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Peptide-based antibiotics (PBAs), including antimicrobial peptides (AMPs) and their synthetic mimics, have received significant interest due to their diverse and unique bioactivities. The integration of high-throughput sequencing and bioinformatics tools has dramatically enhanced the discovery of enzymes, allowing researchers to identify specific genes and metabolic pathways responsible for producing novel PBAs more precisely. Cell-free systems (CFSs) that allow precise control over transcription and translation in vitro are being adapted, which accelerate the identification, characterization, selection, and production of novel PBAs. Furthermore, these platforms offer an ideal solution for overcoming the limitations of small-molecule antibiotics, which often lack efficacy against a broad spectrum of pathogens and contribute to the development of antibiotic resistance. In this review, we highlight recent examples of how CFSs streamline these processes while expanding our ability to access new antimicrobial agents that are effective against antibiotic-resistant infections.

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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 broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase, and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, demonstrate complex formation between TclI, TclJ, and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core. IMPORTANCE: Thiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin.

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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, we demonstrate complex formation between TclI, TclJ and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core.

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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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Pyritides belong to the ribosomally synthesized and post-translationally modified peptide class of natural products that were recently genome-predicted and are structurally defined by unique pyridine-containing macrocycles. Inspired by their biosynthesis, proceeding through peptide modification and cycloaddition to form the heterocyclic core, we report the chemical synthesis of pyritide A2 involving pyridine ring synthesis from an amino acid precursor through aza-Diels-Alder reaction. This strategy permitted the preparation of the decorated pyridine core with an appended amino acid residue in two steps from a commercially available arginine derivative and secured pyritide A2 in ten steps. Moreover, the synthetic logic enables efficient preparation of different pyridine subunits associated with pyritides, allowing rapid and convergent access to this new class of natural products and analogues thereof.

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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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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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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products that benefited from genome sequencing technology in the past two decades. RiPPs are widely distributed in nature and show diverse chemical structures and rich biological activities. Despite the various structural characteristic of RiPPs, they follow a common biosynthetic logic: a precursor peptide containing an N-terminal leader peptide and a C-terminal core peptide; in some cases,a follower peptide is after the core peptide. The precursor peptide undergoes a series of modification, transport, and cleavage steps to form a mature natural product with specific activities. Sactipeptides (Sulfur-to-alpha carbon thioether cross-linked peptides) belong to RiPPs that show various biological activities such as antibacterial, spermicidal and hemolytic properties. Their common hallmark is an intramolecular thioether bond that crosslinks the sulfur atom of a cysteine residue to the α-carbon of an acceptor amino acid, which is catalyzed by a rSAM enzyme. This review summarizes recent achievements concerning the discovery, distribution, structural elucidation, biosynthesis and application prospects of sactipeptides.

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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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Lasso peptides belong to the natural product superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). They are defined by an N-terminal macrolactam ring that is threaded by the C-terminal tail. In class II lasso peptides, this fold is maintained only through steric hindrance. Nonetheless, this fold can often withstand prolonged incubation at highly elevated temperatures. However, some lasso peptides will irreversibly unthread into their branched-cyclic counterparts upon heating. In recent years, an increasing number of research studies have focused on studying the factors that govern the thermal stability (or the lack thereof) of lasso peptides by using in vitro stability assays, mutational analysis, and molecular dynamics simulations. In this review, the current state of understanding the physicochemical parameters deciding the fate of a lasso peptide at elevated temperatures is discussed, and an overview is given of the techniques developed to streamline the separation and discrimination of lasso peptides from their branched-cyclic topoisomers.

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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an important class of natural products that contain antibiotics and a variety of other bioactive compounds. The existing methods for discovery of RiPPs by combining genome mining and computational mass spectrometry are limited to discovering specific classes of RiPPs from small datasets, and these methods fail to handle unknown post-translational modifications. Here, we present MetaMiner, a software tool for addressing these challenges that is compatible with large-scale screening platforms for natural product discovery. After searching millions of spectra in the Global Natural Products Social (GNPS) molecular networking infrastructure against just eight genomic and metagenomic datasets, MetaMiner discovered 31 known and seven unknown RiPPs from diverse microbial communities, including human microbiome and lichen microbiome, and microorganisms isolated from the International Space Station.

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Natural products have played indispensable roles in drug development and biomedical research. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of fast-expanding natural products attribute to genome mining efforts in recent years. Most RiPP natural products were discovered from bacteria, yet many eukaryotic cyclic peptides turned out to be of RiPP origin. This review article presents recent advances in the discovery of eukaryotic RiPP natural products, the elucidation of their biosynthetic pathways, and the molecular basis for their biosynthetic enzyme catalysis.

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Lasso peptides are ribosomally synthesized and post-translationally modified natural products with a characteristic slipknot-like structure, which confers these peptides remarkable stability and diverse pharmacologically relevant bioactivities. Among all the reported lasso peptides, lassomycin and lariatins are unique lasso peptides that exhibit noticeable anti-tuberculosis (TB) activity. Due to the unique threaded structure and the unusual bactericidal mechanism toward Mycobacterium tuberculosis, these peptides have drawn considerable interest, not only in the field of total synthesis but also in several other fields including biosynthesis, bioengineering, and structure-activity studies. During the past few years, significant progress has been made in understanding the biosynthetic mechanism of these intriguing compounds, which has provided a solid foundation for future work. This review highlights recent achievements in the discovery, structure elucidation, biological activity, and the unique anti-TB mechanism of lasso peptides. Moreover, the discovery of their biosynthetic pathway has laid the foundation for combinatorial biosynthesis of their analogs, which provides new perspectives for the production of novel anti-TB lasso peptides.

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Lasso peptides belong to a peculiar family of ribosomally synthesized and post-translationally modified peptides (RiPPs)-natural products with an unusual isopeptide-bonded slipknot structure. Except for assembling of this unusual lasso fold, several further post-translational modifications of lasso peptides, including C-terminal methylation, phosphorylation/poly-phosphorylation, citrullination, and acetylation, have been reported recently. However, most of their biosynthetic logic have not been elucidated except the phosphorylated paeninodin lasso peptide. Herein, we identified two novel lassomycin-like lasso peptide biosynthetic pathways and, for the first time, characterized a novel C-terminal peptide carboxyl methyltransferase involved in these pathways. Our investigations revealed that this new family of methyltransferase could specifically methylate the C terminus of precursor peptide substrates, eventually leading to lassomycin-like C-terminal methylated lasso peptides. Our studies offer another rare insight into the extraordinary strategies of chemical diversification adopted by lasso peptide biosynthetic machinery and predicated two valuable sources for methylated lasso peptide discovery.

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Genetically encoded and ribosomally synthesised peptides and small proteins act as important regulators in fundamental cellular processes, including gene expression, development, signalling and metabolism. Moreover, they also play a crucial role in eukaryotic and prokaryotic defence against microorganisms. Extremely diverse in size and structure, they are often subject to extensive post-translational modification. Recent technological advances are now allowing the analysis of the whole cellular transcriptome and proteome, revealing the presence of hundreds of long-overlooked alternative and short open reading frames (short ORFs, or sORFs) in mRNA and supposedly noncoding RNAs. However, in many instances the biological roles of their translational products remain to be elucidated. Here we provide an overview on the intriguing structural and functional diversity of ribosomally synthesised peptides and newly discovered peptides and small proteins.

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