Re-engineering the building blocks of bacterial natural product assembly lines

Type I polyketide synthases (PKS) are highly complex biosynthetic machines that produce valuable bioactive natural products. Rational engineering and manipulation of PKS building blocks could provide a route to the production of novel high value chemical entities and this project aims to advance our understanding of these fundamental pathways. Kalimantacin is a polyketide assembled by a hybrid type I trans-acyltransferase (AT) PKS/non-ribosomal peptide synthase (NRPS) and displays potent antibacterial properties, including high selectivity for staphylococcal species. Its -branches are critical for its antibiotic activity and incorporated by a multi-protein 3-hydroxy-3-methylglutaryl synthase (HMGS) cassette. Further understanding of how these enzymes tightly control the incorporation of -branches is essential for the generation of novel chemicals with potent biological activities. Studying the kalimantacin assembly line therefore provides an excellent opportunity to achieve this. Key components of the HMGS cassette (enoyl-CoA hydratase domains, both modular and trans-acting) that form either an endo--methyl or exo--methylene will be studied to understand their structure and function and determine if the order of -branch incorporation can be reversed by logical protein engineering. X-ray crystallography, site-directed mutagenesis, Cryo-EM and NMR assays are just some of the techniques that will be employed to explore this exciting avenue.