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Natural product discovery is currently undergoing a transformation from a phenotype-driven field to a genotype-driven one. The increasing availability of genome sequences, coupled with improved techniques for identifying biosynthetic gene clusters, has revealed that secondary metabolomes are strikingly vaster than previously thought. New approaches to correlate biosynthetic gene clusters with the compounds they produce have facilitated the production and isolation of a rapidly growing collection of what we refer to as 'reverse-discovered' natural products, in analogy to reverse genetics. In this review, we present an extensive list of reverse-discovered natural products and discuss seven important lessons for natural product discovery by genome-guided methods: structure prediction, accurate annotation, continued study of model organisms, avoiding genome-size bias, genetic manipulation, heterologous expression, and potential engineering of natural product analogs. [ABSTRACT FROM AUTHOR]

Background: Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a burgeoning class of natural products with diverse activity that share a similar origin and common features in their biosynthetic pathways. The precursor peptides of these natural products are ribosomally produced, upon which a combination of modification enzymes installs diverse functional groups. This genetically encoded peptide-based strategy allows for rapid diversification of these natural products by mutation in the precursor genes merged with unique combinations of modification enzymes. Thiazole/oxazole-modified microcins (TOMMs) are a class of RiPPs defined by the presence of heterocycles derived from cysteine, serine, and threonine residues in the precursor peptide. TOMMs encompass a number of different families, including but not limited to the linear azol(in)e-containing peptides (streptolysin S, microcin B17, and plantazolicin), cyanobactins, thiopeptides, and bottromycins. Although many TOMMs have been explored, the increased availability of genome sequences has illuminated several unexplored TOMM producers. Methods: All YcaO domain-containing proteins (D protein) and the surrounding genomic regions were were obtained from the European Molecular Biology Laboratory (EMBL) and the European Bioinformatics Institute (EBI). MultiGeneBlast was used to group gene clusters contain a D protein. A number of techniques were used to identify TOMM biosynthetic gene clusters from the D protein containing gene clusters. Precursor peptides from these gene clusters were also identified. Both sequence similarity and phylogenetic analysis were used to classify the 20 diverse TOMM clusters identified. Results: Given the remarkable structural and functional diversity displayed by known TOMMs, a comprehensive bioinformatic study to catalog and classify the entire RiPP class was undertaken. Here we report the bioinformatic characterization of nearly 1,500 TOMM gene clusters from genomes in the European Molecular Biology Laboratory (EMBL) and the European Bioinformatics Institute (EBI) sequence repository. Genome mining suggests a complex diversification of modification enzymes and precursor peptides to create more than 20 distinct families of TOMMs, nine of which have not heretofore been described. Many of the identified TOMM families have an abundance of diverse precursor peptide sequences as well as unfamiliar combinations of modification enzymes, signifying a potential wealth of novel natural products on known and unknown biosynthetic scaffolds. Phylogenetic analysis suggests a widespread distribution of TOMMs across multiple phyla; however, producers of similar TOMMs are generally found in the same phylum with few exceptions. Conclusions: The comprehensive genome mining study described herein has uncovered a myriad of unique TOMM biosynthetic clusters and provides an atlas to guide future discovery efforts. These biosynthetic gene clusters are predicted to produce diverse final products, and the identification of additional combinations of modification enzymes could expand the potential of combinatorial natural product biosynthesis. [ABSTRACT FROM AUTHOR]

With billions of years of evolution under its belt, Nature has been expanding and optimizing its biosynthetic capabilities. Chemically complex secondary metabolites continue to challenge and inspire today''s most talented synthetic chemists. A brief glance at these natural products, especially the substantial structural variation within a class of compounds, clearly demonstrates that Nature has long played the role of medicinal chemist. The recent explosion in genome sequencing has expanded our appreciation of natural product space and the vastness of uncharted territory that remains. One small corner of natural product chemical space is occupied by the recently dubbed thiazole/oxazole-modified microcins (TOMMs), which are ribosomally produced peptides with posttranslationally installed heterocycles derived from cysteine, serine and threonine residues. As with other classes of natural products, the genetic capacity to synthesize TOMMs has been widely disseminated among bacteria. Over the evolutionary timescale, Nature has tested countless random mutations and selected for gain of function in TOMM biosynthetic gene clusters, yielding several privileged molecular scaffolds. Today, this burgeoning class of natural products encompasses a structurally and functionally diverse set of molecules (i.e. microcin B17, cyanobactins, and thiopeptides). TOMMs presumably provide their producers with an ecological advantage. This advantage can include chemical weapons wielded in the battle for nutrients, disease-promoting virulence factors, or compounds presumably beneficial for symbiosis. Despite this plethora of functions, many TOMMs await experimental interrogation. This review will focus on the biosynthesis and natural combinatorial diversity of the TOMM family. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The hygrolides, a family of 16-member-ring-containing plecomacrolides produced by Actinobacteria, exhibit numerous reported bioactivities. Using HR-MS/MS, nucleophilic 1,4-addition-based labeling, NMR, and bioinformatic analysis, we identified Streptomyces varsoviensis as a novel producer of JBIR-100, a fumarate-containing hygrolide, and elucidated the previously unknown stereochemistry of the natural product. We investigated the antimicrobial activity of JBIR-100, with preliminary insight into mode of action indicating that it perturbs the membrane of Bacillus subtilis . S. varsoviensis is known to produce compounds from multiple hygrolide sub-families, namely hygrobafilomycins (JBIR-100 and hygrobafilomycin) and bafilomycins (bafilomycin C 1 and D). In light of this, we identified the biosynthetic gene cluster for JBIR-100, which, to our knowledge, represents the first reported for a hygrobafilomycin. Finally, we performed a bioinformatic analysis of the hygrolide family, describing clusters from known and predicted producers. Our results indicate that potential remains for the Actinobacteria to yield novel hygrolide congeners, perhaps with differing biological activities. [ABSTRACT FROM AUTHOR]

Natural products (NPs) serve important roles as drug candidates and as tools for chemical biology. However, traditional NP discovery, largely based on bioassay-guided approaches, is biased toward abundant compounds and rediscovery rates are high. Orthogonal methods to facilitate discovery of new NPs are thus needed, and herein we describe an isotope tag-based expansion of reactivity-based NP screening to address these shortcomings. Reactivity-based screening is a directed discovery approach in which a specific reactive handle on the NP is targeted by a chemoselective probe to enable its detection by mass spectrometry. In this study, we have developed an aminooxy-containing probe to guide the discovery of aldehyde- and ketone-containing NPs. To facilitate the detection of labeling events, the probe was dibrominated, imparting a unique isotopic signature to distinguish labeled metabolites from spectral noise. As a proof of concept, the probe was then utilized to screen a collection of bacterial extracts, leading to the identification of a new analogue of antipain, deimino-antipain. The bacterial producer of deimino-antipain was sequenced and the responsible biosynthetic gene cluster was identified by bioinformatic analysis and heterologous expression. These data reveal the previously undetermined genetic basis for a well-known family of aldehyde-containing, peptidic protease inhibitors, including antipain, chymostatin, leupeptin, elastatinal, and microbial alkaline protease inhibitor, which have been widely used for over 40 years. [ABSTRACT FROM AUTHOR]

Thiazole/oxazole-modified microcins (TOMMs) are a class of post-translationally modified peptide natural products bearing azole and azoline heterocycles. The first step in heterocycle formation is carried out by a two component cyclodehydratase comprised of an El ubiquitin- activating and a YcaO superfamily member. Recent studies have demonstrated that the YcaO domain is responsible for cyclodehydration, while the TOMM El homologue is responsible for peptide recognition during azoline formation. Although all characterized TOMM biosynthetic clusters contain this canonical TOMM El homologue (C domain), we also identified a second, highly divergent El superfamily member, annotated as an Ocin-ThiF-like protein (F protein), associated with more than 300 TOMM biosynthetic clusters. Here we describe the in vitro reconstitution of a novel TOMM cyclodehydratase from such a cluster and demonstrate that this auxiliary protein is required for cyclodehydration. Using a combination of biophysical techniques, we demonstrate that the F protein, rather than the C domain, is responsible for engaging the peptide substrate. The C domain instead appears to serve as a scaffolding protein, bringing the catalytic YcaO domain and the peptide binding Ocin-ThiF-like protein into proximity. Our findings provide an updated biosynthetic framework that provides a foundation for the characterization and reconstitution of approximately 25% of bioinformatically identifiable TOMM synthetases. [ABSTRACT FROM AUTHOR]

Summary Natural products are the most historically significant source of compounds for drug development. However, unacceptably high rates of compound rediscovery associated with large-scale screening of common microbial producers have resulted in the abandonment of many natural product drug discovery efforts, despite the increasing prevalence of clinically problematic antibiotic resistance. Screening of underexplored taxa represents one strategy to avoid rediscovery. Herein we report the discovery, isolation, and structural elucidation of streptomonomicin (STM), an antibiotic lasso peptide from Streptomonospora alba, and report the genome for its producing organism. STM-resistant clones of Bacillus anthracis harbor mutations to walR , the gene encoding a response regulator for the only known widely distributed and essential two-component signal transduction system in Firmicutes. To the best of our knowledge, Streptomonospora had been hitherto biosynthetically and genetically uncharacterized, with STM being the first reported compound from the genus. Our results demonstrate that understudied microbes remain fruitful reservoirs for the rapid discovery of novel, bioactive natural products. [ABSTRACT FROM AUTHOR]