Catching antibiotic factories in action

Polyketide biosynthetic pathways generate vast numbers of diverse natural products and encompassing numerous chemical structures that are exploited for applications including pharmaceuticals, animal health and agrochemical products. Polyketides are generated by modular polyketide synthases, sophisticated biosynthetic multi-domain megaenzymes which act essentially like assembly lines and which may be rationally manipulated to deliver functionally optimised products. The chemical structure of each molecule produced is determined by the enzymes present at each tage of the assembly line, rather like a blueprint. We understand some rules for building these factories and can rearrange the order of modules to produce new compounds, but sometimes this just breaks the assembly line, or produces an unexpected compound. Due to the complexity of the biological "factories", no single technique provides the whole picture, but this PhD project will be part of our wider effort to bring together important skills and scientific expertise to focus different "lenses" on the problem. An understanding of how these systems work will help answer important questions about their design principles so new pathways to novel compounds can be built in a rational way. To provide insights into the molecular mechanisms, the PhD project will we aim to solve cryo-EM and crystal structures of polyketide synthase proteins complexes along the pathway of the anti-MRSA antibiotic kalimantacin (Figure 1A). The dynamic nature of these systems makes studying them by cryo-EM challenging (Figure 1B) but this will be solved by using specific chemical substrate-mimetic probes (i.e. fragments of kalimantacin) in combination with Cryo-EM to capture intermediate conformations of the synthase. You will also work with NMR spectroscopists to use labelled probes (eg 13C or 19F) to follow processing of intermediates in real time. Together this real time monitoring and the Cryo-EM structures will provide a unique spatiotemporal picture of how these antibiotic factories work. Crucially, with this synthetic biology project you will learn techniques encompassing Cryo-EM, X-ray crystallography, NMR, microbiology and molecular modelling/design as well as work with synthetic chemists, offering a wide array of avenues to explore.