EVOLUTION OF THE GLYCOPEPTIDE BIOSYNTHETIC GENE CLUSTER FAMILY

Abstract

The serendipitous discovery of antibiotics in the 20th century paved the way for safer, modern medical interventions. Bacteria in the phylum Actinobacteria are the most prolific producers of natural products, including antibiotics. The availability of low-cost, high-throughput generation of bacterial genome sequence data transforms natural product discovery, making it possible to judge the biosynthetic capacity of a strain based on its genome sequence. I developed a software tool to compare biosynthetic gene clusters (BGCs) to show that streptothricin production is distributed among Streptomyces and that the capacity to produce common natural products does not predict the remaining potential of Streptomyces species. This approach provides a way to consider the rarity of particular natural products and grounded a biotechnological approach using CRISPR/Cas9 engineering to facilitates the identification of rare natural products in these strains. Glycopeptide antibiotics (GPAs) are encoded by BGCs in several genera of Actinobacteria. Their diversity is the product of an intricate evolutionary history. We show that GPA biosynthesis and resistance maps to approximately 150-400 million years ago, from an older, pre-existing pool of components. We find that resistance appeared contemporaneously with biosynthetic genes, raising the possibility that the mechanism of action of glycopeptides was a driver of diversification in these gene clusters. In a set of GPA BGCs, we identify several scaffolds distinct from the traditional D-Ala-D-Ala binding antibiotics. while complestatin, kistamicin, and longer peptides like enduracidin and ramoplanin are known, others are uncharacterized. Through phylogenetic analysis of these BGCs we develop a new classification scheme to organize these BGCs into four major classes. Structural predictions led us to purify complestatin and a novel compound we named corbomycin. Both possess antibacterial activity. Mutations conferring decreased susceptibility to these compounds suggest a novel mechanism of action distinct from known compounds in the GPA family.