similar to those in the bottromycin cluster. These clusters will be probed by gene deletion and heterologous expression. Metabolic or biological changes will be examined following genetic manipulation, and novel compounds will be chemically characterised.

Summary

unavailable

Committee	Not funded via Committee
Research Topics	Microbiology, Structural Biology, Synthetic Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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