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Evolution-guided engineering of bacterial NRPS systems

Principal Supervisor: Dr Alex Mullins

Secondary Supervisor(s): Professor Greg Challis and Dr Lona Alkhalaf

University of Registration: University of Warwick

BBSRC Research Themes:

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Deadline: 4 January, 2024


Project Outline

Background

Bacteria supply the world with an extraordinary number of useful bioactive compounds with uses in agriculture as pesticides and herbicides, and medicine as antimicrobials and pharmaceuticals. This breadth of functionality stems from the impressive structural diversity of the compounds influencing their size, composition, stereochemistry, and cyclisation. Non-ribosomal peptides represent a well-known class of natural product that are synthetised by large multi-enzyme complexes known as non-ribosomal peptide synthetases (NRPSs). NRPS protein architecture consists of modules responsible for the recruitment and condensation of amino acids to the growing peptide to synthetise the final product. Given the modular architecture of NRPS systems, there is considerable interest in manipulating these assembly lines to engineer novel compound derivatives with improved efficacy, specificity or stability.

Conventional techniques of NRPS engineering are often highly case-dependent with restricted broader applicability and create systems with low product tires. An alternative approach applied in my research aims to exploit Nature through an evolution-inspired approach to NRPS engineering. Multiple examples of evolutionarily related biosynthetic gene clusters (BGCs) responsible for NRPS enzymes show evidence of recombination events that lead to the structural diversity observed in Nature. By identifying these events through genomic analysis, we can understand how to successfully engineer NRPS systems in the lab to produce desired natural product derivatives. NRPS BGCs have a high prevalence in diverse bacteria such as Pseudomonas, Burkholderia, Streptomyces, and Bacillus. The huge expansion of available bacterial genomes in recent years, accurate genome-based taxonomy, and ability to rapidly predict BGCs provides an exciting opportunity to understand the natural variations that exist in NRPS systems.

Aim

This genomics-driven PhD project will exploit advances in bacterial genomics to identify and understand the recombination events in NRPS systems. These events will then be re-created in the lab and expanded to engineer novel NRPS systems and synthesise new natural product derivatives.

Techniques

This is an interdisciplinary project that will combine the following techniques:

  • Bacterial genomics (bioinformatics)
  • Microbiology (aseptic technique, antimicrobial activity assays)
  • Molecular genetics (cloning, TAR)
  • Metabolic profiling (LC-MS)
  • Metabolite purification (HPLC)
  • Structural elucidation (NMR)