Cell-free biosynthesis and engineering of ribosomally synthesized lanthipeptides

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Май 21, 2024

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with diverse chemical structures potent biological activities. A vast majority RiPP gene clusters remain unexplored in microbial genomes, which is partially due to the lack rapid efficient heterologous expression systems for characterization biosynthesis. Here, we report unified biocatalysis (UniBioCat) system based on cell-free biosynthesis engineering RiPPs. We demonstrate UniBioCat by reconstituting full biosynthetic pathway de novo salivaricin B, lanthipeptide RiPP. Next, delete several protease/peptidase genes from source strain enhance performance UniBioCat, then can synthesize screen B variants enhanced antimicrobial activity. Finally, show that generalizable synthesizing evaluating bioactivity ten uncharacterized lanthipeptides. expect accelerate discovery, characterization, synthesis

Язык: Английский

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Activity of Gut-Derived Nisin-like Lantibiotics against Human Gut Pathogens and Commensals

ACS Chemical Biology, Год журнала: 2024, Номер 19(2), С. 357 - 369

Опубликована: Янв. 31, 2024

Recent advances in sequencing techniques unveiled the vast potential of ribosomally synthesized and post-translationally modified peptides (RiPPs) encoded microbiomes. Class I lantibiotics such as nisin A, widely used a food preservative, have been investigated for their efficacy killing pathogens. However, impact nisin-like class on commensal bacteria residing human gut remains unclear. Here, we report six gut-derived that are close homologues nisin, four which novel. We applied an improved lantibiotic expression platform to produce purify these antimicrobial assays. determined minimal inhibitory concentration (MIC) against both Gram-positive pathogens commensals profiled resistance genes commensals. Structure–activity relationship (SAR) studies with analogs revealed key regions residues properties. Our characterization SAR could shed light future development lantibiotic-based therapeutics preservatives.

Язык: Английский

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Nisin-relevant antimicrobial peptides: synthesis strategies and applications

Food & Function, Год журнала: 2024, Номер unknown

Опубликована: Янв. 1, 2024

Small pentacyclic peptides, represented by nisin, have been successfully utilized as preservatives in the food industry and evolved into a paradigm for understanding genetic structure, expression, control of genes created lantibiotics. Due to ever-increasing antibiotic resistance, nisin-relevant antimicrobial peptides received much attention, which calls summarization their synthesis, modification applications. In this review, we first provided timeline select highlights nisin biosynthesis engineering. Then, outlined current developments synthesis. We also an overview engineering, screening, production based on enzyme alteration, substrate modification, sequence mining. Furthermore, updated summary applications has developed Finally, study offers insights emerging technologies, limitations future development pathogen inhibition, preservatives, improved health.

Язык: Английский

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The nisin O cluster: species dissemination, candidate leader peptide proteases and the role of regulatory systems

Microbiology, Год журнала: 2025, Номер 171(2)

Опубликована: Фев. 10, 2025

Nisin O is an antimicrobial peptide encoded by the human gut bacterium Blautia obeum A2-162 which has activity against clinically relevant organisms. The nisin biosynthetic gene cluster (BGC) varies from other BGCs as it lacks a leader-peptide cleaving protease and contains two bacterial two-component response regulator–histidine kinase (RK) systems. dissemination of cluster, final proteolytic biosynthesis step regulation are currently unknown foci this study. We identified six O-like across , Dorea Ruminococcus species using comparative genomics. These show evidence genetic transfer between genera, with genes involved in transposition discovered up- downstream BGCs. All contained RK systems but no protease. Mining B. genome candidate proteases that were cloned used pre-nisin leader cleavage assays. None removed leader; however, was achieved trypsin. To maximize expression nsoA1-4 peptides, interactions predicted promoters assessed PepI reporter assay. observed P nsoR2K2 promoter constitutively expressed, NsoR1K1 increasing its activity, there increased when A system present. Long-read cDNA sequencing confirmed nso transcription heterologous novel, highly expressed gene. This study provides BGC been transferred different gut-associated all clusters lacking containing hypothesize lost due to negative selection result high trypsin concentrations gut. Further work required for be potential therapy.

Язык: Английский

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Discovery, biosynthesis, and characterization of a lanthipeptide from Bacillus subtilis EH11 with a unique lanthionine ring pattern

Cell Reports Physical Science, Год журнала: 2023, Номер 4(8), С. 101524 - 101524

Опубликована: Авг. 1, 2023

Lanthipeptide biosynthetic genes are present in a wide range of bacteria, providing convenient tools for engineering bioactive lanthipeptides. Here, we report class I lanthipeptide gene cluster (lanBTC) Bacillus strain, involved the biosynthesis novel that termed balucin. Balucin was active against food-borne pathogens such as cereus and Listeria monocytogenes. The unusual structural features balucin showed presence aspartic acid residues C-terminally flanking lanthionine rings. Such negatively charged amino acids directly modifiable cysteines at C-terminal side have not been reported precursors modified by other LanBCs. Heterologous functional expression dedicated E. coli system achieved, protease responsible cleaving leader peptide identified. With this system, enzymes were successfully employed to modify antimicrobial peptides lanthionine-forming cysteines.

Язык: Английский

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Investigation into the mechanism of action of the antimicrobial peptide epilancin 15X

Frontiers in Microbiology, Год журнала: 2023, Номер 14

Опубликована: Ноя. 2, 2023

Addressing the current antibiotic-resistance challenge would be aided by identification of compounds with novel mechanisms action. Epilancin 15X, a lantibiotic produced Staphylococcus epidermidis 15 × 154, displays antimicrobial activity in submicromolar range against subset pathogenic Gram-positive bacteria. S. is common member human skin or mucosal microbiota. We here investigated mechanism action epilancin 15X. The compound bactericidal carnosus as well Bacillus subtilis and appears to kill these bacteria membrane disruption. Structure–activity relationship studies using engineered analogs show that its conserved positively charged residues dehydroamino acids are important for bioactivity, but N-terminal lactyl group tolerant changes. 15X treatment negatively affects fatty acid synthesis, RNA translation, DNA replication transcription without affecting cell wall biosynthesis. localized surface most potent disrupting membranes liposomes composed lipids lipid II independent manner. does not elicit LiaRS response B. did upregulate VraRS . Treatment resulted an aggregation phenotype microscopy experiments. Collectively provide new information on activity.

Язык: Английский

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Mechanistic insights into lanthipeptide modification by a distinct subclass of LanKC enzyme that forms dimers

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Авг. 17, 2024

Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the events preceding peptide modification remain poorly understood. Here, we identify a distinct subclass of lanthionine synthetase KC (LanKC) enzymes with functional characteristics. We show that PneKC, member this subclass, forms dimer possesses GTPase activity. Through three cryo-EM structures illustrate different stages PneA binding, from initial recognition to full binding. Our kinase domain complexed core GTPγS, phosphate-bound lyase domain, an unconventional cyclase domain. The leader interact gate loop, transitioning extended helical conformation. dimerization hot spot propose "negative cooperativity" mechanism toggling enzyme between tense relaxed Additionally, important salt bridge in differing those conventional domains. These residues are highly conserved LanKC part two signature motifs. results unveil potential differences lanthipeptide assembly deepen our understanding allostery these multifunctional enzymes.

Язык: Английский

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Heterologous Expression Facilitates the Production and Characterization of a Class III Lanthipeptide with Coupled Labionin Cross-Links in Sponge-Associated Streptomyces rochei MB037

ACS Chemical Biology, Год журнала: 2024, Номер 19(9), С. 2060 - 2069

Опубликована: Авг. 15, 2024

Cyclic peptides, with remarkable stability, cellular permeability, and proteolysis resistance, display promising potential in pharmaceutical applications. Labionin (Lab), a unique bicyclic cross-link containing both C–C C–S bonds, provides high rigidity better control of conformation compared to monocyclic cross-links. To discover more Lab-containing scaffolds highly rigid for cyclic peptide drug development, herein, cryptic class III lanthipeptide biosynthetic gene cluster (BGC) (i.e., rcs) was identified the sponge-associated Streptomyces rochei MB037 expressed Escherichia coli, incorporating an N-terminal SUMO-tag on RcsA precursor prevent proteolysis. Subsequently, novel lanthipeptide, i.e., rochsin A, exhibiting coupled Lab cross-links crowded by bulky aromatic amino acids, produced. Three AplP-like proteases outside rcs BGC were proven remove leader A through their dual endo- aminopeptidase activities, resulting mature vitro. Ala mutation experiments revealed C N cyclization direction, like most lanthipeptides. However, RcsKC displays substrate breadth, enabling various ring topologies that are rarely observed other Overall, established expression system broadens chemical diversity peptides offers scaffold development.

Язык: Английский

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Structure-Activity Relationship Studies of Novel Gut-derived Lantibiotics Against Human Gut Commensals

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Сен. 17, 2023

Abstract Recent advances in sequencing techniques unveiled the vast potential of ribosomally synthesized and post-translationally modified peptides (RiPPs) encoded microbiomes. Class I lantibiotics such as nisin A, widely used a food preservative, have been investigated for their efficacy killing pathogens. However, impact nisin-like class on commensal bacteria residing human gut remains unclear. Here, we report six gut-derived that are close homologs nisin, four which novel. We applied an improved lantibiotic expression platform to produce purify these antimicrobial assays. determined minimal inhibitory concentration (MIC) against both Gram-positive pathogens commensals, profiled resistance genes commensals. SAR studies with variants revealed key regions residues properties. Our characterization commensals could shed light future development lantibiotic-based therapeutics preservatives.

Язык: Английский

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Exposure and resistance to lantibiotics impact microbiota composition and function

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Дек. 30, 2023

The intestinal microbiota is composed of hundreds distinct microbial species that interact with each other and their mammalian host. Antibiotic exposure dramatically impacts compositions leads to acquisition antibiotic-resistance genes. Lantibiotics are ribosomally synthesized post-translationally modified peptides produced by some bacterial strains inhibit the growth competing bacteria. Nisin A a lantibiotic Lactococcus lactis commonly added food products reduce contamination Gram-positive pathogens. Little known, however, about lantibiotic-resistance commensal bacteria inhabiting human intestine. Herein, we demonstrate administration mice alters fecal microbiome concentration taurine-conjugated primary bile acids. Lantibiotic Resistance System genes (LRS) encoded lantibiotic-producing but, show, also prevalent in microbiomes across cohorts spanning vastly different lifestyles 5 continents. Bacterial encoding LRS have enhanced vivo fitness upon dietary but reduced absence pressure. Differential binding host derived, secreted IgA contributes discordance between or lacking LRS. Although associated mobile genetic elements, sequence comparisons suggest they been long-term components respective genomes. Our study reveals prevalence, abundance physiologic significance an underappreciated subset antimicrobial resistance constituting gut microbiome, provides insights will guide development augmenting strategies.

Язык: Английский

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