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Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a significant potential for novel therapeutic applications because of their bioactive properties, stability, and specificity. RiPPs are synthesized on ribosomes, followed by intricate post-translational modifications (PTMs), crucial for their diverse structures and functions. PTMs, such as cyclization, methylation, and proteolysis, play crucial roles in enhancing RiPP stability and bioactivity. Advances in synthetic biology and bioinformatics have significantly advanced the field, introducing new methods for RiPP production and engineering. These methods encompass strategies for heterologous expression, genetic refactoring, and exploiting the substrate tolerance of tailoring enzymes to create novel RiPP analogs with improved or entirely new functions. Furthermore, the introduction and implementation of cutting-edge screening methods, including mRNA display, surface display, and two-hybrid systems, have expedited the identification of RiPPs with significant pharmaceutical potential. This comprehensive review not only discusses the current advancements in RiPP research but also the promising opportunities that leveraging these bioactive peptides for therapeutic applications presents, illustrating the synergy between traditional biochemistry and contemporary synthetic biology and genetic engineering approaches.

AbstractPeptide-based medications hold immense potential in addressing a wide range of human disorders and discomforts. However, their widespread utilization encounters two major challenges: preservation and production efficiency. Cyclotides, a class of ribosomally synthesized and post-translationally modified peptides (RiPPs), exhibit unique characteristics, such as a cyclic backbone and cystine knot, enhancing their stability and contributing to a wide range of pharmacological properties exhibited by these compounds. Cyclotides are efficient in the biomedical (e.g., antitumor, antidiabetic, antimicrobial, antiviral) and agrochemical fields by exhibiting activity against pests and plant diseases. Furthermore, their structural attributes make them suitable as molecular scaffolds for grafting and drug delivery. Notably, the mutated variant of kalata B1 cyclotide ([T20K] kalata B1) has recently entered phase 1 of human clinical trials for multiple sclerosis, building upon the success observed in animal trials. To enable large-scale production of cyclotides, it is crucial to further explore their remarkable structural and bioactive properties. This necessitates extensive research focused on enhancing the efficiency of the processes required for their production. This study provides a comprehensive review of the biological synthesis methods of cyclotides, with particular emphasis on various expression systems, namely bacteria, plants, yeast, and cell-free systems. By investigating these expression systems, it becomes possible to design production systems that are adaptable, economically viable, and efficient for generating active and pure cyclotides at an industrial scale. The advantages of biological synthesis over chemical synthesis are thoroughly explored, highlighting the potential of these expression systems in meeting the demands of large-scale cyclotide production. [ABSTRACT FROM AUTHOR]

• Lasso peptides are diverse macrolactam-containing ribosomal peptide natural products distinguished by a kinetically trapped [1]rotaxane fold. • While leader peptidolysis is reasonably well understood, comparatively little is known about how lasso cyclases tie their substrates into the characteristic lariat knot. • Genome mining has enabled the accurate prediction of thousands of lasso peptides. Coupled with new expression methods, it has dramatically expanded our understanding of their diversification potential. • While some lasso peptides have established biological activities, most have elusive functions. Collaboration between natural product chemists, specialists in omics, and microbiologists will be essential to diversify bioactivity screens and understand the role of lasso peptides in the environment. Lasso peptides are a large and sequence-diverse class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products characterized by their slip knot-like shape. These unique, highly stable peptides are produced by bacteria for various purposes. Their stability and sequence diversity make them a potentially useful scaffold for biomedically relevant folded peptides. However, many questions remain about lasso peptide biosynthesis, ecological function, and diversification potential for biomedical and agricultural applications. This review discusses new insights and open questions about lasso peptide biosynthesis and biological function. The role that genome mining has played in the development of new methodologies for discovering and diversifying lasso peptides is also discussed. [ABSTRACT FROM AUTHOR]

Lasso peptides, ribosomally synthesized and post-translationally modified peptides, are primarily produced by bacteria and some archaea. Streptomyces lasso peptides have been known for their antimicrobial, anticancer, and antiviral properties. However, understanding their role in the morphology and production of secondary metabolites remains limited. We identified a previously unknown lasso peptide gene cluster in the genome of Streptomyces sp. L06. This gene cluster (LASS) produces two distinct lasso peptides, morphosin-1 and − 2. Notably, morphosin-2 is a member of a new subfamily of lasso peptides, with BGCs exhibiting a similar structure. When LASS was expressed in different Streptomyces hosts, it led to exciting phenotypic changes, including the absence of spores and damage in aerial mycelium development. In one of the hosts, LASS even triggered antibiotic formation. These findings open up a world of possibilities, suggesting the potential role of morphosins in shaping Streptomyces' morphological and biochemical development. [ABSTRACT FROM AUTHOR]

Borosins are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylated backbones. Although the first mature compound of borosin was reported in 1997, the biosynthetic pathway was elucidated 20 years later. Until this work, borosins have been able to be categorized into 11 types based on the features of their protein structure and core peptides. Type III borosins were reported only in fungi initially. In order to explore the sources and potential of type III borosins, a precise genome mining work of type III borosins was conducted in bacteria and KchMA's self-methylation activity was validated by biochemical experiment. Furthermore, a commercial protease and AI-assisted rational design was employed to engineer KchMA for the capacity to produce various N-methylated peptides. Our work demonstrates that type III borosins are abundant not only in eukaryotes but also in bacteria and have immense potential as a tool for synthetic biology. [ABSTRACT FROM AUTHOR]

The rapid evolution of drug resistance is one of the greatest health issues of the 21st century. There is an alarming situation to find new therapeutic strategies or candidate drugs to tackle ongoing multi-drug resistance development. The marine environment is one of the prime natural ecosystems on Earth, the majority of which is still unexplored, especially when it comes to the microbes. A wide variety of bioactive compounds have been obtained from a varied range of marine organisms; however, marine bacteria-produced bacteriocins are still undermined. Owing to the distinct environmental stresses that marine bacterial communities encounter, their bioactive compounds frequently undergo distinct adaptations that confer on them a variety of shapes and functions, setting them apart from their terrestrial counterparts. Bacterially produced ribosomally synthesized and posttranslationally modified peptides (RiPPs), known as bacteriocins, are one of the special interests to be considered as an alternative to conventional antibiotics because of their variety in structure and diverse potential biological activities. Additionally, the gut microbiome of marine creatures are a largely unexplored source of new bacteriocins with promising activities. There is a huge possibility of novel bacteriocins from marine bacterial communities that might come out as efficient candidates to fight against antibiotic resistance, especially in light of the growing pressure from antibiotic-resistant diseases and industrial desire for innovative treatments. The present review summarizes known and fully characterized marine bacteriocins, their evolutionary aspects, challenges, and the huge possibilities of unexplored novel bacteriocins from marine bacterial communities present in diverse marine ecosystems. [ABSTRACT FROM AUTHOR]

Abstract Lasso peptides, ribosomally synthesized and post-translationally modified peptides, are primarily produced by bacteria and some archaea. Streptomyces lasso peptides have been known for their antimicrobial, anticancer, and antiviral properties. However, understanding their role in the morphology and production of secondary metabolites remains limited. We identified a previously unknown lasso peptide gene cluster in the genome of Streptomyces sp. L06. This gene cluster (LASS) produces two distinct lasso peptides, morphosin-1 and − 2. Notably, morphosin-2 is a member of a new subfamily of lasso peptides, with BGCs exhibiting a similar structure. When LASS was expressed in different Streptomyces hosts, it led to exciting phenotypic changes, including the absence of spores and damage in aerial mycelium development. In one of the hosts, LASS even triggered antibiotic formation. These findings open up a world of possibilities, suggesting the potential role of morphosins in shaping Streptomyces’ morphological and biochemical development.

Comprehensive Summary: The first total synthesis of neopetromin, featuring the highly strained Tyr C‐6‐to‐Trp N‐1' linkage, is hereby reported. This modular synthetic strategy, employing C—H arylation and Larock macrocyclization, offers a novel approach to the synthesis of various RiPPs natural product families. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: The first total synthesis of neopetromin, featuring the highly strained Tyr C‐6‐to‐Trp N‐1' linkage, is hereby reported. This modular synthetic strategy, employing C—H arylation and Larock macrocyclization, offers a novel approach to the synthesis of various RiPPs natural product families. [ABSTRACT FROM AUTHOR]

In recent years, genome and transcriptome mining have dramatically expanded the rate of discovering diverse natural products from bacteria and fungi. In plants, this approach is often more limited due to the lack of available annotated genomes and transcriptomes combined with a less consistent clustering of biosynthetic genes. The recently identified burpitide class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products offer a valuable opportunity for bioinformatics-guided discovery in plants due to their short biosynthetic pathways and gene encoded substrates. Using a high-throughput approach to assemble and analyze 700 publicly available raw transcriptomic data sets, we uncover the potential distribution of split burpitide precursor peptides in Streptophyta. Metabolomic analysis of target plants confirms our bioinformatic predictions of new cyclopeptide alkaloids from both known and new sources.

The traditional Mexican fermented beverage pulque has been considered a healthy product for treating gastrointestinal disorders. Lactic acid bacteria (LAB) have been identified as one of the most abundant microbial groups during pulque fermentation. As traditional pulque is consumed directly from the fermentation vessel, the naturally associated LABs are ingested, reaching the consumer's small intestine alive, suggesting their potential probiotic capability. In this contribution, we assayed the probiotic potential of the strain of Lactiplantibacillus plantarum LB1_P46 isolated from pulque produced in Huitzilac, Morelos State, Mexico. The characterization included resistance to acid pH (3.5) and exposure to bile salts at 37 °C; the assay of the hemolytic activity and antibiotic resistance profiling; the functional traits of cholesterol reduction and β-galactosidase activity; and several cell surface properties, indicating that this LAB possesses probiotic properties comparable to other LAB. Additionally, this L. plantarum showed significance in in vitro antimicrobial activity against several Gram-negative and Gram-positive bacteria and in vivo preventive anti-infective capability against Salmonella in a BALB/c mouse model. Several functional traits and probiotic activities assayed were correlated with the corresponding enzymes encoded in the complete genome of the strain. The genome mining for bacteriocins led to the identification of several bacteriocins and a ribosomally synthesized and post-translationally modified peptide encoding for the plantaricin EF. Results indicated that L. plantarum LB1_P46 is a promising probiotic LAB for preparing functional non-dairy and dairy beverages. [ABSTRACT FROM AUTHOR]

Abstract Background Microbial secondary metabolites play a crucial role in the intricate interactions within the natural environment. Among these metabolites, ribosomally synthesized and post-translationally modified peptides (RiPPs) are becoming a promising source of therapeutic agents due to their structural diversity and functional versatility. However, their biosynthetic capacity and ecological functions remain largely underexplored. Results Here, we aim to explore the biosynthetic profile of RiPPs and their potential roles in the interactions between microbes and viruses in the ocean, which encompasses a vast diversity of unique biomes that are rich in interactions and remains chemically underexplored. We first developed TrRiPP to identify RiPPs from ocean metagenomes, a deep learning method that detects RiPP precursors in a hallmark gene-independent manner to overcome the limitations of classic methods in processing highly fragmented metagenomic data. Applying this method to metagenomes from the global ocean microbiome, we uncover a diverse array of previously uncharacterized putative RiPP families with great novelty and diversity. Through correlation analysis based on metatranscriptomic data, we observed a high prevalence of antiphage defense-related and phage-related protein families that were co-expressed with RiPP families. Based on this putative association between RiPPs and phage infection, we constructed an Ocean Virus Database (OVD) and established a RiPP-involving host-phage interaction network through host prediction and co-expression analysis, revealing complex connectivities linking RiPP-encoding prokaryotes, RiPP families, viral protein families, and phages. These findings highlight the potential of RiPP families involved in prokaryote-phage interactions and coevolution, providing insights into their ecological functions in the ocean microbiome. Conclusions This study provides a systematic investigation of the biosynthetic potential of RiPPs from the ocean microbiome at a global scale, shedding light on the essential insights into the ecological functions of RiPPs in prokaryote-phage interactions through the integration of deep learning approaches, metatranscriptomic data, and host-phage connectivity. This study serves as a valuable example of exploring the ecological functions of bacterial secondary metabolites, particularly their associations with unexplored microbial interactions. Video Abstract

Background: Microbial secondary metabolites play a crucial role in the intricate interactions within the natural environment. Among these metabolites, ribosomally synthesized and post-translationally modified peptides (RiPPs) are becoming a promising source of therapeutic agents due to their structural diversity and functional versatility. However, their biosynthetic capacity and ecological functions remain largely underexplored. Results: Here, we aim to explore the biosynthetic profile of RiPPs and their potential roles in the interactions between microbes and viruses in the ocean, which encompasses a vast diversity of unique biomes that are rich in interactions and remains chemically underexplored. We first developed TrRiPP to identify RiPPs from ocean metagenomes, a deep learning method that detects RiPP precursors in a hallmark gene-independent manner to overcome the limitations of classic methods in processing highly fragmented metagenomic data. Applying this method to metagenomes from the global ocean microbiome, we uncover a diverse array of previously uncharacterized putative RiPP families with great novelty and diversity. Through correlation analysis based on metatranscriptomic data, we observed a high prevalence of antiphage defense-related and phage-related protein families that were co-expressed with RiPP families. Based on this putative association between RiPPs and phage infection, we constructed an Ocean Virus Database (OVD) and established a RiPP-involving host-phage interaction network through host prediction and co-expression analysis, revealing complex connectivities linking RiPP-encoding prokaryotes, RiPP families, viral protein families, and phages. These findings highlight the potential of RiPP families involved in prokaryote-phage interactions and coevolution, providing insights into their ecological functions in the ocean microbiome. Conclusions: This study provides a systematic investigation of the biosynthetic potential of RiPPs from the ocean microbiome at a global scale, shedding light on the essential insights into the ecological functions of RiPPs in prokaryote-phage interactions through the integration of deep learning approaches, metatranscriptomic data, and host-phage connectivity. This study serves as a valuable example of exploring the ecological functions of bacterial secondary metabolites, particularly their associations with unexplored microbial interactions. CSrTVep6AzTb_yem78LPYE Video Abstract [ABSTRACT FROM AUTHOR]

Sponges (Porifera) contain many peptide-specialized metabolites with potent biological activities and significant roles in shaping marine ecology. It is well established that symbiotic bacteria produce bioactive "sponge" peptides, both on the ribosome (RiPPs) and nonribosomally. Here, we demonstrate that sponges themselves also produce many bioactive macrocyclic peptides, such as phakellistatins and related proline-rich macrocyclic peptides (PRMPs). Using the Stylissa carteri sponge transcriptome, methods were developed to find sequences encoding 46 distinct RiPP-type core peptides, of which ten encoded previously identified PRMP sequences. With this basis set, the genome and transcriptome of the sponge Axinella corrugata was interrogated to find 35 PRMP precursor peptides encoding 31 unique core peptide sequences. At least 11 of these produced cyclic peptides that were present in the sponge and could be characterized by mass spectrometry, including stylissamides A-D and seven previously undescribed compounds. Precursor peptides were encoded in the A. corrugata genome, confirm-ing their animal origin. The peptides contained signal peptide sequences and highly repetitive recognition sequence-core peptide elements with up to 25 PRMP copies in a single precursor. In comparison to sponges without PRMPs, PRMP sponges are incredibly enriched in potentially secreted polypeptides, with >23,000 individual signal peptide encoding genes found in a single transcriptome. The similarities between PRMP biosynthetic genes and neuropeptides in terms of their biosynthetic logic suggest a fundamental biology linked to circular peptides, possibly indicating a widespread and underappreciated diversity of signaling peptide post-translational modifications across the animal kingdom. [ABSTRACT FROM AUTHOR]

Cyclic peptides with cyclophane linkers are an attractive compound type owing to the fine‐tuned rigid three‐dimensional structures and unusual biophysical features. Cytochrome P450 enzymes are capable of catalyzing not only the C−C and C−O oxidative coupling reactions found in vancomycin and other nonribosomal peptides (NRPs), but they also exhibit novel catalytic activities to generate cyclic ribosomally synthesized and post‐translationally modified peptides (RiPPs) through cyclophane linkage. To discover more P450‐modified multicyclic RiPPs, we set out to find cryptic and unknown P450‐modified RiPP biosynthetic gene clusters (BGCs) through genome mining. Synergized bioinformatic analysis reveals that P450‐modified RiPP BGCs are broadly distributed in bacteria and can be classified into 11 classes. Focusing on two classes of P450‐modified RiPP BGCs where precursor peptides contain multiple conserved aromatic amino acid residues, we characterized 11 novel P450‐modified multicyclic RiPPs with different cyclophane linkers through heterologous expression. Further mutation of the key ring‐forming residues and combinatorial biosynthesis study revealed the order of bond formation and the specificity of P450s. This study reveals the functional diversity of P450 enzymes involved in the cyclophane‐containing RiPPs and indicates that P450 enzymes are promising tools for rapidly obtaining structurally diverse cyclic peptide derivatives. [ABSTRACT FROM AUTHOR]

The rapid evolution of drug resistance is one of the greatest health issues of the 21st century. There is an alarming situation to find new therapeutic strategies or candidate drugs to tackle ongoing multi-drug resistance development. The marine environment is one of the prime natural ecosystems on Earth, the majority of which is still unexplored, especially when it comes to the microbes. A wide variety of bioactive compounds have been obtained from a varied range of marine organisms; however, marine bacteria-produced bacteriocins are still undermined. Owing to the distinct environmental stresses that marine bacterial communities encounter, their bioactive compounds frequently undergo distinct adaptations that confer on them a variety of shapes and functions, setting them apart from their terrestrial counterparts. Bacterially produced ribosomally synthesized and posttranslationally modified peptides (RiPPs), known as bacteriocins, are one of the special interests to be considered as an alternative to conventional antibiotics because of their variety in structure and diverse potential biological activities. Additionally, the gut microbiome of marine creatures are a largely unexplored source of new bacteriocins with promising activities. There is a huge possibility of novel bacteriocins from marine bacterial communities that might come out as efficient candidates to fight against antibiotic resistance, especially in light of the growing pressure from antibiotic-resistant diseases and industrial desire for innovative treatments. The present review summarizes known and fully characterized marine bacteriocins, their evolutionary aspects, challenges, and the huge possibilities of unexplored novel bacteriocins from marine bacterial communities present in diverse marine ecosystems.

Borosins are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylated backbones. Although the first mature compound of borosin was reported in 1997, the biosynthetic pathway was elucidated 20 years later. Until this work, borosins have been able to be categorized into 11 types based on the features of their protein structure and core peptides. Type III borosins were reported only in fungi initially. In order to explore the sources and potential of type III borosins, a precise genome mining work of type III borosins was conducted in bacteria and KchMA’s self-methylation activity was validated by biochemical experiment. Furthermore, a commercial protease and AI-assisted rational design was employed to engineer KchMA for the capacity to produce various N-methylated peptides. Our work demonstrates that type III borosins are abundant not only in eukaryotes but also in bacteria and have immense potential as a tool for synthetic biology.

Addressing the current antibiotic-resistance challenge would be aided by the identification of compounds with novel mechanisms of action. Epilancin 15X, a lantibiotic produced by Staphylococcus epidermidis 15x154, displays antimicrobial activity in the submicromolar range against a subset of pathogenic Gram-positive bacteria. S. epidermidis is a common member of the human skin or mucosal microbiota. We here investigated the mechanism of action of epilancin 15X. The compound is bactericidal against Staphylococcus carnosus as well as Bacillus subtilis and appears to kill these bacteria by membrane disruption. Structure-activity relationship studies using engineered analogs show that its conserved positively charged residues and dehydroamino acids are important for bioactivity, but the N-terminal lactyl group is tolerant of changes. Epilancin 15X treatment negatively affects fatty acid synthesis, RNA translation, and DNA replication and transcription without affecting cell wall biosynthesis. The compound appears localized to the surface of bacteria and is most potent in disrupting the membranes of liposomes composed of negatively charged membrane lipids in a lipid II independent manner. Epilancin 15X does not elicit a LiaRS response in B. subtilis but did upregulate VraRS in S. carnosus. Treatment of S. carnosus or B. subtilis with epilancin 15X resulted in an aggregation phenotype in microscopy experiments. Collectively these studies provide new information on epilancin 15X activity. [ABSTRACT FROM AUTHOR]

Ribosomally synthesized and post‐translationally modified peptides (RiPPs) are a fascinating group of natural products that exhibit diverse structural features and bioactivities. P450‐catalyzed RiPPs stand out as a unique but underexplored family. Herein, we introduce a rule‐based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co‐occurrence and co‐conservation of precursors and P450s and interactions between them, successfully facilitating the identification of diverse P450‐catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can catalyze the formation of Trp‐Trp‐Tyr (one C−C and two C−N bonds), Tyr‐Trp (C−C bond), Trp‐Trp (C−N bond), and His‐His (ether bond) crosslinks, respectively, within three or four residues. KstB, ScnB, and MciB could accept non‐native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s to expand the chemical diversity of strained macrocyclic peptides and the range of biocatalytic tools available for peptide macrocyclization. [ABSTRACT FROM AUTHOR]

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a significant potential for novel therapeutic applications because of their bioactive properties, stability, and specificity. RiPPs are synthesized on ribosomes, followed by intricate post-translational modifications (PTMs), crucial for their diverse structures and functions. PTMs, such as cyclization, methylation, and proteolysis, play crucial roles in enhancing RiPP stability and bioactivity. Advances in synthetic biology and bioinformatics have significantly advanced the field, introducing new methods for RiPP production and engineering. These methods encompass strategies for heterologous expression, genetic refactoring, and exploiting the substrate tolerance of tailoring enzymes to create novel RiPP analogs with improved or entirely new functions. Furthermore, the introduction and implementation of cutting-edge screening methods, including mRNA display, surface display, and two-hybrid systems, have expedited the identification of RiPPs with significant pharmaceutical potential. This comprehensive review not only discusses the current advancements in RiPP research but also the promising opportunities that leveraging these bioactive peptides for therapeutic applications presents, illustrating the synergy between traditional biochemistry and contemporary synthetic biology and genetic engineering approaches.

LanD flavoproteins catalyze oxidative decarboxylation of the C‐terminal Cys residue of a peptide to produce an enethiol. This enethiol is highly reactive and can be coupled with an upstream dehydroamino acid through Michael addition to form S‐[2‐aminovinyl](3‐methyl)cysteine, an unsaturated thioether residue known to be characteristic of an array of C‐terminally macrocyclized, ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on a two‐stage bioinformatics mining of posttranslational modifications (PTMs) related to C‐terminal Cys processing, we report herein that LanD activity can couple with radical S‐adenosylmethionine chemistry to provide a new unsaturated thioether residue, S‐[2‐aminovinyl]‐3‐carbamoylcysteine, by conjugating the resultant enethiol with Cβ of the Asn residue in the C‐terminal NxxC motif of a peptide for macrocyclization. This study furthers our understanding of the variety of PTMs involved in creating the structure diversity of macrocyclic RiPPs. [ABSTRACT FROM AUTHOR]

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products biosynthesized from a genetically encoded precursor peptide. The enzymes that install the post-translational modifications on these peptides have the potential to be useful catalysts in the production of natural-product-like compounds and can install non-proteogenic amino acids in peptides and proteins. However, engineering these enzymes has been somewhat limited, due in part to limited structural information on enzymes in the same families that nonetheless exhibit different substrate selectivities. Despite AlphaFold2's superior performance in single-chain protein structure prediction, its multimer version lacks accuracy and requires high-end GPUs, which are not typically available to most research groups. Additionally, the default parameters of AlphaFold2 may not be optimal for predicting complex structures like RiPP biosynthetic enzymes, due to their dynamic binding and substrate-modifying mechanisms. This study assessed the efficacy of the structure prediction program ColabFold (a variant of AlphaFold2) in modeling RiPP biosynthetic enzymes in both monomeric and dimeric forms. After extensive benchmarking, it was found that there were no statistically significant differences in the accuracy of the predicted structures, regardless of the various possible prediction parameters that were examined, and that with the default parameters, ColabFold was able to produce accurate models. We then generated additional structural predictions for select RiPP biosynthetic enzymes from multiple protein families and biosynthetic pathways. Our findings can serve as a reference for future enzyme engineering complemented by AlphaFold-related tools. [ABSTRACT FROM AUTHOR]

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. Ribosomally synthesized and post-translationally modified peptides (RiPPs) from microorganisms show high chemical diversity and exhibit potent biological properties. The computational detection of novel classes of RiPPs is hampered by their short length and the lack of universally conserved genes. The high false-positive rate of class-independent computational detection approaches can be addressed by validation via mass spectrometry-based metabolomics. [ABSTRACT FROM AUTHOR]

Cihunamides A–D (1–4), novel antibacterial RiPPs, were isolated from volcanic‐island‐derived Streptomyces sp. The structures of 1–4 were elucidated by 1H, 13C, and 15N NMR, MS, and chemical derivatization; they contain a tetrapeptide core composed of WNIW, cyclized by a unique C−N linkage between two Trp units. Genome mining of the producer strain revealed two biosynthetic genes encoding a cytochrome P450 enzyme and a precursor peptide. Heterologous co‐expression of the core genes demonstrated the biosynthesis of cihunamides through P450‐mediated oxidative Trp‐Trp cross‐linking. Further bioinformatic analysis uncovered 252 homologous gene clusters, including that of tryptorubins, which possess a distinct Trp‐Trp linkage. Cihunamides do not display the non‐canonical atropisomerism shown in tryptorubins, which are the founding members of the "atropitide" family. Therefore, we propose to use a new RiPP family name, "bitryptides", for cihunamides, tryptorubins, and their congeners, wherein the Trp‐Trp linkages define the structural class rather than non‐canonical atropisomerism. [ABSTRACT FROM AUTHOR]

Lanthipeptides are ribosomally‐synthesized natural products from bacteria featuring stable thioether‐crosslinks and various bioactivities. Herein, we report on a new clade of tricyclic class‐IV lanthipeptides with curvocidin from Thermomonospora curvata as its first representative. We obtained crystal structures of the corresponding lanthipeptide synthetase CuvL that showed a circular arrangement of its kinase, lyase and cyclase domains, forming a central reaction chamber for the iterative substrate processing involving nine catalytic steps. The combination of experimental data and artificial intelligence‐based structural models identified the N‐terminal subdomain of the kinase domain as the primary site of substrate recruitment. The ribosomal precursor peptide of curvocidin employs an amphipathic α‐helix in its leader region as an anchor to CuvL, while its substrate core shuttles within the central reaction chamber. Our study thus reveals general principles of domain organization and substrate recruitment of class‐IV and class‐III lanthipeptide synthetases. [ABSTRACT FROM AUTHOR]

Natural bioactive peptide discovery is a challenging and time-consuming process. However, advances in synthetic biology are providing promising new avenues in peptide engineering that allow for the design and production of a large variety of new-to-nature peptides with enhanced or new bioactivities, using known peptides as templates. Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs). The modularity of post-translational modification (PTM) enzymes and ribosomal biosynthesis inherent to lanthipeptides enables their engineering and screening in a high-throughput manner. The field of RiPPs research is rapidly evolving, with many novel PTMs and their associated modification enzymes being identified and characterized. The modularity presented by these diverse and promiscuous modification enzymes has made them promising tools for further in vivo engineering of lanthipeptides, allowing for the diversification of their structures and activities. In this review, we explore the diverse modifications occurring in RiPPs and discuss the potential applications and feasibility of combining various modification enzymes for lanthipeptide engineering. We highlight the prospect of lanthipeptide- and RiPP-engineering to produce and screen novel peptides, including mimics of potent non-ribosomally produced antimicrobial peptides (NRPs) such as daptomycin, vancomycin, and teixobactin, which offer high therapeutic potential. [ABSTRACT FROM AUTHOR]

Aromatic prenyltransferases from cyanobactin biosynthetic pathways catalyse the chemoselective and regioselective intramolecular transfer of prenyl/geranyl groups from isoprene donors to an electron‐rich position in these macrocyclic and linear peptides. These enzymes often demonstrate relaxed substrate specificity and are considered useful biocatalysts for structural diversification of peptides. Herein, we assess the isoprene donor specificity of the N1‐tryptophan prenyltransferase AcyF from the anacyclamide A8P pathway using a library of 22 synthetic alkyl pyrophosphate analogues, of which many display reactive groups that are amenable to additional functionalization. We further used AcyF to introduce a reactive moiety into a tryptophan‐containing cyclic peptide and subsequently used click chemistry to fluorescently label the enzymatically modified peptide. This chemoenzymatic strategy allows late‐stage modification of peptides and is useful for many applications. [ABSTRACT FROM AUTHOR]

Cyclopeptide alkaloids are an abundant class of plant cyclopeptides with over 200 analogs described and bioactivities ranging from analgesic to antiviral. While these natural products have been known for decades, their biosynthetic basis remains unclear. Using a transcriptome‐mining approach, we link the cyclopeptide alkaloids from Ceanothus americanus to dedicated RiPP precursor peptides and identify new, widely distributed split BURP peptide cyclase containing gene clusters. Guided by our bioinformatic analysis, we identify and isolate new cyclopeptides from Coffea arabica, which we named arabipeptins. Reconstitution of the enzyme activity for the BURP found in the biosynthesis of arabipeptin A validates the activity of the newly discovered split BURP peptide cyclases. These results expand our understanding of the biosynthetic pathways responsible for diverse cyclic plant peptides and suggest that these side chain cross‐link modifications are widely distributed in eudicots. [ABSTRACT FROM AUTHOR]

Thiopeptides are macrocyclic natural products with potent bioactivity. Nine new natural thiopeptides (1–9) were obtained from a Nonomuraea jiangxiensis isolated from a terrestrial soil sample collected in Singapore. Even though some of these compounds were previously synthesized or isolated from engineered strains, herein we report the unprecedented isolation of these thiopeptides from a native Nonomuraea jiangxiensis. A comparison with the literature and a detailed analysis of the NMR and HRMS of compounds 1–9 was conducted to assign their chemical structures. The structures of all new compounds were highly related to the thiopeptide antibiotics GE2270, with variations in the substituents on the thiazole and amino acid moieties. Thiopeptides 1–9 exhibited a potent antimicrobial activity against the Gram-positive bacteria, Staphylococcus aureus with MIC90 values ranging from 2 µM to 11 µM. In addition, all compounds were investigated for their cytotoxicity against the human cancer cell line A549, none of the compounds were cytotoxic. [ABSTRACT FROM AUTHOR]

Abstract Background Bacteria and archaea produce an enormous diversity of modified peptides that are involved in various forms of inter-microbial conflicts or communication. A vast class of such peptides are Ribosomally synthesized, Postranslationally modified Peptides (RiPPs), and a major group of RiPPs are graspetides, so named after ATP-grasp ligases that catalyze the formation of lactam and lactone linkages in these peptides. The diversity of graspetides, the multiple proteins encoded in the respective Biosynthetic Gene Clusters (BGCs) and their evolution have not been studied in full detail. In this work, we attempt a comprehensive analysis of the graspetide-encoding BGCs and report a variety of novel graspetide groups as well as ancillary proteins implicated in graspetide biosynthesis and expression. Results We compiled a comprehensive, manually curated set of graspetides that includes 174 families including 115 new families with distinct patterns of amino acids implicated in macrocyclization and further modification, roughly tripling the known graspetide diversity. We derived signature motifs for the leader regions of graspetide precursors that could be used to facilitate graspetide prediction. Graspetide biosynthetic gene clusters and specific precursors were identified in bacterial divisions not previously known to encode RiPPs, in particular, the parasitic and symbiotic bacteria of the Candidate phyla radiation. We identified Bacteroides-specific biosynthetic gene clusters (BGC) that include remarkable diversity of graspetides encoded in the same loci which predicted to be modified by the same ATP-grasp ligase. We studied in details evolution of recently characterized chryseoviridin BGCs and showed that duplication and horizonal gene exchange both contribute to the diversification of the graspetides during evolution. Conclusions We demonstrate previously unsuspected diversity of graspetide sequences, even those associated with closely related ATP-grasp enzymes. Several previously unnoticed families of proteins associated with graspetide biosynthetic gene clusters are identified. The results of this work substantially expand the known diversity of RiPPs and can be harnessed to further advance approaches for their identification.

[Display omitted] The total synthesis of (±)-3-thiaglutamate is reported. Central to our strategy is an thiol addition to an imine to form the thioaminal of the natural product. The resulting thioaminal product is then subjected to triflic acid global deprotection to produce 3-thiaglutamate as a triflate salt. This work constitutes the first total synthesis of 3-thiaglutamate and demonstrates that the hemithioaminal group in 3-thiaglutamate can be stabilized under acidic conditions. [ABSTRACT FROM AUTHOR]

Objective: Thioalbamide is a ribosomally synthesized and post-translationally modified peptide (RiPP) belonging to the family of thioamitides, a rare class of microbial specialized metabolites with unusual post-translational modifications and promising biological activities. Recent studies have demonstrated the ability of thioalbamide to exert highly selective cytotoxic effects on tumor cells by affecting their energy metabolism, thus causing abnormal ROS production and triggering apoptosis. This study is aimed to investigate the molecular mechanisms underlying the antitumor activity of thioalbamide in order to identify its exact molecular target. Methods: Wild type MCF-7 and MDA-MB-231 breast cancer cell lines as well as cancer cells deprived of mitochondrial DNA (ρ0 cells) were employed in order to assess thioalbamide effects on tumor bioenergetics. In this regard, metabolic profile was evaluated by a Seahorse XFe96 analyzer, and the activity of the enzyme complexes involved in oxidative phosphorylation was quantified by spectrophotometric assays. Thioalbamide effects on tumor invasiveness were assessed by gelatin zymography experiments and invasion assays. In vivo experiments were carried out on breast cancer xenograft and “experimental metastasis” mouse models. Results: Experiments carried out on ρ0 breast cancer cells, together with Seahorse analysis and the application of spectrophotometric enzymatic assays, highlighted the ability of thioalbamide to affect the mitochondrial respiration process, and allowed to propose the FoF1-ATPase complex as its main molecular target in breast cancer cells. Additionally, thioalbamide-mediated OXPHOS inhibition was shown, for the first time, to reduce tumor invasiveness by inhibiting metalloproteinase-9 secretion. Furthermore, this study has confirmed the antitumor potential of thioalbamide in two different in vivo models. In particular, experiments on MCF-7 and MDA-MB-231 xenograft mouse models have confirmed in vivo its high anti-proliferative and pro-apoptotic activity, while experiments on MDA-MB-231 ″experimental metastasis” mouse models have highlighted its ability to inhibit breast cancer cell invasiveness. Conclusions: Overall, our results shed more light on the molecular mechanisms underlying the pharmacological potential of thioamidated peptides, thus reducing the gap that separates this rare class of microbial metabolites from clinical studies, which could validate them as effective tools for cancer treatment.

ABSTRACT Bioactivities of fungal peptides are of interest for basic research and therapeutic drug development. Some of these peptides are derived from “KEX2-processed repeat proteins” (KEPs), a recently defined class of precursor proteins that contain multiple peptide cores flanked by KEX2 protease cleavage sites. Genome mining has revealed that KEPs are widespread in the fungal kingdom. Their functions are largely unknown. Here, we present the first in-depth structural and functional analysis of KEPs in a basidiomycete. We bioinformatically identified KEP-encoding genes in the genome of the model agaricomycete Coprinopsis cinerea and established a detection protocol for the derived peptides by overexpressing the C. cinerea KEPs in the yeast Pichia pastoris. Using this protocol, which includes peptide extraction and mass spectrometry with data analysis using the search engine Mascot, we confirmed the presence of several KEP-derived peptides in C. cinerea, as well as in the edible mushrooms Lentinula edodes, Pleurotus ostreatus, and Pleurotus eryngii. While CRISPR-mediated knockout of C. cinerea kep genes did not result in any detectable phenotype, knockout of kex genes caused defects in mycelial growth and fruiting body formation. These results suggest that KEP-derived peptides may play a role in the interaction of C. cinerea with the biotic environment and that the KEP-processing KEX proteases target a variety of substrates in agaricomycetes, including some important for mycelial growth and differentiation. IMPORTANCE Two recent bioinformatics studies have demonstrated that KEX2-processed repeat proteins are widespread in the fungal kingdom. However, despite the prevalence of KEPs in fungal genomes, only few KEP-derived peptides have been detected and studied so far. Here, we present a protocol for the extraction and structural characterization of KEP-derived peptides from fungal culture supernatants and tissues. The protocol was successfully used to detect several linear and minimally modified KEP-derived peptides in the agaricomycetes C. cinerea, L. edodes, P. ostreatus, and P. eryngii. Our study establishes a new protocol for the targeted search of KEP-derived peptides in fungi, which will hopefully lead to the discovery of more of these interesting fungal peptides and allow a further characterization of KEPs.

Ribosomally synthesized and post‐translationally modified peptides (RiPPs) represent one of the largest but primarily underexplored natural product families in bacteria. The genetically encoded nature of RiPPs simplifies the prediction and prioritization of their biosynthetic gene clusters (BGCs). We report a small peptide and enzyme co‐occurrence analysis workflow (SPECO), which allowed us to identify 32 220 prospective rSAM‐catalyzed RiPP BGCs from 161 954 bacterial genomes and prioritize 25 families with new biosynthetic architectures or precursor patterns. We characterized three new enzymes that respectively catalyze cysteine‐glycine (BlaB), histidine‐aliphatic side chain (ScaB), and tyrosine/histidine‐arginine (VguB) cross‐links. The cyclophane‐forming enzyme ScaB exhibits broad substrate selectivity, allowing it to catalyze diverse triceptide formation. These results demonstrate the strength of the SPECO workflow in discovering new enzymes for peptide macrocyclization. [ABSTRACT FROM AUTHOR]

Circular bacteriocins, also known as bacterial head-to-tail cyclized peptides, are a subgroup of ribosomally synthesized and post-translationally modified peptides (RiPPs). Compared with their conventional linear counterparts, circular bacteriocins are highly stable over a broad temperature and pH range, and circularization decreases proteolytic degradation by exopeptidases. These features render them great potential as scaffold candidates to withstand strident conditions in food- and pharmaceutical applications. However, the biosynthesis and bioactivity of circular bacteriocins still remain largely unknown. To investigate and gain more insights into the biosynthesis of circular bacteriocins and to achieve efficient production and characterization of bacteriocin variants, we developed an efficient cloning and heterologous expression system for clostridial circularin A and successfully produced this circular peptide in Lactococcus lactis NZ9000. We report three system formats with single plasmid or plasmid combinations to achieve successful cloning and functional production of circularin A in L. lactis. These systematic varieties enabled us to choose the appropriate method to efficiently obtain various constructs with desired properties. With the established heterologous systems in L. lactis, we performed several mutagenesis studies in the precursor peptide to study its structure/function relationships. The overlay activity assay revealed that these mutant variants had variable effects on different indicator strains: lysine substitution for certain glutamine residue(s) greatly decreased its bioactivity against Clostridium perfringens and L. lactis NZ9000, and alanine replacement for the cationic residues significantly reduced the activity against Lactobacillus sake ATCC 15521, whereas alanine substitution for the aromatic residues decreased its bioactivity against all three testing strains dramatically. Moreover, the conditions for bacteriocin production were optimized. Results show that supplementing the minimal medium with extra glucose (or sucrose) and immediate nisin-induction improved the peptide yield significantly. Briefly, we developed an excellent system for the production of circularin A and a wide range of variant peptides in a convenient host, as well as a method for fast detection of peptide production and activity. This system facilitated our mutagenesis studies which provided valuable insights into the effects of mutating specific residues on its biosynthesis and bioactivity, and will eventually enable more complex research into the biosynthesis of circularin A. [ABSTRACT FROM AUTHOR]

Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α‐carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in‐depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible modification site within the sactipeptide subtilosin A allowing for peptide engineering without compromising formation of thioether bridges. A panel of natural and hybrid sactipeptides was produced to study the AlbA‐mediated formation of thioether bridges, which were identified mass‐spectrometrically. In a proof‐of‐principle study, we re‐engineered subtilosin A to a thioether‐bridged, specific streptavidin targeting peptide, opening the door for the functional engineering of sactipeptides. [ABSTRACT FROM AUTHOR]

Sactipeptide sind ribosomal synthetisierte Peptide, die eine einzigartige Verknüpfung von Schwefel und α‐Kohlenstoffen enthalten. Die Bildung von Thioetherbrücken wird in diesen Molekülen durch Sactisynthasen katalysiert. Diese spezielle Art der Verknüpfung verleiht Sactipeptiden eine erhöhte strukturelle, thermische und proteolytische Stabilität, was sie zu attraktiven Gerüsten für die Entwicklung neuer Biotherapeutika macht. In diesem Artikel berichten wir über eine Studie zur Substrattoleranz der Sactisynthase AlbA, die die Bildung von Thioetherbrücken im Sactipeptid Subtilosin A katalysiert. Wir haben eine Modifikationsstelle innerhalb dieses Sactipeptids identifiziert, die ein Peptid‐Engineering ohne Beeinträchtigung der Bildung von Thioetherbrücken ermöglicht. Eine Reihe von natürlichen und hybriden Sactipeptidkonstrukten wurde hergestellt, um die AlbA vermittelte Bildung von Thioetherbrücken zu untersuchen und diese massenspektrometrisch zu identifizieren. In einer Proof‐of‐Principle‐Studie haben wir Subtilosin A mit einer neue Funktion ausgestattet, ein Thioether‐verbrücktes Streptavidin‐bindendes Peptid generiert und damit die Tür für das funktionelle Engineering von Sactipeptiden weiter geöffnet. [ABSTRACT FROM AUTHOR]

Biomacromolecules are known to feature complex three‐dimensional shapes that are essential for their function. Among natural products, ambiguous molecular shapes are a rare phenomenon. The hexapeptide tryptorubin A can adopt one of two unusual atropisomeric configurations. Initially hypothesized to be a non‐ribosomal peptide, we show that tryptorubin A is the first characterized member of a new family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) that we named atropopeptides. The sole modifying enzyme encoded in the gene cluster, a cytochrome P450 monooxygenase, is responsible for the atropospecific formation of one carbon‐carbon and two carbon‐nitrogen bonds. The characterization of two additional atropopeptide biosynthetic pathways revealed a two‐step maturation process. Atropopeptides promote pro‐angiogenic cell functions as indicated by an increase in endothelial cell proliferation and undirected migration. Our study expands the biochemical space of RiPP‐modifying enzymes and paves the way towards the chemoenzymatic utilization of atropopeptide‐modifying P450s. [ABSTRACT FROM AUTHOR]

The rapid emergence of antibiotic-resistant strains of Staphylococcus aureus presents a substantial challenge to global public health, underscoring the urgent need for novel antibiotics with diverse mechanisms of action. In this study, we conducted mutagenesis on the C -terminal region of the lantibiotic ripcin C to enhance its antimicrobial efficacy against S. aureus . The resulting optimized variant, ripcin C P23A , demonstrated potent and selective antimicrobial activity, with a minimal inhibitory concentration of 2-4 mg/L against S. aureus . Beyond its strong antimicrobial properties, ripcin C P23A exhibited significant antibiofilm activity against methicillin-resistant S. aureus (MRSA). Mechanistic studies revealed that, in addition to targeting lipid II, ripcin C P23A disrupts bacterial membranes, a capability absent in ripcin C, which may contribute to its superior antimicrobial and antibiofilm effects. Moreover, ripcin C P23A displayed favorable biosafety and plasma stability profiles. Notably, in a mouse model of MRSA-induced mastitis, ripcin C P23A effectively reduced bacterial load, alleviated inflammation, and preserved the normal histomorphology of mammary glands. This study introduces ripcin C P23A as a promising antibiotic candidate for the treatment of MRSA-related infections.

Isoprenoid modifications of proteins and peptides serve fundamental biological functions and are of therapeutic interest. While C 15 (farnesyl) and C 20 (geranylgeranyl) moieties are prevalent among proteins, known ribosomal peptide prenylations involve shorter-chain units not exceeding farnesyl in size. To our knowledge, cyclized terpene moieties have not been reported from either biomolecule class. Here we used targeted genome mining and heterologous pathway reconstitution to identify ribosomally synthesized and post-translationally modified peptides (RiPPs) with elaborate, cyclized geranylgeranyl modifications. The installing maturases commonly feature fused prenyltransferase-terpene cyclase architectures. We characterized two bifunctional maturases with distinct prenyltransferase folds and identified the terminal product of a cyanobacterial proteusin as an exceptionally complex pseudosteroid-annelated polycyclic peptide. Bioassays suggest modest anti-cyanobacterial activity with the modification being crucial for activity. Genome data predict cyclic isoprenoid units for various RiPP families including proteusin, Nif11, and lasso peptides and thus broader natural and biotechnological compatibility of the maturase system., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products biosynthesized from a genetically encoded precursor peptide. The enzymes that install the post-translational modifications on these peptides have the potential to be useful catalysts in the production of natural-product-like compounds and can install non-proteogenic amino acids in peptides and proteins. However, engineering these enzymes has been somewhat limited, due in part to limited structural information on enzymes in the same families that nonetheless exhibit different substrate selectivities. Despite AlphaFold2’s superior performance in single-chain protein structure prediction, its multimer version lacks accuracy and requires high-end GPUs, which are not typically available to most research groups. Additionally, the default parameters of AlphaFold2 may not be optimal for predicting complex structures like RiPP biosynthetic enzymes, due to their dynamic binding and substrate-modifying mechanisms. This study assessed the efficacy of the structure prediction program ColabFold (a variant of AlphaFold2) in modeling RiPP biosynthetic enzymes in both monomeric and dimeric forms. After extensive benchmarking, it was found that there were no statistically significant differences in the accuracy of the predicted structures, regardless of the various possible prediction parameters that were examined, and that with the default parameters, ColabFold was able to produce accurate models. We then generated additional structural predictions for select RiPP biosynthetic enzymes from multiple protein families and biosynthetic pathways. Our findings can serve as a reference for future enzyme engineering complemented by AlphaFold-related tools.

The wide range of moieties installed in ribosomally synthesized and post‐translationally modified peptides (RiPPs) suggests largely untapped potential for protein engineering. However, many RiPP maturases recognize target peptide precursors through an N‐terminal leader sequence that is challenging to adapt to proteins. We have recently reported a family of enzymes that splice XYG sites in RiPPs to install α‐keto‐β‐amino acids. Backbone modifications influence diverse protein properties, yet the toolkit to install β‐amino acids is limited. Here we report their leader‐independent incorporation into proteins in E. coli. Integrating an 11‐residue splice tag into six different proteins permitted the site‐selective introduction of β‐residues in vivo. The motif fusion at C‐, N‐terminal, and internal positions yielded various β‐residues. Our approach complements the few existing methods to introduce β‐amino acids or ketone‐bearing moieties, suggesting diverse applications in chemical biology. [ABSTRACT FROM AUTHOR]

Lasso peptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs) and feature the threaded, lariat knot-like topology. The basic post-translational modifications (PTMs) of lasso peptide contain two steps, including the leader peptide removal of the ribosome-derived linear precursor peptide by an ATP-dependent cysteine protease, and the macrolactam cyclization by an ATP-dependent macrolactam synthetase. Recently, advanced bioinformatic tools combined with genome mining have paved the way to uncover a rapidly growing number of lasso peptides as well as a series of PTMs other than the general class-defining processes. Despite abundant reviews focusing on lasso peptide discoveries, structures, properties, and physiological functionalities, few summaries concerned their unique PTMs. In this review, we summarized all the unique PTMs of lasso peptides uncovered to date, shedding light on the related investigations in the future. [ABSTRACT FROM AUTHOR]

Nature has evolved a remarkable array of biosynthetic enzymes that install diverse chemistries into natural products (NPs), bestowing them with a range of important biological properties that are of considerable therapeutic value. This is epitomized by the ribosomally synthesized and post-translationally modified peptides (RiPPs), a class of peptide natural products that undergo extensive post-translational modifications to produce structurally diverse bioactive peptides. In this review, we provide an overview of our research into the proteusin RiPP family, describing characterized members and the maturation enzymes responsible for their unique chemical structures and biological activities. The diverse enzymology identified in the first two proteusin pathways highlights the enormous potential of the RiPP class for new lead structures and novel pharmacophore-installing maturases as biocatalytic tools for drug discovery efforts.

Radical S-adenosylmethionine (RS) enzymology has emerged as a major biochemical strategy for the homolytic cleavage of unactivated C-H bonds. At the same time, the post-translational modification of ribosomally synthesized peptides is a rapidly expanding area of investigation. We discuss the functional cross-section of these two disciplines, highlighting the recently uncovered importance of protein-protein interactions, especially between the peptide substrate and its chaperone, which functions either as a stand-alone protein or as an N-terminal fusion to the respective RS enzyme. The need for further work on this class of enzymes is emphasized, given the poorly understood roles performed by multiple, auxiliary iron-sulfur clusters and the paucity of protein X-ray structural data.

ABSTRACT Microcin C (McC)-like compounds are natural Trojan horse peptide-nucleotide antibiotics produced by diverse bacteria. The ribosomally synthesized peptide parts of these antibiotics are responsible for their facilitated transport into susceptible cells. Once inside the cell, the peptide part is degraded, releasing the toxic payload, an isoaspartyl-nucleotide that inhibits aspartyl-tRNA synthetase, an enzyme essential for protein synthesis. Bacteria that produce microcin C-like compounds have evolved multiple ways to avoid self-intoxication. Here, we describe a new strategy through the action of S51 family peptidases, which we name MccG. MccG cleaves the toxic isoaspartyl-nucleotide, rendering it inactive. While some MccG homologs are encoded by gene clusters responsible for biosynthesis of McC-like compounds, most are encoded by standalone genes whose products may provide a basal level of resistance to peptide-nucleotide antibiotics in phylogenetically distant bacteria. IMPORTANCE Here, we identified a natural substrate for a major phylogenetic clade of poorly characterized S51 family proteases from bacteria. We show that these proteins can contribute to a basal level of resistance to an important class of natural antibiotics.

ABSTRACT The advent of multidrug-resistant bacteria has hampered the development of new antibiotics, exacerbating their morbidity and mortality. In this context, the gastrointestinal tract reveals a valuable source of novel antimicrobials. Microcins are bacteriocins produced by members of the family Enterobacteriaceae, which are endowed with a wide diversity of structures and mechanisms of action, and exert potent antibacterial activity against closely related bacteria. In this study, we investigated the antibacterial activities of four microcins against 54 Enterobacteriaceae isolates from three species (Escherichia coli, Klebsiella pneumoniae, and Salmonella enterica). The selected microcins, microcin C (McC, nucleotide peptide), microcin J25 (MccJ25, lasso peptide), microcin B17 (MccB17, linear azol(in)e-containing peptide), and microcin E492 (MccE492, siderophore peptide) carry different post-translational modifications and have distinct mechanisms of action. MICs and minimal bactericidal concentrations (MBC) of the microcins were measured and the efficacy of combinations of the microcins together or with antibiotics was assessed to identify potential synergies. Every isolate showed sensitivity to at least one microcin with MIC values ranging between 0.02 μM and 42.5 μM. Among the microcins tested, McC exhibited the broadest spectrum of inhibition with 46 strains inhibited, closely followed by MccE492 with 38 strains inhibited, while MccJ25 showed the highest activity. In general, microcin activity was observed to be independent of antibiotic resistance profile and strain genus. Of the 42 tested combinations, 20 provided enhanced activity (18 out of 20 being microcin–antibiotic combinations), with two being synergetic. IMPORTANCE With their wide range of structures and mechanisms of action, microcins are shown to exert antibacterial activities against Enterobacteriaceae resistant to antibiotics together with synergies with antibiotics and in particular colistin.

Radical SAM enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides catalyze unusual transformations that lead to unique peptide scaffolds and building blocks. Several natural products from these pathways show encouraging antimicrobial activities and represent next-generation therapeutics for infectious diseases. These systems are uniquely configured to benefit from genome-mining approaches because minimal substrate and cognate modifying enzyme expression can reveal unique, chemically complex transformations that outperform late-stage chemical reactions. This report highlights the main strategies used to reveal these enzymatic transformations, which have relied mainly on genome mining using enzyme-first approaches. We describe the general biosynthetic components for rSAM enzymes and highlight emerging approaches that may broaden the discovery and study of rSAM-RiPP enzymes. The large number of uncharacterized rSAM proteins, coupled with their unpredictable transformations, will continue to be an essential and exciting resource for enzyme discovery. [ABSTRACT FROM AUTHOR]