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The prevalence of antimicrobial resistance in Gram-negative bacteria poses a greater challenge due to their intrinsic resistance to many antibiotics. Recently, darobactins have emerged as a novel class of antibiotics originating from previously unexplored Gram-negative bacterial species such as Photorhabdus, Vibrio, Pseudoalteromonas and Yersinia. Darobactins belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) class of antibiotics, exhibiting selective activity against Gram-negative bacteria. They target the ß-barrel assembly machinery (BAM), which is crucial for the maturation and insertion of outer membrane proteins in Gram-negative bacteria. The dar operon in the producer's genome encodes for the synthesis of darobactins, which are characterized by a fused ring system connected via an alkyl-aryl ether linkage (C-O-C) and a C-C cross-link. The enzyme DarE, using the radical S-adenosyl-l-methionine (rSAM), facilitates the formation of these bonds. Biosynthetic manipulation of the darobactin gene cluster, along with its expression in a surrogate host, has enabled access to diverse darobactin analogues with variable antibiotic activities. Recently, two independent research groups successfully achieved the total synthesis of darobactin, employing Larock heteroannulation to construct the bicyclic structure. This paper presents a comprehensive review of darobactins, encompassing their discovery through to the most recent advancements.

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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a chemically diverse class of metabolites. Many RiPPs show potent biological activities that make them attractive starting points for drug development. A promising approach for the discovery of new classes of RiPPs is genome mining. However, the accuracy of genome mining is hampered by the lack of signature genes shared across different RiPP classes. One way to reduce false-positive predictions is by complementing genomic information with metabolomics data. In recent years, several new approaches addressing such integrative genomics and metabolomics analyses have been developed. In this review, we provide a detailed discussion of RiPP-compatible software tools that integrate paired genomics and metabolomics data. We highlight current challenges in data integration and identify opportunities for further developments targeting new classes of bioactive RiPPs.

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Here we provide a review on TldD/TldE family proteins, summarizing current knowledge and outlining further research perspectives. Despite being widely distributed in bacteria and archaea, TldD/TldE proteins have been escaping attention for a long time until several recent reports pointed to their unique features. Specifically, TldD/TldE generally act as peptidases, though some of them turned out to be N-deacetylases. Biological function of TldD/TldE has been extensively described in bacterial specialized metabolism, in which they participate in the biosynthesis of lincosamide antibiotics (as N-deacetylases), and in the biosynthesis of ribosomally synthesized and post-translationally modified bioactive peptides (as peptidases). These enzymes possess special position in the relevant biosynthesis since they convert non-bioactive intermediates into bioactive metabolites. Further, based on a recent study of Escherichia coli TldD/TldE, these heterodimeric metallopeptidases possess a new protein fold exhibiting several structural features with no precedent in the Protein Data Bank. The most interesting ones are structural elements forming metal-containing active site on the inner surface of the catalytically active subunit TldD, in which substrates bind through ß sheet interactions in the sequence-independent manner. It results in relaxed substrate specificity of TldD/TldE, which is counterbalanced by enclosing the active centre within the hollow core of the heterodimer and only appropriate substrates can entry through a narrow channel. Based on the published data, we hypothesize a yet unrecognized central metabolic function of TldD/TldE in the degradation of (partially) unfolded proteins, i.e., in protein quality control.

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In silico genome mining provides easy access to secondary metabolite biosynthetic gene clusters (BGCs) encoding the biosynthesis of many bioactive compounds, which are the basis for many important drugs used in human medicine. However, the association between BGCs and other functions encoded in the genomes of producers have remained elusive. Here, we present a systems biology workflow that integrates genome mining with a detailed pangenome analysis for detecting genes associated with a particular BGC. We analyzed 3,889 enterobacterial genomes and found 13,266 BGCs, represented by 252 distinct BGC families and 347 additional singletons. A pangenome analysis revealed 88 genes putatively associated with a specific BGC coding for the colon cancer-related colibactin that code for diverse metabolic and regulatory functions. The presented workflow opens up the possibility to discover novel secondary metabolites, better understand their physiological roles, and provides a guide to identify and analyze BGC associated gene sets.

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In recent years, a variety of fungal cyclic peptides with interesting bioactivities have been discovered. For many of these peptides, the biosynthetic pathways are unknown and their elucidation often holds surprises. The cyclic and backbone N-methylated omphalotins from Omphalotus olearius were recently shown to constitute a novel class (borosins) of ribosomally synthesized and posttranslationally modified peptides, members of which are produced by many fungi, including species of the genus Rhizopogon. Other recently discovered fungal peptide macrocycles include the mariannamides from Mariannaea elegans and the backbone N-methylated verrucamides and broomeanamides from Myrothecium verrucaria and Sphaerostilbella broomeana, respectively. Here, we present draft genome sequences of four fungal species Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria, and Sphaerostilbella broomeana. We screened these genomes for precursor proteins or gene clusters involved in the mariannamide, verrucamide, and broomeanamide biosynthesis including a general screen for borosin-producing precursor proteins. While our genomic screen for potential ribosomally synthesized and posttranslationally modified peptide precursor proteins of mariannamides, verrucamides, broomeanamides, and borosins remained unsuccessful, antiSMASH predicted nonribosomal peptide synthase gene clusters that may be responsible for the biosynthesis of mariannamides, verrucamides, and broomeanamides. In M. verrucaria, our antiSMASH search led to a putative NRPS gene cluster with a predicted peptide product of 20 amino acids, including multiple nonproteinogenic isovalines. This cluster likely encodes a member of the peptaibols, an antimicrobial class of peptides previously isolated primarily from the Genus Trichoderma. The nonribosomal peptide synthase gene clusters discovered in our screenings are promising candidates for future research.

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Ustiloxin B is a ribosomally synthesized and post-translationally modified peptide (RiPP) first reported in Ascomycetes. Its biosynthetic pathway was recently identified in the filamentous fungus Aspergillus flavus. The precursor protein of ustiloxin B, UstA, has a signal peptide to the endoplasmic reticulum at its N-terminal and a subsequent tandemly highly repeated segment cleaved at Lys-Arg dipeptides by Kex2 protease; such proteins are called Kex2-processed repeat proteins (KEPs). RiPP biosynthetic pathways using KEPs as precursor proteins are widely distributed in the Fungi kingdom, with high diversity of precursor protein sequences. UstA in A. flavus has a 16-fold tandemly repeated segment containing the core peptide Tyr-Ala-Ile-Gly, which forms the ustiloxin B backbone structure, but it is unknown why such a costly-to-maintain highly repeated sequence is retained. Here, we replaced ustA, the gene encoding the ustiloxin B precursor protein, with synthetic genes encoding 1-, 3-, 5-, 7-, and 11-fold tandem-repeat segments in A. flavus, to investigate the relationship between the repeat number and ustiloxin B production. Ustiloxin B production increased quadratically with increasing repeat number in ustA variants, although it dropped in a previously constructed ustA variant that had a substituted synthetic gene encoding a 16-fold repeat segment probably because of the presence of the many rare codons in the sequence. We also examined the transcript levels of substituted synthetic genes in ustA variants, and surprisingly we found that the transcript levels of the synthetic genes increased linearly with increasing repeat number. This result implies that an unknown mechanism stabilizes ustA transcripts via the highly repeated structure in a feedback manner. We also constructed a transformant without the intron in native ustA, but no effect of intron removal was observed on either ustiloxin B production or the precursor gene transcript level. The costly-to-maintain highly repeated sequence in KEPs probably serves the purpose of maintaining stable transcripts and thus increasing the amount of substrate.

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Peptide-derived natural products are a large class of bioactive molecules that often contain chemically challenging modifications. In the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs), radical-SAM (rSAM) enzymes have been shown to catalyze the formation of ether, thioether, and carbon-carbon bonds on the precursor peptide. The installation of these bonds typically establishes the skeleton of the mature RiPP. To facilitate the search for unexplored rSAM-dependent RiPPs for the community, we employed a bioinformatic strategy to screen a subfamily of peptide-modifying rSAM enzymes which are known to bind up to three [4Fe-4S] clusters. A sequence similarity network was used to partition related families of rSAM enzymes into >250 clusters. Using representative sequences, genome neighborhood diagrams were generated using the Genome Neighborhood Tool. Manual inspection of bacterial genomes yielded numerous putative rSAM-dependent RiPP pathways with unique features. From this analysis, we identified and experimentally characterized the rSAM enzyme, TvgB, from the tvg gene cluster from Halomonas anticariensis. In the tvg gene cluster, the precursor peptide, TvgA, is comprised of a repeating TVGG motif. Structural characterization of the TvgB product revealed the repeated formation of cyclopropylglycine, where a new bond is formed between the γ-carbons on the precursor valine. This novel RiPP modification broadens the functional potential of rSAM enzymes and validates the proposed bioinformatic approach as a practical broad search tool for the discovery of new RiPP topologies.

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Complex peptide natural products exhibit diverse biological functions and a wide range of physico-chemical properties. As a result, many peptides have entered the clinics for various applications. Two main routes for the biosynthesis of complex peptides have evolved in nature: ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic pathways and non-ribosomal peptide synthetases (NRPSs). Insights into both bioorthogonal peptide biosynthetic strategies led to the establishment of universal principles for each of the two routes. These universal rules can be leveraged for the targeted identification of novel peptide biosynthetic blueprints in genome sequences and used for the rational engineering of biosynthetic pathways to produce non-natural peptides. In this review, we contrast the key principles of both biosynthetic routes and compare the different biochemical strategies to install the most frequently encountered peptide modifications. In addition, the influence of the fundamentally different biosynthetic principles on past, current and future engineering approaches is illustrated. Despite the different biosynthetic principles of both peptide biosynthetic routes, the arsenal of characterized peptide modifications encountered in RiPP and NRPS systems is largely overlapping. The continuous expansion of the biocatalytic toolbox of peptide modifying enzymes for both routes paves the way towards the production of complex tailor-made peptides and opens up the possibility to produce NRPS-derived peptides using the ribosomal route and vice versa.

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The increasing length and complexity of peptide drug candidates foster the development of novel strategies for their manufacture, which should include sustainable and efficient technologies. In this context, including enzymatic catalysis in the production of peptide molecules has gained interest. Here, several enzymes from ribosomally synthesized and post-translationally modified peptides biosynthesis pathways are reviewed, with attention to their capacity to introduce stability-promoting structural features on peptides, providing an initial framework towards their use in therapeutic peptide production processes.

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Peptidyl compounds produced by filamentous fungi, which are nonribosomal peptides (NRPs) and ribosomally synthesized and post-translationally modified peptides (RiPPs), are rich sources of bioactive compounds with a wide variety of structures. Some of these peptidyl compounds are useful as pharmaceuticals and pesticides. However, for industrial use, their low production often becomes an obstacle, and various approaches have been challenged to overcome this weakness. In this article, we summarize the successful attempts to increase the production of NRPs and RiPPs in filamentous fungi and present our perspectives on how to improve it further.

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Microviridins, tricyclic peptide natural products originally isolated from cyanobacteria, function as inhibitors of diverse serine-type proteases. Here we report the structure and biochemical characterization of AMdnB, a unique iterative macrocyclase involved in a microviridin biosynthetic pathway from Anabaena sp. PCC 7120. The ATP-dependent cyclase, along with the homologous AMdnC, introduce up to nine macrocyclizations on three distinct core regions of a precursor peptide, AMdnA. The results presented here provide structural and mechanistic insight into the iterative chemistry of AMdnB. In vitro AMdnB-catalyzed cyclization reactions demonstrate the synthesis of the two predicted tricyclic products from a multi-core precursor peptide substrate, consistent with a distributive mode of catalysis. The X-ray structure of AMdnB shows a structural motif common to ATP-grasp cyclases involved in RiPPs biosynthesis. Additionally, comparison with the noniterative MdnB allows insight into the structural basis for the iterative chemistry. Overall, the presented results provide insight into the general mechanism of iterative enzymes in ribosomally synthesized and post-translationally modified peptide biosynthetic pathways.

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Methanobactins (MBs) are ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The posttranslational modification that defines MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b is mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons and, in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the ΔmbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry, and solution nuclear magnetic resonance (NMR) spectroscopy. MB-OB3b from the ΔmbnC mutant was missing the C-terminal Met and was also found to contain a Pro and a Cys in place of the pyrrolidinyl-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals and nanoparticle formation, as well as the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for posttranslational modification(s) of the two oxazolone groups are not identical.

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BACKGROUND: Soil bacteria are a major source of specialized metabolites including antimicrobial compounds. Yet, one of the most diverse genera of bacteria ubiquitously present in soil, Clostridium, has been largely overlooked in bioactive compound discovery. As Clostridium spp. thrive in extreme environments with their metabolic mechanisms adapted to the harsh conditions, they are likely to synthesize molecules with unknown structures, properties, and functions. Therefore, their potential to synthesize small molecules with biological activities should be of great interest in the search for novel antimicrobial compounds. The current study focused on investigating the antimicrobial potential of four soil Clostridium isolates, FS01, FS2.2 FS03, and FS04, using a genome-led approach, validated by culture-based methods. RESULTS: Conditioned/spent media from all four Clostridium isolates showed varying levels of antimicrobial activity against indicator microorganism; all four isolates significantly inhibited the growth of Pseudomonas aeruginosa. FS01, FS2.2, and FS04 were active against Bacillus mycoides and FS03 reduced the growth of Bacillus cereus. Phylogenetic analysis together with DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and functional genome distribution (FGD) analyses confirmed that FS01, FS2.2, and FS04 belong to the species Paraclostridium bifermentans, Clostridium cadaveris, and Clostridium senegalense respectively, while FS03 may represent a novel species of the genus Clostridium. Bioinformatics analysis using antiSMASH 5.0 predicted the presence of eight biosynthetic gene clusters (BGCs) encoding for the synthesis of ribosomally synthesized post-translationally modified peptides (RiPPs) and non-ribosomal peptides (NRPs) in four genomes. All predicted BGCs showed no similarity with any known BGCs suggesting novelty of the molecules from those predicted gene clusters. In addition, the analysis of genomes for putative virulence factors revealed the presence of four putative Clostridium toxin related genes in FS01 and FS2.2 genomes. No genes associated with the main Clostridium toxins were identified in the FS03 and FS04 genomes. CONCLUSIONS: The presence of BGCs encoding for uncharacterized RiPPs and NRPSs in the genomes of antagonistic Clostridium spp. isolated from farm soil indicated their potential to produce novel secondary metabolites. This study serves as a basis for the identification and characterization of potent antimicrobials from these soil Clostridium spp. and expands the current knowledge base, encouraging future research into bioactive compound production in members of the genus Clostridium.

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Prenyltransferase (PTase) enzymes play crucial roles in natural product biosynthesis by transferring isoprene unit(s) to target substrates, thereby generating prenylated compounds. The prenylation step leads to a diverse group of natural products with improved membrane affinity and enhanced bioactivity, as compared to the non-prenylated forms. The last two decades have witnessed increasing studies on the identification, characterization, enzyme engineering, and synthetic biology of microbial PTase family enzymes. We herein summarize several examples of microbial soluble aromatic PTases for chemoenzymatic syntheses of unnatural novel prenylated compounds.

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Mycofactocin (MFT) is a recently discovered glycosylated redox cofactor, which has been associated with the detoxification of antibiotics in pathogenic mycobacteria, and, therefore, of potential medical interest. The MFT biosynthetic gene cluster is commonly found in mycobacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Since the MFT molecule is highly interesting for basic research and could even serve as a potential drug target, large-scale production of the molecule is highly desired. However, conventional shake flask cultivations failed to produce enough MFT for further biochemical characterization like kinetic studies and structure elucidation, and a more comprehensive study of cultivation parameters is urgently needed. Being a redox cofactor, it can be hypothesized that the oxygen transfer rate (OTR) is a critical parameter for MFT formation. Using the non-pathogenic strain Mycobacterium smegmatis mc2 155, shake flask experiments with online measurement of the oxygen uptake and the carbon dioxide formation, were conducted under different levels of oxygen supply. Using liquid chromatography and high-resolution mass spectrometry, a 4-8 times increase of MFT production was identified under oxygen-limited conditions, in both complex and mineral medium. Moreover, the level of oxygen supply modulates not only the overall MFT formation but also the length of the glycosidic chain. Finally, all results were scaled up into a 7 L stirred tank reactor to elucidate the kinetics of MFT formation. Ultimately, this study enables the production of high amounts of these redox cofactors, to perform further investigations into the role and importance of MFTs.

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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of cyclic or linear peptidic natural products with remarkable structural and functional diversity. Recent advances in genomics and synthetic biology, are facilitating us to discover a large number of new ribosomal natural products, including lanthipeptides, lasso peptides, sactipeptides, thiopeptides, microviridins, cyanobactins, linear thiazole/oxazole-containing peptides and so on. In this review, we summarize bioinformatic strategies that have been developed to identify and prioritize biosynthetic gene clusters (BGCs) encoding RiPPs, and the genome mining-guided discovery of novel RiPPs. We also prospectively provide a vision of what genomics-guided discovery of RiPPs may look like in the future, especially the discovery of RiPPs from dominant but uncultivated microbes, which will be promoted by the combinational use of synthetic biology and metagenome mining strategies.

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Ustiloxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) first reported in Ascomycetes. Originally identified as metabolites of the rice pathogenic fungus Ustilaginoidea virens, they were recently identified among the metabolites of the mold Aspergillus flavus, along with their corresponding biosynthetic gene cluster. Ustilaginoidea virens produces ustiloxins A and B, whereas A. flavus produces only ustiloxin B. Correspondingly, in U. virens, the ustiloxin precursor peptide, from which the compound backbone is cleaved and cyclized, contains the core peptides Tyr(Y)-Val(V)-Ile(I)-Gly(G) and Tyr(Y)-Ala(A)-Ile(I)-Gly(G) for ustiloxins A and B, respectively, whereas that of A. flavus contains only the YAIG motif for ustiloxin B. In this study, the gene that encodes the precursor peptide in A. flavus, ustA, was replaced with synthetic genes encoding the core peptides YVIG or FAIG, to investigate their compatibility with the ustiloxin biosynthetic machinery. We also examined the importance of the hydroxyl group on the aromatic ring of Tyr for cyclization of the YAIG core peptide. Against our expectation, the ustA variant possessing YVIG core peptides did not produce a detectable amount of ustiloxin A, even though the ustiloxin biosynthetic gene clusters of A. flavus and U. virens both contain 13 homologous genes. We confirmed that the lack of ustiloxin A production was not due to lack or insufficient expression of the substituted synthetic gene. This result, along with the differences between the primary sequences of UstYa and UstYb in A. flavus and U. virens, suggests that the ustiloxin biosynthetic machinery is optimized for the native core peptide sequences. The synthetic FAIG-encoding ustA did not yield any compounds specific to the FAIG core peptide, suggesting that the hydroxyl group on the aromatic ring of Tyr in the core peptide is indispensable for cyclization of the core peptide, even though it is not structurally involved in the cyclization.

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BACKGROUND: Recently, a gene cluster responsible for biosynthesis of ustiloxin in Aspergillus flavus was identified as the first case of a ribosomally synthesized and post-translationally modified peptide (RiPP) synthetic pathway in Ascomycota. RiPPs are biosynthesized from precursor peptides, which are processed to produce the RiPP backbone (core peptides) for further modifications such as methylation and cyclization. Ustiloxin precursor peptide has two distinctive features: a signal peptide for translocation into the endoplasmic reticulum and highly repeated core sequences cleaved by Kex2 protease in the Golgi apparatus. On the basis of these characteristics, the ustiloxin-type RiPP precursor peptides or Kex2-processed repeat proteins (KEPs) in strains belonging to the Fungi kingdom were computationally surveyed, in order to investigate the distribution and putative functions of KEPs in fungal ecology. RESULTS: In total, 7878 KEPs were detected in 1345 of 1461 strains belonging to 8 phyla. The average number of KEPs per strain was 5.25 in Ascomycota and 5.30 in Basidiomycota, but only 1.35 in the class Saccharomycetes (Ascomycota) and 1.00 in the class Tremellomycetes (Basidiomycota). The KEPs were classified into 838 types and 2560 stand-alone ones, which had no homologs. Nearly 200 types were distributed in more than one genus, and 14 types in more than one phylum. These types included yeast α-mating factors and fungal pheromones. Genes for 22% KEPs were accompanied by genes for DUF3328-domain-containing proteins, which are indispensable for cyclization of the core peptides. DUF3328-domain-containing protein genes were located at an average distance of 3.09 genes from KEP genes. Genes for almost all (with three exceptions) KEPs annotated as yeast α-mating factors or fungal pheromones were not accompanied by DUF3328-domain-containing protein genes. CONCLUSION: KEPs are widely distributed in the Fungi kingdom, but their repeated sequences are highly diverse. From these results and some examples, a hypothesis was raised that KEPs initially evolved as unmodified linear peptides (e.g., mating factors), and then those that adopted a modified cyclic form emerged (e.g., toxins) to utilize their strong bioactivity against predators and competitive microorganisms.

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Ranthipeptides, defined as radical non-α thioether-containing peptides, are a newly emerging class of natural products belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. Ranthipeptides are shown to be widespread in the bacterial kingdom, whereas heretofore their biological functions remain completely elusive. In this work, putative ranthipeptides are investigated from two solventogenic clostridia, Clostridium beijerinckii and Clostridium ljungdahlii, which are derived from the so-called six Cys in forty-five residues (SCIFF) family of precursor peptides. A series of analysis show that these two ranthipeptides participate in quorum sensing and controlling cellular metabolism. These results highlight the diverse biological functions of the ever-increasing family of RiPP natural products and showcase the potential to engineer industrially interesting organisms by manipulating their RiPP biosynthetic pathways.

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Microbes are talented chemists with the ability to generate tremendously complex and diverse natural products which harbor potent biological activities. Natural products are produced using sets of specialized biosynthetic enzymes encoded by secondary metabolism pathways. Here, we present a two-step evolutionary model to explain the diversification of biosynthetic pathways that account for the proliferation of these molecules. We argue that the appearance of natural product families has been a slow and infrequent process. The first step led to the original emergence of bioactive molecules and different classes of natural products. However, much of the chemical diversity observed today has resulted from the endless modification of the ancestral biosynthetic pathways. The second step rapidly modulates the pre-existing biological activities to increase their potency and to adapt to changing environmental conditions. We highlight the importance of enzyme promiscuity in this process, as it facilitates both the incorporation of horizontally transferred genes into secondary metabolic pathways and the functional differentiation of proteins to catalyze novel chemistry. We provide examples where single point mutations or recombination events have been sufficient for new enzymatic activities to emerge. A unique feature in the evolution of microbial secondary metabolism is that gene duplication is not essential but offers opportunities to synthesize more complex metabolites. Microbial natural products are highly important for the pharmaceutical industry due to their unique bioactivities. Therefore, understanding the natural mechanisms leading to the formation of diverse metabolic pathways is vital for future attempts to utilize synthetic biology for the generation of novel molecules.

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