Abstract 2219 Exploring uncharted territory in RiPP biosynthesis: studies on curacozole, a cyanobactin from Streptomyces curacoi

The majority of antibiotic, antiviral, and anticancer drugs used in modern medicine are natural products and their derivatives. Ribosomally synthesized and post translationally modified peptides (RiPPs) form a major class of bioactive natural products, comprising over 20 structurally distinct families. To unlock the full potential of RiPPs and opportunities for pathway engineering, we must develop a deeper understanding of their biosynthetic principles.1,2 The cyanobactins are a class of RiPP first discovered in cyanobacteria. They are small cyclic peptides that are highly modified through side chain cyclization, epimerization, prenylation, and oxidation.3 In recent years, similar compounds have been discovered in Streptomyces. This project aims to characterize the biosynthetic pathway of curacozole, a cyanobactin produced by the soil bacterium Streptomyces curacoi. A putative gene cluster has been proposed, but the functions of the many encoded enzymes are unknown as they share little similarity to the known biosynthetic gene clusters of cyanobactins, such as the patellamides and trunkamide.4,5 Enzymes and peptides were purified by nickel column chromatography. In vitro assays were performed and analyzed by LC-MS and MS/MS. So far, cyclodehydratase and dehydrogenase enzymes have been biochemically characterized by the formation of one heterocycle. Investigations of a putative Ile epimerase in the cluster show evidence of activity. Finally, a novel cupin-superfamily enzyme adjacent to the cluster shows oxidative activity and dependence on alpha-ketoglutarate. References 1. Hudson, G. A.; Mitchell, D. A. RiPP Antibiotics: Biosynthesis and Engineering Potential. Current Opinion in Microbiology 2018, 45, 61–69. 2. Montalbán-López, M.; Scott, T. A.; Ramesh, S.; Rahman et. al. New Developments in RiPP Discovery, Enzymology and Engineering. Nat. Prod. Rep. 2021, 38 (1), 130–239. 3. Sivonen, K.; Leikoski, N.; Fewer, D. P.; Jokela, J. Cyanobactins-Ribosomal Cyclic Peptides Produced by Cyanobacteria. Appl Microbiol Biotechnol 2010, 86 (5), 1213–1225. 4. Kaweewan, I.; Komaki, H.; Hemmi et. al. Isolation and Structure Determination of a New Cytotoxic Peptide, Curacozole, from Streptomyces Curacoi Based on Genome Mining. J Antibiot 2019, 72 (1), 1–7. 5. Schmidt, E. W.; Nelson, J. T.; Rasko et. al. Patellamide A and C Biosynthesis by a Microcin-like Pathway in Prochloron Didemni, the Cyanobacterial Symbiont of Lissoclinum Patella. Proc Natl Acad Sci U S A2005, 102 (20), 7315–7320. We would like to acknowledge NSERC for funding the research.