Isolation of aflatoxin biosynthetic inhibitor from Chondrostereum purpureum mushroom culture filtrate.

Aflatoxins (AFs) are highly toxic mycotoxins produced by the fungi, Aspergillus flavus and Aspergillus parasiticus. AFs pose severe health risks owing to their acute toxicity and carcinogenic properties. The control of AF contamination remains significantly challenging despite the extensive efforts toward controlling it. Here, we investigated the potential of mushroom extracts as a source of AF biosynthetic inhibitors. The A. parasiticus mutant strain, NFRI-95, that accumulates an AF biosynthesis intermediate, norsolorinic acid, was used in the bioassay to detect the inhibitory activity against AF biosynthesis. The screening of 195 mushroom extracts revealed that the culture filtrate extract of Chondrostereum purpureum exhibited strong inhibitory activity against AF biosynthesis. Next, large-scale culturing of C. purpureum was performed to isolate the compounds accounting for the inhibitory activity. The culture filtrate was extracted with ethyl acetate, after which the active compound was isolated by silica gel column chromatography and preparative high performance liquid chromatography (HPLC). The active compound was identified as cyclo(Val-Pro) by spectroscopic analyses. Further, four stereoisomers of cyclo(Val-Pro) were synthesized by the condensation of the N-Boc derivatives of d- and l-valine with the methyl esters of d- and l-proline. The naturally isolated compound was identified as cyclo(l-Val-l-Pro) by comparing its retention time with those of synthetic compounds by chiral HPLC analysis and CD spectra. The IC50 value of cyclo(L-Val-L-Pro) was 2.4 mM, whereas the LD, DL, and DD isomers exhibited weaker activities, with IC50 values of >5 mM.

Cyclic Peptides from Graspetide Biosynthesis and Native Chemical Ligation.

The ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily of natural products includes many examples of cyclic peptides with diverse macrocyclization chemistries. The graspetides, one family of macrocyclized RiPPs, harbor side chain-side chain ester or amide linkages. We recently reported the structure and biosynthesis of the graspetide pre-fuscimiditide, a 22-amino-acid (aa) peptide with two ester cross-links forming a stem-loop structure. These cross-links are introduced by a single graspetide synthetase, the ATP-grasp enzyme ThfB. Here we show that ThfB can also catalyze the formation of amide or thioester cross-links in prefuscimiditide, with thioester formation being especially efficient. We further show that upon proteolysis to reveal an N-terminal cysteine residue, the thioester-linked peptide rapidly and quantitatively rearranges via native chemical ligation into an isopeptide-bonded head-to-tail cyclic peptide. The solution structure of this rearranged peptide was determined by using 2D NMR spectroscopy experiments. Our methodology offers a straightforward recombinant route to head-to-tail cyclic peptides.

Scalable and Efficient In Planta Biosynthesis of Sunflower Trypsin Inhibitor-1 (SFTI) Peptide Therapeutics.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide which has been effectively employed as a scaffold for engineering a range of peptide therapeutic candidates. Typically, synthesis of SFTI-1-based therapeutics is performed via solid-phase peptide synthesis and native chemical ligation, with significant financial and environmental costs associated. In planta synthesis of SFTI-1 based therapeutics serves as a greener approach for environmentally sustainable production. Here, we detail the methods for the transient expression, production, and purification of SFTI-1-based therapeutic peptides in Nicotiana benthamiana using a scalable and high-throughput approach. We demonstrate that a prerequisite for this is the co-expression of specialized asparaginyl endopeptidases (AEPs) that perform the backbone cyclization of SFTI-1. In our founding study, we were able to achieve in planta yields of a plasmin inhibitor SFTI-1 peptide at yields of ~60 µg/g of dried plant material.

Discovery and biosynthesis of cyclic plant peptides via autocatalytic cyclases.

Many bioactive plant cyclic peptides form side-chain-derived macrocycles. Lyciumins, cyclic plant peptides with tryptophan macrocyclizations, are ribosomal peptides (RiPPs) originating from repetitive core peptide motifs in precursor peptides with plant-specific BURP (BNM2, USP, RD22 and PG1beta) domains, but the biosynthetic mechanism for their formation has remained unknown. Here, we characterize precursor-peptide BURP domains as copper-dependent autocatalytic peptide cyclases and use a combination of tandem mass spectrometry-based metabolomics and plant genomics to systematically discover five BURP-domain-derived plant RiPP classes, with mono- and bicyclic structures formed via tryptophans and tyrosines, from botanical collections. As BURP-domain cyclases are scaffold-generating enzymes in plant specialized metabolism that are physically connected to their substrates in the same polypeptide, we introduce a bioinformatic method to mine plant genomes for precursor-peptide-encoding genes by detection of repetitive substrate domains and known core peptide features. Our study sets the stage for chemical, biosynthetic and biological exploration of plant RiPP natural products from BURP-domain cyclases.

Influence of Culture Substrates on Morphology and Function of Pulmonary Alveolar Cells In Vitro.

Cell's microenvironment has been shown to exert influence on cell behavior. In particular, matrix-cell interactions strongly impact cell morphology and function. The purpose of this study was to analyze the influence of different culture substrate materials on phenotype and functional properties of lung epithelial adenocarcinoma (A549) cells. A549 cells were seeded onto two different biocompatible, commercially available substrates: a polyester coverslip (Thermanox™ Coverslips), that was used as cell culture plate control, and a polydimethylsiloxane membrane (PDMS, Elastosil® Film) investigated in this study as alternative material for A549 cells culture. The two substrates influenced cell morphology and the actin cytoskeleton organization. Further, the Yes-associated protein (YAP) and its transcriptional coactivator PDZ-binding motif (TAZ) were translocated to the nucleus in A549 cells cultured on polyester substrate, yet it remained mostly cytosolic in cells on PDMS substrate. By SEM analysis, we observed that cells grown on Elastosil® Film maintained an alveolar Type II cell morphology. Immunofluorescence staining for surfactant-C revealing a high expression of surfactant-C in cells cultured on Elastosil® Film, but not in cells cultured on Thermanox™ Coverslips. A549 cells grown onto Elastosil® Film exhibited morphology and functionality that suggest retainment of alveolar epithelial Type II phenotype, while A549 cells grown onto conventional plastic substrates acquired an alveolar Type I phenotype.

Activation, Structure, Biosynthesis and Bioactivity of Glidobactin-like Proteasome Inhibitors from Photorhabdus laumondii.

The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881-1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.

Engineering of a genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production.

BACKGROUND: Genome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Surfactin, a lipopeptide-type biosurfactant with broad spectrum antibiotic activity, has wide application prospects in anticancer therapy, biocontrol and bioremediation. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis. RESULTS: In this study, a genome-reduced strain GR167 lacking ~ 4.18% of the B. amyloliquefaciens LL3 genome was constructed by deleting some unnecessary genomic regions. Compared with the strain NK-1 (LL3 derivative, ΔuppΔpMC1), GR167 exhibited faster growth rate, higher transformation efficiency, increased intracellular reducing power level and higher heterologous protein expression capacity. Furthermore, the chassis strain GR167 was engineered for enhanced surfactin production. Firstly, the iturin and fengycin biosynthetic gene clusters were deleted from GR167 to generate GR167ID. Subsequently, two promoters PRsuc and PRtpxi from LL3 were obtained by RNA-seq and promoter strength characterization, and then they were individually substituted for the native srfA promoter in GR167ID to generate GR167IDS and GR167IDT. The best mutant GR167IDS showed a 678-fold improvement in the transcriptional level of the srfA operon relative to GR167ID, and it produced 311.35 mg/L surfactin, with a 10.4-fold increase relative to GR167. CONCLUSIONS: The genome-reduced strain GR167 was advantageous over the parental strain in several industrially relevant physiological traits assessed and it was highlighted as a chassis strain for further genetic modification. In future studies, further reduction of the LL3 genome can be expected to create high-performance chassis for synthetic biology applications.

Genomic insights into a plant growth-promoting Pseudomonas koreensis strain with cyclic lipopeptide-mediated antifungal activity.

Strain S150 was isolated from the tobacco rhizosphere as a plant growth-promoting rhizobacterium. It increased plant fresh weight significantly and lateral root development, and it antagonized plant pathogenic fungi but not phytobacteria. Further tests showed that strain S150 solubilized organic phosphate and produced ammonia, siderophore, protease, amylase, and cellulase, but it did not produce indole-3-acetic acid. Using morphology, physiological characteristics, and multi-locus sequence analysis, strain S150 was identified as Pseudomonas koreensis. The complete genome of strain S150 was sequenced, and it showed a single circular chromosome of 6,304,843 bp with a 61.09% G + C content. The bacterial genome contained 5,454 predicted genes that occupied 87.7% of the genome. Venn diagrams of the identified orthologous clusters of P. koreensis S150 with the other three sequenced P. koreensis strains revealed up to 4,167 homologous gene clusters that were shared among them, and 21 orthologous clusters were only present in the genome of strain S150. Genome mining of the bacterium P. koreensis S150 showed that the strain possessed 10 biosynthetic gene clusters for secondary metabolites, which included four clusters of non-ribosomal peptide synthetases (NRPSs) involved in the biosynthesis of cyclic lipopeptides (CLPs). One of the NRPSs possibly encoded lokisin, a cyclic lipopeptide produced by fluorescent Pseudomonas. Genomic mutation of the lokA gene, which is one of the three structural NRPS genes for lokisin in strain S150, led to a deficiency in fungal antagonism that could be restored fully by gene complementation. The results suggested that P. koreensis S150 is a novel plant growth-promoting agent with specific cyclic lipopeptides and contains a lokisin-encoding gene cluster that is dominant against plant fungal pathogens.

Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression.

Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH-VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.

Different Reaction Specificities of F420H2-Dependent Reductases Facilitate Pyrrolobenzodiazepines and Lincomycin To Fit Their Biological Targets.

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of l-tyrosine- or l-leucine-derived 4-alkyl-l-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, our bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated l-proline-like precursors and likely in the microbial central metabolism.

Potential Probiotics Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 Co-Aggregate with Clinical Isolates of Proteus mirabilis and Prevent Biofilm Formation.

A urinary tract infection (UTI) is a multi-factorial disease including cystitis, pyelonephritis, and pyelitis. After Escherichia coli, Proteus mirabilis is the most common UTI-associated opportunistic pathogen. Antibiotic resistance of bacteria and infection recurrence can be connected to biofilm formation by P. mirabilis. In this study, human and sheep isolates of P. mirabilis were investigated for antibiotic sensitivity using an antibiotic disk test. Co-aggregation of the tested potential probiotic bacilli, Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933, with the isolated pathogen was also evaluated. Then, the anti-biofilm activity of naturally derived metabolites, such as subtilin and subtilosin, in the bacilli-free supernatants was assessed against biofilms of P. mirabilis isolates. The isolated pathogens were sensitive to 30 µg of amikacin and 5 µg of ciprofloxacin but resistant to other tested antibiotics. After 24 h, auto-aggregation of B. amyloliquefaciens B-1895 was at 89.5% and higher than auto-aggregation of B. subtilis KATMIRA1933 (59.5%). B. amyloliquefaciens B-1895 strongly co-aggregated with P. mirabilis isolates from human UTIs. Cell-free supernatants of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 showed higher antimicrobial activity against biofilms of P. mirabilis isolated from humans as compared with biofilms of sheep isolates. According to our knowledge, this is the first report evaluating the anti-biofilm activity of probiotic spore-forming bacilli against clinical and animal UTI isolates of P. mirabilis. Further studies are recommended to investigate the anti-biofilm activity and the mode of action for the antimicrobial substances produced by these bacilli, subtilosin and subtilin.

A minimalistic hydrolase based on co-assembled cyclic dipeptides.

The self-assembly of small peptides into larger aggregates is an important process for the fundamental understanding of abiogenesis. In this article we demonstrate that blends of cyclic dipeptides (2,5-diketopiperazines - DKPs) bearing either histidine or cysteine in combination with a lipophilic amino acid form highly stable aggregates in aqueous solution with esterase-like activity. We demonstrate that the catalytic activity is based on an intermolecular cooperative behavior between histidine and cysteine. A high control of the molecular arrangement of the peptide assemblies was gained by C-H-π interactions between Phe and Leu or Val sidechains, resulting in a significant increase in catalytic activity. These interactions were strongly supported by Hartree-Fock calculations and finally confirmed via1H-NMR HRMAS NOE spectroscopy.

A comprehensive genomic and growth proteomic analysis of antitumor lipopeptide bacillomycin Lb biosynthesis in Bacillus amyloliquefaciens X030.

Lipopeptides (such as iturin, fengycin, and surfactin) from Bacillus possess antibacterial, antifungal, and antiviral activities and have important application in agriculture and pharmaceuticals. Although unremitting efforts have been devoted to improve lipopeptide production by designing gene regulatory circuits or optimizing fermentation process, little attention has been paid to utilizing multi-omics for systematically mining core genes and proteins during the bacterial growth cycle. Here, lipopeptide bacillomycin Lb from new Bacillus amyloliquefaciens X030 was isolated and first found to have anticancer activity in various cancer cells (such as SMMC-7721 and MDA-MB-231). A comprehensive genomic and growth proteomic analysis of X030 revealed bacillomycin Lb biosynthetic gene cluster, key enzymes and potential regulatory proteins (PerR, PhoP, CcpA, and CsfB), and novel links between primary metabolism and bacillomycin Lb production in X030. The antitumor activity of the fermentation supernatant supplemented with amino acids (such as glutamic acid) and sucrose was significantly increased, verifying the role of key metabolic switches in the metabolic regulatory network. Quantitative real-time PCR analysis confirmed that 7 differential expressed genes exhibited a positive correlation between changes at transcriptional and translational levels. The study not only will stimulate the deeper and wider antitumor study of lipopeptides but also provide a comprehensive database, which promotes an in-depth analysis of pathways and networks for complex events in lipopeptide biosynthesis and regulation and gives great help in improving the yield of bacillomycin Lb (media optimization, genetic modification, or pathway engineering).

Astin C Production by the Endophytic Fungus Cyanodermella asteris in Planktonic and Immobilized Culture Conditions.

The fungal endophyte Cyanodermella asteris (C. asteris) has been recently isolated from the medicinal plant Aster tataricus (A. tataricus). This fungus produces astin C, a cyclic pentapeptide with anticancer and anti-inflammatory properties. The production of this secondary metabolite is compared in immobilized and planktonic conditions. For immobilized cultures, a stainless steel packing immersed in the culture broth is used as a support. In these conditions, the fungus exclusively grows on the packing, which provides a considerable advantage for astin C recovery and purification. C. asteris metabolism is different according to the culture conditions in terms of substrate consumption rate, cell growth, and astin C production. Immobilized-cell cultures yield a 30% increase of astin C production, associated with a 39% increase in biomass. The inoculum type as spores rather than hyphae, and a pre-inoculation washing procedure with sodium hydroxide, turns out to be beneficial both for astin C production and fungus development onto the support. Finally, the influence of culture parameters such as pH and medium composition on astin C production is evaluated. With optimized culture conditions, astin C yield is further improved reaching a five times higher final specific yield compared to the value reported with astin C extraction from A. tataricus (0.89 mg g-1 and 0.16 mg g-1 respectively).

Characterization of the FMN-Dependent Cysteine Decarboxylase from Thioviridamide Biosynthesis.

The biosynthesis of thioviridamide-like compounds has not been elucidated. Herein, we report that TvaF from the thioviridamide biosynthetic gene cluster is an FMN-dependent cysteine decarboxylase that transforms the C-terminal cysteine of precursor peptides into a thioenol motif and exhibits high substrate flexibility. We resolved the crystal structure of TvaF bound with FMN at 2.24 Å resolution. Key residues for FMN binding and catalytic activity of TvaF have been identified and evaluated by mutagenesis studies.

Distinct Autocatalytic α- N-Methylating Precursors Expand the Borosin RiPP Family of Peptide Natural Products.

Backbone N-methylations impart several favorable characteristics to peptides including increased proteolytic stability and membrane permeability. Nonetheless, amide bond N-methylations incorporated as post-translational modifications are scarce in nature and were first demonstrated in 2017 for a single set of fungal metabolites. Here we expand on our previous discovery of iterative, autocatalytic α- N-methylating precursor proteins in the borosin family of ribosomally encoded peptide natural products. We identify over 50 putative pathways in a variety of ascomycete and basidiomycete fungi and functionally validate nearly a dozen new self-α- N-methylating catalysts. Significant differences in precursor size, architecture, and core peptide properties subdivide this new peptide family into three discrete structural types. Lastly, using targeted genomics, we link the biosynthetic origins of the potent antineoplastic gymnopeptides to the borosin natural product family. This work highlights the metabolic potential of fungi for ribosomally synthesized peptide natural products.

Role of Lipid Composition, Physicochemical Interactions, and Membrane Mechanics in the Molecular Actions of Microbial Cyclic Lipopeptides.

Several experimental and theoretical studies have extensively investigated the effects of a large diversity of antimicrobial peptides (AMPs) on model lipid bilayers and living cells. Many of these peptides disturb cells by forming pores in the plasma membrane that eventually lead to the cell death. The complexity of these peptide-lipid interactions is mainly related to electrostatic, hydrophobic and topological issues of these counterparts. Diverse studies have shed some light on how AMPs act on lipid bilayers composed by different phospholipids, and how mechanical properties of membranes could affect the antimicrobial effects of such compounds. On the other hand, cyclic lipopeptides (cLPs), an important class of microbial secondary metabolites, have received comparatively less attention. Due to their amphipathic structures, cLPs exhibit interesting biological activities including interactions with biofilms, anti-bacterial, anti-fungal, antiviral, and anti-tumoral properties, which deserve more investigation. Understanding how physicochemical properties of lipid bilayers contribute and determining the antagonistic activity of these secondary metabolites over a broad spectrum of microbial pathogens could establish a framework to design and select effective strategies of biological control. This implies unravelling-at the biophysical level-the complex interactions established between cLPs and lipid bilayers. This review presents, in a systematic manner, the diversity of lipidated antibiotics produced by different microorganisms, with a critical analysis of the perturbing actions that have been reported in the literature for this specific set of membrane-active lipopeptides during their interactions with model membranes and in vivo. With an overview on the mechanical properties of lipid bilayers that can be experimentally determined, we also discuss which parameters are relevant in the understanding of those perturbation effects. Finally, we expose in brief, how this knowledge can help to design novel strategies to use these biosurfactants in the agronomic and pharmaceutical industries.

Piperazic acid-containing natural products: structures and biosynthesis.

Covering: up to the end of 2018 Piperazic acid is a cyclic hydrazine and a non-proteinogenic amino acid found in diverse non-ribosomal peptide (NRP) and hybrid NRP-polyketide (PK) structures. Piperazic acid was first identified as a residue in the monamycins in 1959. Since then, the piperazic acid residue has been found in >30 families of natural products, representing >140 compounds. Many of these compounds have potent biological activity, ranging from anti-malarial to anti-apoptotic to anti-bacterial activity, although high toxicity often accompanies this potent biological activity. Recently, we identified a piperazate synthase, responsible for N-N bond formation to give piperazic acid. Here, we review piperazic acid-containing natural products discovered from 1959 to 2018, with an emphasis on the biosynthetic routes to these natural products.

Biosynthesis and Heterologous Production of Argyrins.

Argyrins represent a family of cyclic octapeptides exhibiting promising antimicrobial, antitumorigenic and immunosuppressant activities. They derive from a nonribosomal peptide synthetase pathway, which was identified and characterized in this study from the myxobacterial producer strain Cystobacter sp. SBCb004. Using the native biosynthetic gene cluster (BGC) sequence as template synthetic BGC versions were designed and assembled from gene synthesis fragments. A heterologous expression system was established after chromosomal deletion of a well-expressed lipopeptide pathway from the host strain Myxococcus xanthus DK1622. Different approaches were applied to engineer and improve heterologous argyrin production, which was finally increased to 160 mg/L, around 20-fold higher yields compared to the native producer. Heterologous production platform also led to identification of several novel argyrin derivatives (A2, F3, G3, I, J, K, and L). The optimized production system provides a versatile platform for future supply of argyrins and novel derivatives thereof.

The role of protein-protein interactions in the biosynthesis of ribosomally synthesized and post-translationally modified peptides.

Covering: up to 02/2019 This review covers the role of protein-protein complexes in the biosynthesis of selected ribosomally synthesized and post-translationally modified peptide (RiPP) classes. The genomic organization of RiPP systems usually allows the expression of each biosynthetic enzyme as an individual unit, which is in stark contrast to the giant assembly lines found in non-ribosomal peptide and polyketide synthesis systems. Evidence is mounting however that the formation of multi-enzyme complexes is critical for efficient RiPPs biosynthesis and that these complexes may be involved in substrate channeling or conformational sampling. In some pathways, polyfunctional enzymes have evolved, which can be viewed as perpetual protein complexes. We summarize what is currently known on enzyme complexes in RiPP systems for lasso peptides, cyanobactins, linear azolic peptides, thiopeptides, and lanthipeptides.