Understanding the link between genetics and natural product biosynthesis informs not only on the types of reactions nature applies for synthesis, but also how biosynthetic enzymes might be used as tools to quickly make new compounds. For ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, linear peptides are chemically modified by tailoring enzymes, generating structurally diverse compounds from simple, genetically encoded templates. Bioinformatic analysis of publically-available open-source genomic data provides a means to identify and even predict novel RiPP tailoring. The cyclodehydratase responsible for RiPP azoline formation was used to search for divergent enzymes of the same protein family (YcaO) on this basis. One such candidate was predicted and confirmed to be responsible for peptide macrocyclization, forming a 12-member amidine ring, the major structural motif of the potent antimicrobial natural product bottromycin. The formation of this product was found to occur through ATP-utilizing backbone activation chemistry, analogous to previously characterized YcaO-family enzymes. Thus, bottromycin’s cyclase illustrates how a similar mechanism can evolve to form dissimilar products, expanding the known biosynthetic capabilities of YcaO proteins. <i>In vitro</i> reactions with mutant precursor peptide substrates show relaxed substrate specificity, forming novel “unnatural natural products”. This is in contrast with previous <i>in vivo</i> heterologous expression systems and suggests that biosynthetic reconstitution may be a better strategy to process mutant substrates and generate new analogs. These enzymes are being used to produce a panel of bottromycin-like derivatives to be screened for similar antibacterial effects and to evaluate the biosynthetic machinery’s promiscuity as a general peptide-macrocyclization reaction.

Natural product engineering enabled by a YcaO-domain bottromycin macrocyclase

251st National Meeting (2016)

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