McrD binds asymmetrically to methyl-coenzyme M reductase improving active-site accessibility during assembly.

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane, and its activity accounts for nearly all biologically produced methane released into the atmosphere. The assembly of MCR is an intricate process involving the installation of a complex set of posttranslational modifications and the unique Ni-containing tetrapyrrole called coenzyme F430. Despite decades of research, details of MCR assembly remain largely unresolved. Here, we report the structural characterization of MCR in two intermediate states of assembly. These intermediate states lack one or both F430 cofactors and form complexes with the previously uncharacterized McrD protein. McrD is found to bind asymmetrically to MCR, displacing large regions of the alpha subunit and increasing active-site accessibility for the installation of F430-shedding light on the assembly of MCR and the role of McrD therein. This work offers crucial information for the expression of MCR in a heterologous host and provides targets for the design of MCR inhibitors.

Tryptophan-Centric Bioinformatics Identifies New Lasso Peptide Modifications.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by a macrolactam linkage between the N-terminus and the side chain of an internal aspartic acid or glutamic acid residue. Instead of adopting a branched-cyclic conformation, lasso peptides are "threaded", with the C-terminal tail passing through the macrocycle to present a kinetically trapped rotaxane conformation. The availability of enhanced bioinformatics methods has led to a significant increase in the number of secondary modifications found on lasso peptides. To uncover new ancillary modifications in a targeted manner, a bioinformatic strategy was developed to discover lasso peptides with modifications to tryptophan. This effort identified numerous putative lasso peptide biosynthetic gene clusters with core regions of the precursor peptides enriched in tryptophan. Parsing of these tryptophan (Trp)-rich biosynthetic gene clusters uncovered several putative ancillary modifying enzymes, including halogenases and dimethylallyltransferases expected to act upon Trp. Characterization of two gene products yielded a lasso peptide with two 5-Cl-Trp modifications (chlorolassin) and another bearing 5-dimethylallyl-Trp and 2,3-didehydro-Tyr modifications (wygwalassin). Bioinformatic analysis of the requisite halogenase and dimethylallyltransferase revealed numerous other putative Trp-modified lasso peptides that remain uncharacterized. We anticipate that the Trp-centric strategy reported herein may be useful in discovering ancillary modifications for other RiPP classes and, more generally, guide the functional prediction of enzymes that act on specific amino acids.

Darobactin Substrate Engineering and Computation Show Radical Stability Governs Ether versus C-C Bond Formation.

The Gram-negative selective antibiotic darobactin A has attracted interest owing to its intriguing fused bicyclic structure and unique targeting of the outer membrane protein BamA. Darobactin, a ribosomally synthesized and post-translationally modified peptide (RiPP), is produced by a radical S-adenosyl methionine (rSAM)-dependent enzyme (DarE) and contains one ether and one C-C cross-link. Herein, we analyze the substrate tolerance of DarE and describe an underlying catalytic principle of the enzyme. These efforts produced 51 enzymatically modified darobactin variants, revealing that DarE can install the ether and C-C cross-links independently and in different locations on the substrate. Notable variants with fused bicyclic structures were characterized, including darobactin W3Y, with a non-Trp residue at the twice-modified central position, and darobactin K5F, which displays a fused diether ring pattern. While lacking antibiotic activity, quantum mechanical modeling of darobactins W3Y and K5F aided in the elucidation of the requisite features for high-affinity BamA engagement. We also provide experimental evidence for ß-oxo modification, which adds support for a proposed DarE mechanism. Based on these results, ether and C-C cross-link formation was investigated computationally, and it was determined that more stable and longer-lived aromatic Cß radicals correlated with ether formation. Further, molecular docking and transition state structures based on high-level quantum mechanical calculations support the different indole connectivity observed for ether (Trp-C7) and C-C (Trp-C6) cross-links. Finally, mutational analysis and protein structural predictions identified substrate residues that govern engagement to DarE. Our work informs on darobactin scaffold engineering and further unveils the underlying principles of rSAM catalysis.

Enterolyin S, a Polythiazole-containing Hemolytic Peptide from Enterococcus caccae.

The ß-hemolytic factor streptolysin S (SLS) is an important linear azol(in)e-containing peptide (LAP) that contributes significantly to the virulence of Streptococcus pyogenes. Despite its discovery 85 years ago, SLS has evaded structural characterizing owing to its notoriously problematic physicochemical properties. Here, we report the discovery and characterization of a structurally analogous hemolytic peptide from Enterococcus caccae, termed enterolysin S (ELS). Through heterologous expression, site-directed mutagenesis, chemoselective modification, and high-resolution mass spectrometry, we found that ELS contains an intriguing contiguous octathiazole moiety. The discovery of ELS expands our knowledge of hemolytic LAPs by adding a new member to this virulence-promoting family of modified peptides.

Mechanism of Action of Ribosomally Synthesized and Post-Translationally Modified Peptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a natural product class that has undergone significant expansion due to the rapid growth in genome sequencing data and recognition that they are made by biosynthetic pathways that share many characteristic features. Their mode of actions cover a wide range of biological processes and include binding to membranes, receptors, enzymes, lipids, RNA, and metals as well as use as cofactors and signaling molecules. This review covers the currently known modes of action (MOA) of RiPPs. In turn, the mechanisms by which these molecules interact with their natural targets provide a rich set of molecular paradigms that can be used for the design or evolution of new or improved activities given the relative ease of engineering RiPPs. In this review, coverage is limited to RiPPs originating from bacteria.

Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element.

The RiPP precursor recognition element (RRE) is a conserved domain found in many prokaryotic ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic gene clusters (BGCs). RREs bind with high specificity and affinity to a recognition sequence within the N-terminal leader region of RiPP precursor peptides. Lasso peptide biosynthesis involves an RRE-dependent leader peptidase, which is discretely encoded or fused to the RRE as a di-domain protein. Here we leveraged thousands of predicted BGCs to define the RRE:leader peptidase interaction through evolutionary covariance analysis. Each interacting domain contributes a three-stranded ß-sheet to form a hydrophobic ß-sandwich-like interface. The bioinformatics-guided predictions were experimentally confirmed using proteins from discrete and fused lasso peptide BGC architectures. Support for the domain-domain interface derived from chemical shift perturbation, paramagnetic relaxation enhancement experiments, and rapid variant activity screening using cell-free biosynthesis. Further validation of selected variants was performed with purified proteins. We developed a p-nitroanilide-based leader peptidase assay to illuminate the role of RRE domains. Our data show that RRE domains play a dual function. RRE domains deliver the precursor peptide to the leader peptidase, and the rate is saturable as expected for a substrate. RRE domains also partially compose the elusive S2 proteolytic pocket that binds the penultimate threonine of lasso leader peptides. Because the RRE domain is required to form the active site, leader peptidase activity is greatly diminished when the RRE domain is supplied at substoichiometric levels. Full proteolytic activation requires RRE engagement with the recognition sequence-containing portion of the leader peptide. Together, our observations define a new mechanism for protease activity regulation.

Functional elucidation of TfuA in peptide backbone thioamidation.

YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser-Lys catalytic pair.

Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans.

The enzyme methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six posttranslationally modified amino acids near the active site. Recent studies have identified enzymes performing two of these modifications (thioglycine and 5-[S]-methylarginine), yet little is known about the formation and function of the remaining posttranslationally modified residues. Here, we provide in vivo evidence that a dedicated S-adenosylmethionine-dependent methyltransferase encoded by a gene we designated methylcysteine modification (mcmA) is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue might play a role in adaption to mesophilic conditions. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. High-resolution crystal structures of inactive MCR lacking the modified amino acids were indistinguishable from the fully modified enzyme, suggesting that interactions between the posttranslationally modified residues do not exert a major influence on the static structure of the enzyme but rather serve to fine-tune the activity and efficiency of MCR.

Accessing Diverse Pyridine-Based Macrocyclic Peptides by a Two-Site Recognition Pathway.

Macrocyclic peptides are sought-after molecular scaffolds for drug discovery, and new methods to access diverse libraries are of increasing interest. Here, we report the enzymatic synthesis of pyridine-based macrocyclic peptides (pyritides) from linear precursor peptides. Pyritides are a recently described class of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are related to the long-known thiopeptide natural products. RiPP precursors typically contain an N-terminal leader region that is physically engaged by the biosynthetic proteins that catalyze modification of the C-terminal core region of the precursor peptide. We demonstrate that pyritide-forming enzymes recognize both the leader region and a C-terminal tripeptide motif, with each contributing to site-selective substrate modification. Substitutions in the core region were well-tolerated and facilitated the generation of a wide range of pyritide analogues, with variations in macrocycle sequence and size. A combination of the pyritide biosynthetic pathway with azole-forming enzymes was utilized to generate a thiazole-containing pyritide (historically known as a thiopeptide) with no similarity in sequence and macrocycle size to the naturally encoded pyritides. The broad substrate scope of the pyritide biosynthetic enzymes serves as a future platform for macrocyclic peptide lead discovery and optimization.

Enzymatic Pyridine Aromatization during Thiopeptide Biosynthesis.

Thiazole-containing pyritides (thiopeptides) are ribosomally synthesized and post-translationally modified peptides (RiPPs) that have attracted interest owing to their potent biological activities and structural complexity. The class-defining feature of a thiopeptide is a six-membered, nitrogenous heterocycle formed by an enzymatic [4 + 2]-cycloaddition. In rare cases, piperidine or dehydropiperidine (DHP) is present; however, the aromatized pyridine is considerably more common. Despite significant effort, the mechanism by which the central pyridine is formed remains poorly understood. Building on our recent observation of the Bycroft-Gowland intermediate (i.e., the direct product of the [4 + 2]-cycloaddition), we interrogated thiopeptide pyridine synthases using a combination of targeted mutagenesis, kinetic assays, substrate analogs, enzyme-substrate cross-linking, and chemical rescue experiments. Collectively, our data delineate roles for several conserved residues in thiopeptide pyridine synthases. A critical tyrosine facilitates the final aromatization step of pyridine formation. This work provides a foundation for further exploration of the [4 + 2]-cycloaddition reaction and future customization of pyridine-containing macrocyclic peptides.

Oil Irradiation Experiments Document Changes in Oil Properties, Molecular Composition, and Dispersant Effectiveness Associated with Oil Photo-Oxidation.

While chemical dispersants are a powerful tool for treating spilled oil, their effectiveness can be limited by oil weathering processes such as evaporation and emulsification. It has been suggested that oil photo-oxidation could exacerbate these challenges. To address the role of oil photo-oxidation in dispersant effectiveness, outdoor mesocosm experiments with crude oil on seawater were performed. Changes in bulk oil properties and molecular composition were quantified to characterize oil photo-oxidation over 11 days. To test relative dispersant effectiveness, oil residues were evaluated using the Baffled Flask Test. The results show that oil irradiation led to oxygen incorporation, formation of oxygenated hydrocarbons, and higher oil viscosities. Oil irradiation was associated with decreased dispersant efficacy, with effectiveness falling from 80 to <50% in the Baffled Flask Test after more than 3 days of irradiation. Increasing photo-oxidation-induced viscosity seems to drive the decreasing dispersant effectiveness. Comparing the Baffled Flask Test results with field data from the Deepwater Horizon oil spill showed that laboratory dispersant tests underestimate the dispersion of photo-oxidized oil in the field. Overall, the results suggest that prompt dispersant application (within 2-4 days), as recommended by current oil spill response guidelines, is necessary for effective dispersion of spilled oil.

Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance.

Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like, threaded conformation. Owing to a locked three-dimensional structure, lasso peptides can be unusually stable toward heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. All known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent class of RiPPs; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address longstanding challenges associated with lasso peptide production. We report the successful use of CFB for the formation of an array of sequence-diverse lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants to evaluate the substrate tolerance of the biosynthetic pathway. By evaluating more than 1000 randomly chosen variants, we show that the lasso-forming cyclase from the fusilassin pathway is capable of producing millions of sequence-diverse lasso peptides via CFB. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.

New developments in RiPP discovery, enzymology and engineering.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.

Enzymatic reconstitution of ribosomal peptide backbone thioamidation.

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.

Precursor peptide-targeted mining of more than one hundred thousand genomes expands the lanthipeptide natural product family.

BACKGROUND: Lanthipeptides belong to the ribosomally synthesized and post-translationally modified peptide group of natural products and have a variety of biological activities ranging from antibiotics to antinociceptives. These peptides are cyclized through thioether crosslinks and can bear other secondary post-translational modifications. While lanthipeptide biosynthetic gene clusters can be identified by the presence of genes encoding characteristic enzymes involved in the post-translational modification process, locating the precursor peptides encoded within these clusters is challenging due to their short length and high sequence variability, which limits the high-throughput exploration of lanthipeptide biosynthesis. To address this challenge, we enhanced the predictive capabilities of Rapid ORF Description & Evaluation Online (RODEO) to identify members of all four known classes of lanthipeptides. RESULTS: Using RODEO, we mined over 100,000 bacterial and archaeal genomes in the RefSeq database. We identified nearly 8500 lanthipeptide precursor peptides. These precursor peptides were identified in a broad range of bacterial phyla as well as the Euryarchaeota phylum of archaea. Bacteroidetes were found to encode a large number of these biosynthetic gene clusters, despite making up a relatively small portion of the genomes in this dataset. A number of these precursor peptides are similar to those of previously characterized lanthipeptides, but even more were not, including potential antibiotics. One such new antimicrobial lanthipeptide was purified and characterized. Additionally, examination of the biosynthetic gene clusters revealed that enzymes installing secondary post-translational modifications are more widespread than initially thought. CONCLUSION: Lanthipeptide biosynthetic gene clusters are more widely distributed and the precursor peptides encoded within these clusters are more diverse than previously appreciated, demonstrating that the lanthipeptide sequence-function space remains largely underexplored.

Microviridin 1777: A Toxic Chymotrypsin Inhibitor Discovered by a Metabologenomic Approach.

The toxicity of the cyanobacterium Microcystis aeruginosa EAWAG 127a was evaluated against the sensitive grazer Thamnocephalus platyurus, and the extract possessed strong activity. To investigate the compounds responsible for cytotoxicity, a series of peptides from this cyanobacterium were studied using a combined genomic and molecular networking approach. The results led to the isolation, structure elucidation, and biological evaluation of microviridin 1777, which represents the most potent chymotrypsin inhibitor characterized from this family of peptides to date. Furthermore, the biosynthetic gene clusters of microviridin, anabaenopeptin, aeruginosin, and piricyclamide were located in the producing organism, and six additional natural products were identified by tandem mass spectrometry analyses. These results highlight the potential of modern techniques for the identification of natural products, demonstrate the ecological role of protease inhibitors produced by cyanobacteria, and raise ramifications concerning the presence of novel, yet uncharacterized, toxin families in cyanobacteria beyond microcystin.

Structural insights into enzymatic [4+2] aza-cycloaddition in thiopeptide antibiotic biosynthesis.

The [4+2] cycloaddition reaction is an enabling transformation in modern synthetic organic chemistry, but there are only limited examples of dedicated natural enzymes that can catalyze this transformation. Thiopeptides (or more formally thiazolyl peptides) are a class of thiazole-containing, highly modified, macrocyclic secondary metabolites made from ribosomally synthesized precursor peptides. The characteristic feature of these natural products is a six-membered nitrogenous heterocycle that is assembled via a formal [4+2] cycloaddition between two dehydroalanine (Dha) residues. This heteroannulation is entirely contingent on enzyme activity, although the mechanism of the requisite pyridine/dehydropiperidine synthase remains to be elucidated. The unusual aza-cylic product is distinct from the more common carbocyclic products of synthetic and biosynthetic [4+2] cycloaddition reactions. To elucidate the mechanism of cycloaddition, we have determined atomic resolution structures of the pyridine synthases involved in the biosynthesis of the thiopeptides thiomuracin (TbtD) and GE2270A (PbtD), in complex with substrates and product analogs. Structure-guided biochemical, mutational, computational, and binding studies elucidate active-site features that explain how orthologs can generate rigid macrocyclic scaffolds of different sizes. Notably, the pyridine synthases show structural similarity to the elimination domain of lanthipeptide dehydratases, wherein insertions of secondary structural elements result in the formation of a distinct active site that catalyzes different chemistry. Comparative analysis identifies other catalysts that contain a shared core protein fold but whose active sites are located in entirely different regions, illustrating a principle predicted from efforts in de novo protein design.

Enzymatic Reconstitution and Biosynthetic Investigation of the Lasso Peptide Fusilassin.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified 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 remains poorly characterized due to the 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 branched-cyclic 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 residues governing an important protein-protein interaction, illustrating how genomic 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.

Bioinformatic Mapping of Radical S-Adenosylmethionine-Dependent Ribosomally Synthesized and Post-Translationally Modified Peptides Identifies New Cα, Cß, and Cγ-Linked Thioether-Containing Peptides.

Recently developed bioinformatic tools have bolstered the discovery of ribosomally synthesized and post-translationally modified peptides (RiPPs). Using an improved version of Rapid ORF Description and 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-α carbon) cross-links 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 six cysteines in forty-five residues (SCIFFs) might not be sactipeptides as previously thought. Indeed, the bioinformatically identified SCIFF peptide "freyrasin" was demonstrated to contain six thioethers linking the ß carbons of six aspartate residues. Another SCIFF, thermocellin, was shown to contain a thioether cross-linked to the γ carbon of threonine. SCIFFs feature a different paradigm of non-α carbon thioether linkages, and they are exclusively formed by radical SAM enzymes, as opposed to the polar chemistry employed during lanthipeptide biosynthesis. Therefore, we propose the renaming of the SCIFF family as radical non-α thioether peptides (ranthipeptides) to better distinguish them from the sactipeptide and lanthipeptide RiPP classes.

A new genome-mining tool redefines the lasso peptide biosynthetic landscape.

Ribosomally synthesized and post-translationally modified peptide (RiPP) natural products are attractive for genome-driven discovery and re-engineering, but limitations in bioinformatic methods and exponentially increasing genomic data make large-scale mining of RiPP data difficult. We report RODEO (Rapid ORF Description and Evaluation Online), which combines hidden-Markov-model-based analysis, heuristic scoring, and machine learning to identify biosynthetic gene clusters and predict RiPP precursor peptides. We initially focused on lasso peptides, which display intriguing physicochemical properties and bioactivities, but their hypervariability renders them challenging prospects for automated mining. Our approach yielded the most comprehensive mapping to date of lasso peptide space, revealing >1,300 compounds. We characterized the structures and bioactivities of six lasso peptides, prioritized based on predicted structural novelty, including one with an unprecedented handcuff-like topology and another with a citrulline modification exceptionally rare among bacteria. These combined insights significantly expand the knowledge of lasso peptides and, more broadly, provide a framework for future genome-mining efforts.