Ringing the changes: how bacterial enzymes convert ribosomally-synthesised peptides into antibiotics

The project aims to investigate the mechanism of peptide transformations carried out by YcaO enzymes and to use this knowledge to create novel antimicrobials. Bacterial YcaO proteins are essential for the biosynthesis of multiple classes of peptide natural products through their ability to introduce important structural modifications including heterocycles, macrocycles, and thioamides. YcaO repurposing and swapping between different pathways offers exciting possibilities to create novel artificial peptides bearing such modifications. Despite much recent progress, we still do not really understand how these proteins work, and how their activities are coordinated within multisubunit enzyme complexes. I will use cryo-EM in combination with X-ray crystallography to solve the structures of YcaOs within their native complex assemblies trapped at different catalytic stages. In parallel, I will discover and characterise new YcaO proteins to further expand their known catalytic repertoire. Finally, the promiscuity of YcaOs allows for the production of large genetically encoded peptide antibiotic libraries. I will generate these by co-expressing randomised precursor genes along with modification systems. A high-throughput fluorescence-based version of a Waksman platform will be used for screening, where nano-sized “Petri dishes” (hydrogel beads) will be inoculated with both producer and reporter strains and sorted by FACS.