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Biosynthetic engineering of nonribosomal lipopeptide antibiotics in the model actinomycete Streptomyces coelicolor

ReferenceBB/C503662/1
Principal Investigator / Supervisor Professor Jason Micklefield
Co-Investigators /
Co-Supervisors		 Professor Colin Smith
Institution The University of Manchester
DepartmentChemistry
Funding typeResearch
Value (£) 246,575
StatusCompleted
TypeResearch Grant
Start date 28/03/2005
End date 27/03/2008
Duration36 months

Abstract

The first major aim of the project is to continue investigating the biosynthetic origin of CDA. In particular we wish to characterise the individual enzymes involved in the biosynthesis of the unusual amino acids (3-methylgutamic acid, Z-dehydrotryptophan, 3-hydroxyasparagine & 3-phosphohydroxyasparagine) as well as the trans-2, 3-epoxyhexanoyl fatty acid side chain in CDA. Similarly we wish to explore the mode of transfer of the fatty acids side chain in the initiation step of lipopeptide assembly. To do this we will delete genes in the cda cluster that are predicted to be involved in CDA biosynthesis. The mutants will then be characterised in order to determine if they produce CDA, or new variants of CDA. We will then feed synthetic precursors in a attempt to re-establish CDA production or to accumulate modified CDAs (mutasynthesis). In addition we will complement the mutants, in trans, with copies of the missing genes or genes encoding similar enzymes from the other lipopeptide gene clusters. Secondly we will overproduce key enzymes in E. coli, and/or Streptomyces lividans and develop in vitro assays with synthetic substrates in order to establish their function and specificity. The second main aim of the project is to develop methods for engineering the biosynthesis of modified lipopeptides in vivo. To do this we will explore three approaches: mutasynthesis, site directed mutagenesis of adenylation domains and module swapping experiments. For example we will feed synthetic glutamic acid derivatives and modified fatty acids to mutants lacking the genes involved in 3-MeGlu or fatty acid biosynthesis in order to generate modified lipopeptides by mutasynthesis. In addition we will replace genes involved in fatty acid biosynthesis and transfer within the CDA cluster with those from other lipopeptides gene clusters (eg. daptomycin and A54145) in order that alternative fatty acid side chains might be used to initiate CDA biosynthesis. We will use site directed mutagenesis of A-domains in vivo to change the amino acid sequence of CDA. We will also explore if similar mutation of condensation domains can be used to improve the efficiency of peptide bond formation with non-cognate amino acids and avoid hydrolysis of upstream peptide intermediates; this was noted in A-domain mutants generated in our earlier work. We will investigate module-swapping experiments using NRPS-encoding genes from the daptomycin and A54145 gene clusters. Here, homologous recombination strategies will be used to replace or add modules to generate lipopeptides of differing size as well as sequence. Finally we will generate large libraries of do novo NRPS modules using the methods of directed evolution (sloppy PCR, DNA shuffling, environmental DNA) and use these to replace existing modules of the CDA NRPS. New NRPSs will be selected that are capable of activating and incorporating unusual amino acids, that are not found in natural nonribosomal peptides. Biotica technologies Ltd will test the antibiotic activity of these compounds. (Joint with BB/C507210/1)

Summary

unavailable
Committee Closed Committee - Biomolecular Sciences (BMS)
Research TopicsX – not assigned to a current Research Topic
Research PriorityX – Research Priority information not available
Research Initiative X - not in an Initiative
Funding SchemeX – not Funded via a specific Funding Scheme
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