Microviridins are ribosomally synthesized tricyclic depsipeptides produced by different genera of cyanobacteria. The prevalence of the microviridin gene clusters and the natural diversity of microviridin precursor sequences are currently unknown. Screening of laboratory strains and field samples of the bloom-forming freshwater cyanobacterium Microcystis via PCR revealed global occurrence of the microviridin pathway and an unexpected natural variety. We could detect 15 new variants of the precursor gene mdnA encoding microviridin backbones that differ in up to 4 amino acid positions from known isoforms of the peptide. The survey not only provides insights into the versatility of the biosynthetic enzymes in a closely related group of cyanobacteria, but also facilitates the discovery and characterization of cryptic microviridin variants. This is demonstrated for microviridin L in Microcystis aeruginosa strain NIES843 and heterologously produced variants.Bloom-forming freshwater cyanobacteria are a rich source of natural products (30). They flourish in lakes and ponds of different temperate zones, where they can eventually form thick scums at the surface. Microcystis is one of the predominant genera composing these blooms, particularly in lakes suffering from eutrophication (17). Like other bloom-forming species, it is infamous for the production of the hepatotoxin microcystin, a nonribosomal peptide toxin that inhibits protein phosphatases in a broad range of eukaryotes from zooplankton to humans (1, 9). Moreover, Microcystis is known to produce a multitude of other peptides that are considered to be potential drug leads (14,30).Microviridins form one of the most intriguing classes of peptides, since they feature a cage-like structure (e.g., microviridin B) (Fig. 1A). The highly unusual tricyclic architecture results from -ester and -amide bonds between amino acid side chains. The past few years have afforded 13 variants of the peptide from both field samples and laboratory strains (5,8,13,15,21,22). Most of the variants show inhibitory activities against serine-type proteases, most notably against elastase, which is a target enzyme in the treatment of lung emphysema (28). One of the peptide isoforms, microviridin J, has been shown to inhibit the molting process of Daphnia, leading to death of the animals (23).Recent studies have revealed a unique biosynthetic mechanism for microviridins in Microcystis and the filamentous cyanobacterial genus Planktothrix (18, 31). The 14-amino-acid (aa) peptide sequence is encoded at the C terminus of the ribosomal precursor peptide MdnA (31). Macrocyclization of the peptide depends on the activities of two ATP grasp-type ligases, MdnB and -C, that are encoded downstream of the precursor gene. Further enzymes encoded by the cluster include the N-acetyltransferase MdnD and the putative transporter-peptidase MdnE (31) (Fig. 1B). The activities of the enzymes MdnB, -C, and -D were confirmed by heterologous production of microviridins in Escherichia coli (31). The MdnB and -C ortholog...

Understanding and controlling proteolysis is an important goal in therapeutic chemistry. Among the natural products specifically inhibiting proteases microviridins are particularly noteworthy. Microviridins are ribosomally produced and posttranslationally modified peptides that are processed into a unique, cagelike architecture. Here, we report a combined rational and random mutagenesis approach that provides fundamental insights into selectivity-conferring moieties of microviridins. The potent variant microviridin J was co-crystallized with trypsin, and for the first time the three-dimensional structure of microviridins was determined and the mode of inhibition revealed.

Microviridins are unique protease inhibitors from bloom-forming cyanobacteria that have both ecological and pharmacological relevance. Their peptide backbones are produced ribosomally, and ATP grasp ligases introduce ω-ester and ω-amide bonds to yield rare cage-like structures. Bioinformatic analysis of the microviridin biosynthesis gene cluster suggests a novel type of processing machinery, which could rationalize the challenging in vivo/in vitro reconstitution of the pathway. In this work, we report the establishment of a minimal expression system for microviridins. Through bioinformatics and mutational analysis of the MdnA leader peptide we identified and characterized a strictly conserved binding motif that is specific for microviridin ligases. Furthermore, we showed that the ABC transporter MdnE is crucial for cyclization and processing of microviridins and demonstrated that MdnE is essential for stability of the microviridin biosynthesis complex.

bMicroviridins represent a unique family of ribosomally synthesized cage-like depsipeptides from cyanobacteria with potent protease-inhibitory activities. The natural diversity of these peptides is largely unexplored. Here, we describe two methodologies that were developed to functionally characterize cryptic microviridin gene clusters from metagenomic DNA. Environmental samples were collected and enriched from cyanobacterial freshwater blooms of different geographical origins containing predominantly Microcystis sp. Microviridins were produced either directly from fosmid clones or after insertion of environmental DNA-derived gene cassettes into a minimal expression platform in Escherichia coli. Three novel microviridin variants were isolated and tested against different serine-type proteases. The comparison of the bioactivity profiles of the new congeners allows deduction of further structure-function relationships for microviridins. Moreover, this study provides new insights into microviridin processing and gene cluster organization.

The cyanobacterial genus Microcystis is known to produce an elaborate array of structurally unique and biologically active natural products, including hazardous cyanotoxins. Cytotoxic aeruginoguanidines represent a yet unexplored family of peptides featuring a trisubstituted benzene unit and farnesylated arginine derivatives. In this study, we aimed at assigning these compounds to a biosynthetic gene cluster by utilizing biosynthetic attributes deduced from public genomes of Microcystis and the sporadic distribution of the metabolite in axenic strains of the Pasteur Culture Collection of Cyanobacteria. By integrating genome mining with untargeted metabolomics using liquid chromatography with mass spectrometry, we linked aeruginoguanidine (AGD) to a nonribosomal peptide synthetase gene cluster and coassigned a significantly smaller product to this pathway, microguanidine (MGD), previously only reported from two Microcystis blooms. Further, a new intermediate class of compounds named microguanidine amides was uncovered, thereby further enlarging this compound family. The comparison of structurally divergent AGDs and MGDs reveals an outstanding versatility of this biosynthetic pathway and provides insights into the assembly of the two compound subfamilies. Strikingly, aeruginoguanidines and microguanidines were found to be as widespread as the hepatotoxic microcystins, but the occurrence of both toxin families appeared to be mutually exclusive. Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site.