Microbial biosynthesis of β-lactone natural products: from mechanisms to machine learning

Abstract

Natural products with β-lactone (2-oxetanone) rings often have potent antibiotic, antifungal and antitumor properties. These reactive pharmacophores are known to covalently inhibit enzymes from over 20 different families including lipases, proteases, and fatty acid synthases. Since the discovery of the first β-lactone natural product, anisatin, in 1952, over 30 compounds with β-lactone moieties have been isolated from bacteria, fungi, plants and insects. Now in the post-genomic era, the field of natural product drug discovery is in the midst of a transformation from traditional ‘grind and find’ methods to targeted genome mining approaches. However, genomics-guided discovery of new β-lactone natural products was hampered by a lack of understanding of the enzymes that catalyze β-lactone ring formation. In 2017, our lab reported the first standalone β-lactone synthetase enzyme, OleC, in a bacterial long-chain hydrocarbon biosynthesis pathway from Xanthomonas campestris. This thesis builds on this initial breakthrough through biochemical characterization of the substrate specificity, kinetics, and mechanism of X. campestris OleC. Using these biochemical data, I trained machine learning classifiers to predict the substrate specificity of β-lactone synthetases and related adenylate-forming enzymes. I developed this into a web-based predictive tool and mapped the biochemical diversity of adenylate-forming enzymes in >50,000 candidate biosynthetic gene clusters across bacterial, plant, and fungal genomes. This global genomic analysis led to my discovery and characterization of the biosynthetic gene cluster for an orphan β-lactone natural product, nocardiolactone. To more broadly investigate enzymatic production of β-lactone compounds, a library of 1,095 distinct enzyme-substrate combinations for OleA family of enzymes upstream in the β-lactone biosynthesis pathway were screened. Overall, this body of work advanced progress towards the discovery of new β-lactone natural products and combinatorial biosynthesis of β-lactone compound libraries.