Dissecting and Harnessing Carrier Protein Interactions in Fungal Megasynth(et)ases

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Fungal polyketide synthases (PKS) are giant multi-domain proteins responsible for the biosynthesis of a vast number of biologically active natural products. Whilst many find important applications as medicinal or agrochemical agents, some are toxins that are harmful to both human health and agriculture. The precise assembly of these complex compounds relies on the highly programmed protein-protein interactions (PPIs) between a small carrier protein (CP) domain, to which biosynthetic intermediates are covalently tethered, and the individual catalytic domains within the PKSs, ensuring that the overall process is efficient and maintains product fidelity. Despite the importance of these interactions, they remain poorly understood, primarily due to their ephemeral nature, requiring a combination of techniques to be studied effectively. Uncovering the molecular factors governing programming is the greatest remaining problem in our understanding of fungal PKSs, and represents a huge obstacle to rewiring these enzymes towards user-designed molecules. This project aims to combine cutting-edge mass spectrometry, structural biology and biochemical techniques to elucidate the molecular details of CP-dependent interactions underpinning fungal PKS machinery. The research will initially focus on characterising PPIs in two similar PKS systems; one involved in the biosynthesis of cyclosporin (clinically used immunosuppressant), the other responsible for the construction of lovastatin (clinically used cholesterol-lowering agent). These PKSs differ by the positioning of a single catalytic domain, and therefore represent excellent model systems to establish the common principles underlying CP-dependent interactions. Taken together, this body of work will significantly deepen our understanding of the roles played by PPIs in fungal PKSs. It will also form a knowledge-base to begin exploiting such interactions to construct engineered systems capable of producing novel natural product analogues.