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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.

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MccY is a novel, structurally stable microcin with antibacterial activity against Enterobacteriaceae. However, the bioavailability of orally administrated MccY is unknown. This study evaluated the effects of MccY as a antimicrobial on pre-digestion in vitro and its intake, digestion and gut metabolism in vivo. The result of pre-digestion results that MccY maintained its biological activity and was resistant to decomposition. The study established a safe threshold of 4.46-9.92 mg/kg for the MccY dosage-body weight relationship in BALB/c mice. Mice fed with MccY demonstrated improved body weight and intestinal barrier function, accompanied with increased IgM immunogenicity and decreased levels of TNF-α, IL-6, and IL-10 in the intestine. MccY significantly facilitates the growth and activity of probiotics including Lactobacillus, Prevotella, and Bacteroides, and leading to the production of SCFAs and MCFAs during bacterial interactions. Furthermore, MccY effectively protects against the inflammatory response caused by Salmonella Typhimurium infection and effectively clears the Salmonella bacteria from the gut. In conclusion, MccY is seen as a promising new therapeutic target drug for enhancing the intestinal microbe-barrier axis and preventing enteritis.

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The probiotic strain Bacillus licheniformis MCC2514 has been shown to produce a strong antibacterial peptide and the whole genome sequence of this strain is also reported in our previous study. The present study is focused on the genome level investigation of this peptide antibiotic and its characterization. Genome mining of the culture revealed the presence of three putative bacteriocin clusters, viz. lichenicidin, sonorensin and lasso peptide. Hence, the mode of action of the peptide was investigated by reporter assay, scanning electron microscopy, and Fourier Transform Infrared spectroscopy. Additionally, the peptide treated groups of Kocuria rhizophila showed a reduction in the fold expression for transcription-related genes. The gene expression studies, quantitative ß-galactosidase induction assay using the RNA stress reporter strain, yvgS along with the homology studies concluded that lasso peptide is responsible for the antibacterial activity of the peptide which acts as an inhibitor of RNA biosynthesis. Gene expression analysis showed a considerable increase in fold expression of lasso peptide genes at various fermentation hours. Also, the peptide was isolated, and its time-kill kinetics and minimum inhibitory concentration against the indicator pathogen K. rhizophila were examined. The peptide was also purified and the molecular weight was determined to be ~ 2 kDa. Our study suggests that this bacteriocin can function as an effective antibacterial agent in food products as well as in therapeutics as it contains lasso peptide, which inhibits the RNA biosynthesis.

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The knotted configuration of lasso peptides confers thermal stability and proteolytic resistance, addressing two shortcomings of peptide-based drugs. However, low isolation yields hinder the discovery and development of lasso peptides. While testing Burkholderia sp. FERM BP-3421 as a bacterial host to produce the lasso peptide capistruin, an overproducer clone was previously identified. In this study, we show that an increase in the plasmid copy number partially contributed to the overproducer phenotype. Further, we modulated the plasmid copy number to recapitulate titers to an average of 160% relative to the overproducer, which is 1000-fold higher than previously reported with E. coli, reaching up to 240 mg/L. To probe the applicability of the developed tools for lasso peptide discovery, we targeted a new lasso peptide biosynthetic gene cluster from endosymbiont Mycetohabitans sp. B13, leading to the isolation of mycetolassin-15 and mycetolassin-18 in combined titers of 11 mg/L. These results validate Burkholderia sp. FERM BP-3421 as a production platform for lasso peptide discovery.

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Superior antagonistic activity against axenic Microcystis aeruginosa PCC7806 was observed with Paucibacter sp. B51 isolated from cyanobacterial bloom samples among 43 tested freshwater bacterial species. Complete genome sequencing, analyzing average nucleotide identity and digital DNA-DNA hybridization, designated the B51 strain as Paucibacter aquatile. Electron and fluorescence microscopic image analyses revealed the presence of the B51 strain in the vicinity of M. aeruginosa cells, which might provoke direct inhibition of the photosynthetic activity of the PCC7806 cells, leading to perturbation of cellular metabolisms and consequent cell death. Our speculation was supported by the findings that growth failure of the PCC7806 cells led to low pH conditions with fewer chlorophylls and down-regulation of photosystem genes (e.g., psbD and psaB) during their 48-h co-culture condition. Interestingly, the concentrated ethyl acetate extracts obtained from B51-grown supernatant exhibited a growth-inhibitory effect on PCC7806. The physical separation of both strains by a filter system led to no inhibitory activity of the B51 cells, suggesting that contact-mediated anti-cyanobacterial compounds might also be responsible for hampering the growth of the PCC7806 cells. Bioinformatic tools identified 12 gene clusters that possibly produce secondary metabolites, including a class II lasso peptide in the B51 genome. Further chemical analysis demonstrated anti-cyanobacterial activity from fractionated samples having a rubrivinodin-like lasso peptide, named paucinodin. Taken together, both contact-mediated inhibition of photosynthesis and the lasso peptide secretion of the B51 strain are responsible for the anti-cyanobacterial activity of P. aquatile B51.

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IMPORTANCE: Diseases caused by Enterobacteriaceae multidrug-resistant strains have become increasingly difficult to manage. It is necessary to verify the new antibacterial drug MccY effect on non-typhoid Salmonella infection in mice since it is regarded as a promising microcin. The results demonstrated that MccY has a potential therapeutic application value in the protection against Salmonella-induced intestinal damage and alleviating related intestinal dysbiosis and metabolic disorders. MccY could be a promising candidate as an antimicrobial or anti-inflammatory agent for treating infectious diseases.

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Lasso peptides are ribosomally synthesized peptides that undergo post-translational modifications including leader peptide removal by B (or the segregated B1 and B2) proteins and core peptide macrolactamization by C proteins to form a unique lariat topology. A conserved threonine residue at the penultimate position of leader peptide is hitherto found in lasso peptide precursors and shown to be a critical recognition element for effective enzymatic processing. We identified a lasso peptide biosynthetic gene cluster (bsf) from Bradymonas sediminis FA350, a Gram-negative and facultatively prey-dependent bacterium that belongs to a novel bacterial order Bradymonadales in the class Deltaproteobacteria. The kinase BsfK specifically catalyzes the phosphorylation of the precursor peptide BsfA on the Ser3 residue. BsfB1 performs dual functions to accelerate the post-translational phosphorylation and assist BsfB2 in leader peptide removal. Most importantly, the penultimate residue of leader peptide is an isoleucine rather than the conserved threonine and this isoleucine has a marked impact on the phosphorylation of Ser3 as well as leader peptide removal, implying that BsfB1 and BsfB2 exhibit a new substrate selectivity for leader peptide binding and excision. This is the first experimentally validated penultimate isoleucine residue in a lasso peptide precursor to our knowledge. In silico analysis reveals that the leader peptide Ile/Val(-2) residue is rare but not uncommon in phosphorylated lasso peptides, as this residue is also discovered in Acidobacteriaceae and Sphingomonadales in addition to Bradymonadales.

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Microcin J25 (MccJ25) and microcin Y (MccY) are lasso peptides and considered potential alternatives to antibiotics and harmful preservatives. The combination of these two microcins can provide a wide antimicrobial spectrum against food-borne Salmonella. Currently, MccJ25 and MccY are produced using Escherichia coli expression systems; however, the entire production process is accompanied by negative effects from endotoxins. In this study, we identified Bacillus subtilis as a suitable host for MccJ25 and MccY production. High-level production of microcins was achieved by promoter optimization, host strain selection, and recombinant expression. The engineered strains produced maximum yields of 2.827 µM MccJ25 and 1.481 µM MccY. This is the first study to demonstrate the expression of MccJ25 and MccY in B. subtilis, and it offers a few engineered strains that are without antibiotic resistance markers, inducer-free, sporulation-deficient, and free of the negative effects of endotoxins for antibacterial therapy and food preservation.

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Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that feature an isopeptide bond and a distinct lariat fold. A growing number of secondary modifications have been described that further decorate lasso peptide scaffolds. Using genome mining, we have discovered a pair of lasso peptide biosynthetic gene clusters (BGCs) that include cytochrome P450 genes. Here, we report the structural characterization of two unique examples of (C-N) biaryl-containing lasso peptides. Nocapeptin A, from Nocardia terpenica, is tailored with Trp-Tyr crosslink while longipepetin A, from Longimycelium tulufanense, features Trp-Trp linkage. Besides the unusual bicyclic frame, longipepetin A receives an S-methylation by a new Met methyltransferase resulting in unprecedented sulfonium-bearing RiPP. Our bioinformatic survey revealed P450(s) and further maturating enzyme(s)-containing lasso BGCs awaiting future characterization.

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Black band disease is a globally distributed and easily recognizable coral disease. Despite years of study, the etiology of this coral disease, which impacts dozens of stony coral species, is not completely understood. Although black band disease mats are predominantly composed of the cyanobacterial species Roseofilum reptotaenium, other filamentous cyanobacterial strains and bacterial heterotrophs are readily detected. Through chemical ecology and metagenomic sequencing, we uncovered cryptic strains of Roseofilum species from Siderastrea siderea corals that differ from those on other corals in the Caribbean and Pacific. Isolation of metabolites from Siderastrea-derived Roseofilum revealed the prevalence of unique forms of looekeyolides, distinct from previously characterized Roseofilum reptotaenium strains. In addition, comparative genomics of Roseofilum strains showed that only Siderastrea-based Roseofilum strains have the genetic capacity to produce lasso peptides, a family of compounds with diverse biological activity. All nine Roseofilum strains examined here shared the genetic capacity to produce looekeyolides and malyngamides, suggesting these compounds support the ecology of this genus. Similar biosynthetic gene clusters are not found in other cyanobacterial genera associated with black band disease, which may suggest that looekeyolides and malyngamides contribute to disease etiology through yet unknown mechanisms.

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Cancer metastasis, including cell invasion, is a major cause of poor clinical outcomes and death in numerous cancer patients. In recent years, many efforts have been made to develop potent therapeutic molecules from naturally derived peptides. Sungsanpin is a naturally derived lasso peptide that inhibits A549 cell invasion. We aimed to evaluate the potential of sungsanpin derivatives as candidates for anti-invasion drugs. We synthesized an analog of sungsanpin (Sun A) using a solid-phase peptide synthesis strategy (SPPS) and further modified its structure to improve its anti-invasion activity. All peptides were tested for their proliferative inhibition and anti-invasion activities in the A549 cell lines. Octapeptide S3 and cyclooctapeptide S4 upregulated the expression of TIMP-1 and TIMP-2 mRNA effectively and thus improved the inhibitory effect on the invasion of A549 cells. The two peptides can inhibit the invasion of A549 cells by up to 60 %, suggesting that they have potential as lead molecules for the development of peptide inhibitors.

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Microcin J25 (MccJ25), a lasso peptide, has a unique 3-D interlocked structure that provides high stability under acidic conditions, at high temperatures, and in the presence of proteases. In this study, we generated a positron emission tomography (PET) probe based on MccJ25 analog with an RGD motif and investigated their pharmacokinetics and utility for integrin αvß3 imaging in tumors. The MccJ25 variant with an RGD motif in the loop region and a lysine substitution at the C-terminus (MccJ25(RGDF)GtoK) was produced in E. coli transfected with plasmid DNA containing the MccJ25 biosynthetic gene cluster (mcjABCD). [64Cu]Cu-MccJ25(RGDF)GtoK was synthesized using the C-terminal lysine labeled with copper-64 (t1/2 = 12.7 h) via a bifunctional chelator; it showed stability in 90% mouse plasma for 45 min. Using PET imaging for integrin αvß3 positive U87MG tumor bearing mice, [64Cu]Cu-MccJ25(RGDF)GtoK could clearly distinguish the tumor, and its accumulation was significantly higher than that of MccJ25(GIGT)GtoK without the binding motif for integrin αvß3. Furthermore, MccJ25(RGDF)GtoK enabled visualization of only U87MG tumors but not MCF-7 tumors with low integrin αvß3 expression in double tumor-bearing mice. In ex vivo biodistribution analysis, the integrin αvß3 non-specific accumulation of [64Cu]Cu-MccJ25(RGDF)GtoK was significantly lower in various tissues, except for the kidneys, as compared to the control probe ([64Cu]Cu-cyclic RGD peptide). These results of the present study indicate that 64Cu-labeling methods are appropriate for the synthesis of MccJ25-based PET probes, and [64Cu]Cu-MccJ25 variants are useful tools for cancer molecular imaging.

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Using genome mining and heterologous expression, we report the discovery and production of a new antimicrobial lasso peptide from species related to the Enterobacter cloacae complex. Using NMR and mass spectrometric analysis, we show that this lasso peptide, named cloacaenodin, employs a threaded lasso fold which imparts proteolytic resistance that its unthreaded counterpart lacks. Cloacaenodin has selective, low micromolar, antimicrobial activity against species related to the E. cloacae complex, including species implicated in nosocomial infections and against clinical isolates of carbapenem-resistant Enterobacterales. We further used site-directed mutagenesis to probe the importance of specific residues to the peptide's biosynthesis, stability, and bioactivity.

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The strain designated as AN120528T was isolated from farmland soil in South Korea. This strain grows well on R2A medium at 28 °C. The cells are an off-white colour and have no hyphae. The phylogenetic analysis indicated that the strain is a member of the genus Shimazuella with a 98.11% similarity to Shimazuella alba KC615T and a 97.05% similarity to S. kribbensis KCTC 9933T, respectively. The strain AN120528T shares common chemotaxonomic features with the other two type strains in the genus. It has MK-9 (H4) and MK-10 (H4) as its predominant menaquinones. The major fatty acids are iso-C14:0, iso-C15:0, anteiso-C15:0 and iso-C16:0. Diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), lipids (L), and aminolipids (AL) were identified as the major cellular polar lipids. Analysis of the peptidoglycan showed the presence of meso-diaminopimelic acid. Whole-genome sequencing revealed that the genome of the strain is approximately 3.3 Mbp in size. The strain showed a 77.5% average nucleotide identity (ANI) with S. alba KC615T. The genomic DNA (gDNA) G + C content is 39.0%. Based on polyphasic taxonomy analysis, it is proposed that this strain, AN120528T, represents a novel species in the genus Shimazuella, designated as Shimazuella soli sp. nov. The type stain is AN120528T (=KCTC 39810T = DSM 103571T). Furthermore, shimazuellin I, a new 15-amino-acid peptide, was discovered in the AN120528T through genome mining; it has the features of a lasso peptide, containing eight amino acids (-G-Q-G-G-S-N-N-D-) that form a macrolactam ring and seven amino acids (-D-G-W-Y-H-S-K-) that form a tail.

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Salmonella bacteria pose a significant threat to animal husbandry and human health due to their virulence and multidrug resistance. The lasso peptide MccY is a recently discovered antimicrobial peptide that acts against various serotypes of Salmonella. In this study, we further explore the resistance mechanism and activity of MccY. Mutants of Ton system genes, including tonB, exbB, and exbD, in Salmonella enterica subsp. enterica serovar Typhimurium were constructed, and the MICs to MccY exhibited significant increases in these deletion mutants compared to the MIC of the parent strain. Subsequently, MccY resistance was quantitatively analyzed, and these mutants also showed greatly reduced rates of killing, even with a high concentration of MccY. In addition, a minimal medium with low iron environment enhanced the sensitivity of these mutants to MccY. Measurements of a series of physiological indicators, including iron utilization, biofilm formation, and motility, demonstrated that MccY may decrease the virulence of S. Typhimurium. Transcriptomic analysis showed that iron utilization, biofilm formation, flagellar assembly, and virulence-related genes were downregulated to varying degrees when S. Typhimurium was treated with MccY. In conclusion, deletion of Ton system genes resulted in resistance to MccY and the susceptibility of these mutants to MccY was increased and differed under a low-iron condition. This lasso peptide can alter multiple physiological properties of S. Typhimurium. Our study will contribute to improve the knowledge and understanding of the mechanism of MccY resistance in Salmonella strains. IMPORTANCE The resistance of Salmonella to traditional antibiotics remains a serious challenge. Novel anti-Salmonella drugs are urgently needed to address the looming crisis. The newly identified antimicrobial peptide MccY shows broad prospects for development and application because of its obvious antagonistic effect on various serotypes of Salmonella. However, our previous study showed that the peptide could confer resistance to Salmonella by disrupting the receptor gene fhuA. In this study, we further explored the potential resistance mechanism of MccY and demonstrated the importance of the Salmonella Ton complex for MccY transport. Disruption in Ton system genes resulted in S. Typhimurium resistance to this peptide, and MccY could alter multiple bacterial physiological properties. In summary, this study further explored the resistance mechanism and antibacterial effect of MccY in S. Typhimurium and provided a scientific basis for its development and application.

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Biochemical and structural analyses of purified proteins are essential for the understanding of their properties. However, many proteins are unstable and difficult to purify, hindering their characterization. The B2 proteins of the lasso peptide biosynthetic pathways are cysteine proteases that cleave precursor peptides during the maturation process. The B2 proteins are poorly soluble, and no experimentally solved structures are available. Here, we performed a rapid semicomprehensive mutational analysis of the B2 protein from the thermophilic actinobacterium, Thermobifida fusca (FusB2), using a cell-free transcription/translation system, and compared the results with the structure prediction by AlphaFold2. Analysis of 34 FusB2 mutants with substitutions of hydrophobic residues confirmed the accuracy of the predicted structure, and revealed a hydrophobic patch on the protein surface, which likely serves as the binding site of the partner protein, FusB1. Our results suggest that the combination of rapid cell-free mutant analyses with precise structure predictions can greatly accelerate structure-function research of proteins for which no structures are available.

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Genome mining is an important tool for discovery of new natural products; however, the number of publicly available genomes for natural product-rich microbes such as actinomycetes, relative to human pathogens with smaller genomes, is small. To obtain contiguous DNA assemblies and identify large (ca. 10 to greater than 100 kb) biosynthetic gene clusters (BGCs) with high GC (>70%) and high-repeat content, it is necessary to use long-read sequencing methods when sequencing actinomycete genomes. One of the hurdles to long-read sequencing is the higher cost. In the current study, we assessed Flongle, a recently launched platform by Oxford Nanopore Technologies, as a low-cost DNA sequencing option to obtain contiguous DNA assemblies and analyze BGCs. To make the workflow more cost-effective, we multiplexed up to four samples in a single Flongle sequencing experiment while expecting low-sequencing coverage per sample. We hypothesized that contiguous DNA assemblies might enable analysis of BGCs even at low sequencing depth. To assess the value of these assemblies, we collected high-resolution mass spectrometry data and conducted a multi-omics analysis to connect BGCs to secondary metabolites. In total, we assembled genomes for 20 distinct strains across seven sequencing experiments. In each experiment, 50% of the bases were in reads longer than 10 kb, which facilitated the assembly of reads into contigs with an average N50 value of 3.5 Mb. The programs antiSMASH and PRISM predicted 629 and 295 BGCs, respectively. We connected BGCs to metabolites for N,N-dimethyl cyclic-di-tryptophan, two novel lasso peptides, and three known actinomycete-associated siderophores, namely, mirubactin, heterobactin, and salinichelin. IMPORTANCE Short-read sequencing of GC-rich genomes such as those from actinomycetes results in a fragmented genome assembly and truncated biosynthetic gene clusters (often 10 to >100 kb long), which hinders our ability to understand the biosynthetic potential of a given strain and predict the molecules that can be produced. The current study demonstrates that contiguous DNA assemblies, suitable for analysis of BGCs, can be obtained through low-coverage, multiplexed sequencing on Flongle, which provides a new low-cost workflow ($30 to 40 per strain) for sequencing actinomycete strain libraries.

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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of natural products that exhibit a range of structures and bioactivities. Initially assembled from the twenty proteinogenic amino acids in a ribosome-dependent manner, RiPPs assume their peculiar bioactive structures through various post-translational modifications. The essential modifications representative of each subfamily of RiPP are performed on a precursor peptide by the so-called processing enzymes; however, various tailoring enzymes can also embellish the precursor peptide or processed peptide with additional functional groups. Lasso peptides are an interesting subfamily of RiPPs characterized by their unique lariat knot-like structure, wherein the C-terminal tail is inserted through a macrolactam ring fused by an isopeptide bond between the N-terminal amino group and an acidic side chain. Until recently, relatively few lasso peptides were found to be tailored with extra functional groups. Nevertheless, the development of new routes to diversify lasso peptides and thus introduce novel or enhanced biological, medicinally relevant, or catalytic properties is appealing. In this review, we highlight several strategies through which lasso peptides have been successfully modified and provide a brief overview of the latest findings on the tailoring of these peptides. We also propose future directions for lasso peptide tailoring as well as potential applications for these peptides in hybrid catalyst design.

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Actinobacteria is a phylum composed of aerobic, Gram-positive, and filamentous bacteria with a broad spectrum of biological activity, including antioxidant, antitumor, and antibiotic. The crude extract of Streptomyces griseocarneus R132 was fractionated on a C18 silica column and the isolated compound was identified by 1H and 13C nuclear magnetic resonance as 3-(phenylprop-2-enoic acid), also known as trans-cinnamic acid. Antimicrobial activity against human pathogens was assayed in vitro (disk-diffusion qualitative test) and in vivo using Galleria mellonella larvae (RT-qPCR). The methanol fractions 132-F30%, 132-F50%, 132-F70%, and 132-F100% inhibited the Escherichia coli (ATCC 25922) and Staphylococcus aureus (MRSA) growth in vitro the most effectively. Compared with the untreated control (60-80% of larvae death), the fractions and isolated trans-cinnamic acid increased the survival rate and modulated the immune system of G. mellonella larvae infected with pathogenic microorganisms. The anti-infection effect of the S. griseocarneus R132 fermentation product led us to sequence its genome, which was assembled and annotated using the Rast and antiSMASH platforms. The assembled genome consisted of 227 scaffolds represented on a linear chromosome of 8.85 Mb and 71.3% of GC. We detected conserved domains typical of enzymes that produce molecules with biological activity, such as polyketides and non-ribosomal and ribosomal peptides, indicating a great potential for obtaining new antibiotics and molecules with biotechnological application. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02942-1.

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Lasso peptides, a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) secreted by bacteria, have antimicrobial activity. Here, a novel lasso peptide, microcin Y (MccY), was discovered and characterized. The gene cluster for MccY synthesis was cloned for expression in Escherichia coli. This peptide was purified by HPLC and characterized by Q-TOF. MIC assays showed that some Bacillus, Staphylococcus, Pseudomonas, Shigella, and Salmonella strains were sensitive to MccY. Interestingly, Salmonellatyphimurium and Salmonella infantis were efficiently inhibited by MccY, while they were not affected by MccJ25, a lasso peptide that has antibacterial effects on many Salmonella strains. Furthermore, MccY-resistant strains of S. typhimurium were screened, and mutations were found in FhuA and SbmA, indicating the importance of these transporters for MccY absorption. This novel peptide can greatly broaden the antimicrobial spectrum of MccJ25 in Salmonella and is expected to be used in food preservation and animal feed additive areas.

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