Discovery and Engineering of RiPP Natural Products

My group studies the genomics, enzymology, structure-function, and engineering of ribosomally synthesized and post-translationally modified peptides (RiPPs). Roughly 50 molecular classes of RiPPs have been described, defined primarily by the type of modification(s) installed. RiPPs are biosynthesized from a bipartite precursor peptide with distinct regions for enzymatic recognition and modification. The physical separation of the substrate specificity and enzymatic modification sites renders RiPPs attractive for the facile evolution of new functions, a strategy already seized by Nature. In learning how to leverage RiPP biosynthesis for various biomedical applications, we discovered a widespread protein domain used by most prokaryotic RiPP pathways to engage the substrate peptide. Coupled with our AI-based tool RODEO, which identifies the often short, hypervariable, and unannotated precursor peptides, we have redefined RiPP genome-mining, illuminated tens of thousands of biosynthetic gene clusters hiding in plain sight, accessed the mature products from scores of pathways, identified robust enzymes for pathway engineering, discovered new enzymatic reactions, and in some instances, predicted enzymatic function. This MIRA application builds on these past achievements and proposes several new innovative directions for our research program. While the pace of genome sequencing accelerates, much of the data remains inaccessible to experimentalists with limited bioinformatics knowledge. Undoubtedly locked away in the Whole- Genome Shotgun database with little/no gene annotation are sequences from metagenomic and microbiome studies that encode countless undiscovered RiPPs formed via entirely new enzymatic transformations. To mine this vast, untapped resource, we are developing MetaRODEO. From the resulting datasets, we will validate the algorithm by experimentally characterizing new classes of RiPPs that are formed via unprecedented enzyme chemistry. As per our past practices, the tool and all datasets will be publicly available. Additionally, this project will elucidate the mechanistic enzymology that guides known and newly discovered RiPP biosynthetic pathways. Lasso peptides, for instance, are RiPPs distinguished by a knotted architecture and remarkable heat and protease stability. We wish to understand the molecular gymnastics performed by lasso cyclases that kinetically trap lasso peptides in their threaded rotaxane conformation. Inspired by their natural diversity and disparate functions, we will exploit our knowledge of lasso peptide biosynthesis to perform structure-guided and directed-evolution studies to engage desired biological targets with high affinity and specificity.