Characterising and engineering enzymes involved in antimycin and surugamide biosynthesis

Increased antimicrobial resistance combined with the lack of new antibiotics, is leading towards a return to a pre-antibiotic world and immense clinical strain. The vast majority of clinically utilised antibiotics are derived from secondary metabolites produced by Streptomyces species. Work into the discovery and biosynthesis of these secondary metabolites has undergone a recent resurgence, particularly due to the observation that most organisms harbour an increased number of biosynthetic gene clusters within their genomes than originally postulated. The often-novel enzymology involved in natural product biosynthesis has also generated interest, with understanding their enzymology a key milestone toward the goal of bioengineering novel natural products. This work was centred around three projects based on the biosynthetic pathways producing the antimycins and surugamides. The antimycins are potent bioactive compounds, chemically composed of a central multi-substituted dilactone ring, bound to a rare 3-formamidosalicylate moiety. The biosynthetic steps involved in compound formation are relatively well characterised. In this work, one uncharacterised aspect of the biosynthesis, the function of a standalone ketoreductase, was studied in detail, with particular emphasis on how the structure of the protein mediates specific spatiotemporal catalysis. Engineering of an antimycin pathway acyltransferase domain, utilising a novel methodology, also gave insights into pathway functionality and potentially yielded novel antimycin-type compounds, demonstrating the utility of engineering workflows in the creation of new metabolites. The surugamides are non- ribosomal peptides, whose biosynthesis employs a novel strategy for chain termination and peptide cyclisation. In this work, SurE was found to be a standalone cyclase, and functional characterisation of the enzyme and its substrate scope gave insights into the nature of this versatile biocatalyst. The insights gained in understanding the enzymology of secondary metabolite biosynthesis, and the subsequent engineering of those enzymes, would be valuable in future attempts to create novel bioactive compounds.