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

Abstract

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.

RRE-Finder: a Genome-Mining Tool for Class-Independent RiPP Discovery

“…The generated pHMMs were considered valid if an hmmsearch of the UniProtKB database (30) with a bit score cutoff of 25 gave only hits within BGCs with architectures similar to those of the target class. In addition, characterized data sets of RiPP proteins (e.g., lanthipeptides [31,32], lasso peptides [22,23], and sactipeptides [24]) were used to test auxiliary models using hmmscan analysis. Models giving few or no hits were considered to have acceptably low false-positive rates.…”

“…When available, these data sets were employed to select seed sequences. The data sets included those describing known gene clusters for lanthipeptides (32), lasso peptides (22), thiopeptides (25), cyanobactins (51), bottromycins (52), linear azol(in)e-containing peptides (LAPs, including heterocycloanthracins, plantazolicins, nitrile hydratase-like leader peptides [NHLP]-derived RiPPs, Nif11-derived RiPPs, goadsporins, and cytolysins) (4), pantocins/microcins (53), and radical S-adenosylmethionine-derived RiPPs (including sactipeptides, ranthipeptides, quinohemoprotein amine dehydrogenases, and streptides). In these cases, sequence diversity was evaluated by generating a sequence similarity network (SSN) using the Enzyme Function Initiative Enzyme Similarity Tool (EFI-EST) (26) and visualizing the SSN with Cytoscape (27).…”

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“…Comparison with RODEO. All positively scored precursor peptides were extracted from genome mining studies done with RODEO [26,27,[29][30][31]. For lasso peptides, only the most recent dataset was used [29].…”

“…Typically, the classifier was still capable of predicting the class that was left out, with an area-under-receiver operating characteristics curve of 0.955. To validate the SVM, we used it to classify precursor hits from the various RiPP mining studies performed using RODEO [26][27][28][29][30][31]. In general, Fig 1. decRiPPter pipeline for the detection of novel RiPP families.…”

“…While all the genes, including the precursors, were well conserved between the 2 gene clusters, the ltnJ gene had been replaced by an sprOR homolog, suggesting that their gene products catalyze similar functions (S17 Fig). A recent paper describes the product of a BGC that contains a gene that similarly encodes a luciferase-like monooxygenase and shows that serine residues are indeed converted to alanine residues [30], further suggesting that this enzyme is responsible for this modification.…”