Mapping Protein-Protein Interactions in Fungal Megasynth(et)ases

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Fungi are responsible for the production of industrially-important and structurally-diverse natural products that have 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). A major class of these natural products 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 can introduce significant 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 provide novel products has attracted intensive research. However, to date, reprogramming efforts have been largely unsuccessful. This is mostly 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 will examine the molecular details of protein-protein interactions (PPIs) in fungal PKSs using state-of-the-art biochemical and biophysical techniques. These include: design and synthesis of novel mechanism-based crosslinkers 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 to allow effective 'reprogramming' of the PKS machinery and yield novel products.