Principal Supervisor: Dr. Matthew JennerLink opens in a new window

Co-supervisor: Dr. Mark Greenhalgh

PhD project title: Engineering Biosynthetic Assembly Lines for the Incorporation of Photoreactive Diazirines

University of Registration: University of Warwick

Project outline:

Polyketides are an industrially-important family of natural products, which find a wealth of applications in medicine (e.g. the treatment of infectious diseases, cancer, high cholesterol and transplant rejection). Many of these molecules are constructed in a stepwise manner from simple acyl building blocks by multi-domain enzymatic assembly lines known as polyketide synthases (PKSs). The biosynthetic logic employed by PKSs utilises pantetheine (Pant) groups appended to acyl carrier protein (ACP) domains to shuttle thioester intermediates between catalytic domains, similar to that of fatty acid biosynthesis. The complex and diverse structures exhibited by polyketide natural products are challenging to produce and/or modify via chemical synthesis alone. However, given the modular nature of their biosynthetic construction, these pathways are excellent targets for biosynthetic engineering efforts to generate natural product analogues.

While many polyketide compounds exhibit excellent bioactivity profiles, identification of their molecular target can be difficult, yet this knowledge is essential for their development as drug leads. Diazirine-based photoactivatable probes can aid this process by covalently capturing transient molecular interactions via in situ formation of a highly reactive carbene upon exposure to long-wave UV radiation (~350 nm). This allows target proteins to be identified and the ‘interactome’ of the compound to be established using standard proteomics-based methodologies. Furthermore, this approach has the potential to identify the location of binding sites once a molecular target has been established. Diazirine incorporation into natural products has been achieved via total synthesis and semi-synthesis approaches. This was particularly successful for polymyxin B, where solid-phase peptide synthesis allowed incorporation of a diazirine-containing leucine residue (photo-leucine) into the natural product scaffold. Photolabelling experiments in E. coli allowed the membrane-bound protein targets of polymyxin B to be identified. However, (semi)synthetic approaches are generally challenging, costly, and often unselective.

AIM: This PhD project aims to engineer a catalytic domain from a PKS pathway to selectively incorporate a diazirine moity into a polyketide natural product, allowing covalent modification of natural product target(s) through photochemical labelling experiments (Fig. 1). The project will use a PKS system for which the molecular target is already established to validate the methodology, laying the foundations for a versatile approach to molecular target identification.

Fig. 1 | Engineering of a single PKS / NRPS catalytic domain for the biosynthetic incorporation of diazirine functionality into natural product skeletons.

This project will be jointly supervised by Dr. Matthew Jenner (Protein Biochemistry, Mass Spectrometry, Structural Biology) and Dr. Mark Greenhalgh (Diazirine Synthesis)
 

BBSRC Strategic Research Priority: Understanding the rules of life – Structural Biology, and Microbiology, and Renewable Resources and Clean Growth - Industrial Biotechnology.

 

Techniques that will be undertaken during the project:

• Bioinformatics.
• Molecular cloning.
• Recombinant protein overproduction and purification.
• Organic synthesis.
• Intact/structural protein mass spectrometry techniques.
• Protein biochemistry.
• X-ray crystallography of proteins.
  
  

Contact: Dr. Matthew JennerLink opens in a new window