Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a rapidly growing natural product class. RiPP precursor peptides can undergo extensive enzymatic tailoring, yielding structurally and functionally diverse products, and their biosynthetic logic makes them attractive bioengineering targets. Recent work suggests that unrelated RiPP modifying enzymes contain structurally similar precursor peptide-binding domains. Using profile hidden Markov model comparisons, we discovered related and previously unrecognized peptide-binding domains in proteins spanning the majority of known prokaryotic RiPP classes; thus, we named this conserved domain the RiPP precursor peptide recognition element (RRE). Through binding studies, we verify the role of the RRE for three distinct RiPP classes: linear azole-containing peptides, thiopeptides, and lasso peptides. Because numerous RiPP biosynthetic enzymes act on peptide substrates, our findings have powerful predictive value as to which protein(s) drive substrate binding, laying a foundation for further characterization of RiPP biosynthetic pathways and the rational engineering of new peptide-binding activities.

Thiopeptides are potent antibiotics that inhibit protein synthesis. They are made by a remarkable post-translational modification process that transforms a linear peptide into a polycyclic structure. We present here the in vitro biosynthesis of the core scaffold of thiomuracin catalyzed by six proteins. We show that cyclodehydration precedes dehydration, and that dehydration is catalyzed by two proteins in a tRNAGlu-dependent manner. The enzyme that generates the pyridine core from two dehydroalanines ejects the leader peptide as a C-terminal carboxamide. Mutagenesis studies of the enzyme TbtD identified important residues for a formal [4+2] cycloaddition process. The core structure of thiomuracin exhibits similar antimicrobial activity to other known congeners, illustrating that in vitro biosynthesis is a viable route to potent antibiotics that can be explored for the rapid and renewable generation of analogues.

Recently developed bioinformatic tools have bolstered the discovery of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Using an improved version of Rapid ORF Description & Evaluation Online (RODEO 2.0), a biosynthetic gene cluster mining algorithm, we bioinformatically mapped the sactipeptide RiPP class via the radical S-adenosylmethionine (SAM) enzymes that form the characteristic sactionine (sulfur-to-alpha carbon) crosslinks between cysteine and acceptor residues. Hundreds of new sactipeptide biosynthetic gene clusters were uncovered and a novel sactipeptide "huazacin" with growth-suppressive activity against Listeria monocytogenes was characterized. Bioinformatic analysis further suggested that a group of sactipeptide-like peptides heretofore referred to as SCIFFs (six cysteines in forty-five residues) might not be sactipeptides as previously thought. Indeed, the bioinformatically-identified SCIFF

Lasso peptides are a class of ribosomally synthesized natural product which possess a unique lariat knot conformation. The low entropy "threaded" conformation endows lasso peptides with considerable resistance to heat and proteolytic degradation, which are attractive properties for the development of peptide-based therapeutics. Despite their discovery nearly 30 years ago, the molecular mechanism underlying lasso peptide biosynthesis remain poorly characterized due to low stability of the purified biosynthetic enzymes. Here, we report the biosynthetic reconstitution of a lasso peptide derived from Thermobifida fusca, termed fusilassin. Beyond robust catalytic activity, the fusilassin enzymes demonstrate extraordinary substrate tolerance during heterologous expression in E. coli and upon purification in cell-free biosynthetic reconstitution reactions. We provide evidence that the fusilassin biosynthetic enzymes are not capable of forming branchedcyclic products but can produce entirely unrelated lasso peptides. Finally, we leveraged our bioinformatic survey of all lasso peptides identified in GenBank to perform coevolutionary analysis of two requisite biosynthetic proteins. This effort correctly identified an important protein-protein interaction site, illustrating how genomics insight can accelerate the characterization of natural product biosynthetic pathways. The fusilassin enzymes described within represent a model system for both designing future lasso peptides of biomedical importance and also for elucidating the molecular mechanisms that govern lasso peptide biosynthesis.

Thiopeptides are members of the ribosomally synthesized and post-translationally modified peptide family of natural products. Most characterized thiopeptides display nanomolar potency toward Gram-positive bacteria by blocking protein translation with several being produced at the industrial scale for veterinary and livestock applications. Employing our custom bioinformatics program, RODEO, we expand the thiopeptide family of natural products by a factor of four. This effort revealed many new thiopeptide biosynthetic gene clusters with products predicted to be distinct from characterized thiopeptides and identified gene clusters for previously characterized molecules of unknown biosynthetic origin. To further validate our data set of predicted thiopeptide biosynthetic gene clusters, we isolated and characterized a structurally unique thiopeptide featuring a central piperidine and rare thioamide moiety. Termed saalfelduracin, this thiopeptide displayed potent antibiotic activity toward several drug-resistant Gram-positive pathogens. A combination of whole-genome sequencing, comparative genomics, and heterologous expression experiments confirmed that the thioamide moiety of saalfelduracin is installed post-translationally by the joint action of two proteins, TfuA and YcaO. These results reconcile the previously unknown origin of the thioamide in two long-known thiopeptides, thiopeptin and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic space, we provide a roadmap for the discovery of additional members of this natural product family.