About the Project
Fungi are responsible for production of industrially-important and structurally-diverse natural products with a wealth of applications in medicine (e.g. the treatment of infectious diseases, cancer, high cholesterol and transplant rejection) and agriculture (e.g. crop protection and animal health). Of which, a major class of these compounds are polyketides. In fungi, polyketides are synthesised by giant multi-domain ‘megasynthase’ proteins known as polyketide synthases (PKSs), which closely resemble the mammalian fatty acid synthase (FAS) in both domain architecture and catalysis. However, unlike fatty acid biosynthesis, fungal PKSs introduce structural complexity by programming the degree of chain processing. This ultimately dictates the structural elements introduced into the final natural product.
Since the discovery of PKSs in fungi, the desire to manipulate the programming of the biosynthetic machinery to produce novel structures has attracted intensive research. However, to date, reprogramming efforts have been largely unsuccessful. This is largely due to incompatible domain combinations resulting in erroneous products and dramatically reduced activity and yields, limiting the engineering potential of PKSs, and preventing access to further potential therapeutics. Different regions of a given PKS domain regulate how it interacts with both its substrate and with other domains. Within the PKS itself, these interactions are critical for the correct ordering of reactions and efficient polyketide construction. Achieving control over domain function via an in-depth understanding of PKS systems, at the molecular level, is essential for achieving and enhancing these bioengineering strategies.
This project aims to examine the molecular details of protein-protein interactions (PPIs) in fungal PKSs using state-of-the-art biochemical and biophysical techniques. These include: mechanism-based crosslinking to trap protein complexes; alanine scanning mutagenesis and carbene footprinting to pin-point interaction sites; intact protein mass spectrometry to visualise the biosynthetic process; and X-ray crystallography to obtain atomic-level structures of individual domains within the PKS. These data will guide mutations/domain switches in the PKS allowing effective ‘reprogramming’ of the PKS machinery to yield novel products.
BBSRC Strategic Research Priority: Renewable Resources and Clean Growth: Industrial Biotechnology. Understanding the Rules of Life: Microbiology & Structural Biology
Techniques that will be undertaken during the project:
• Molecular cloning.
• Recombinant protein overproduction and purification.
• Intact/structural protein mass spectrometry techniques.
• Protein biochemistry.
• X-ray crystallography of proteins.
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