Exploiting the Natural Diversity of Microviridin Gene Clusters for Discovery of Novel Tricyclic Depsipeptides

Abstract

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...

Functional Analysis of Environmental DNA-Derived Microviridins Provides New Insights into the Diversity of the Tricyclic Peptide Family

“…We have established two different heterologous production approaches based on entirely different vectors and insert sizes. Our analysis was motivated by the enormous sequence diversity observed for mdnA prepeptide sequences in a recent field survey (12). During their long evolutionary history, microviridins have gone through a diversification-and-selection process, the analysis of which can help one to understand the traits and constraints of the microviridin RiPP system.…”

“…Protease inhibition assays. Serine protease-inhibitory assays, except those for subtilisin, were carried out according to methods described previously (12). The subtilisin-inhibitory assay was performed according to the modified method of Taichi et al (19).…”

“…A PCR survey for microviridin biosynthesis genes in laboratory strains of Microcystis and in environmental DNA (eDNA) revealed a widespread occurrence of this pathway (12). Moreover, each of the strains analyzed encoded a novel variant of the 12-to 14-membered peptides (12). From this analysis, one could anticipate a large and unexplored library of natural microviridins in cyanobacterial field strains and culture collections with an as-yet-unknown range of bioactivities.…”

“…Microviridins were successfully produced by using either a fosmid-based system or a minimal expression platform (6,11). A PCR survey for microviridin biosynthesis genes in laboratory strains of Microcystis and in environmental DNA (eDNA) revealed a widespread occurrence of this pathway (12). Moreover, each of the strains analyzed encoded a novel variant of the 12-to 14-membered peptides (12).…”

Health Effects of Toxic Cyanobacteria in U.S. Drinking and Recreational Waters: Our Current Understanding and Proposed Direction

“…In the same study, microcystin was shown to intracellularly bind and protect proteins from the damaging effects of free oxygen radicals-providing the first evidence that this metabolite may be of primary importance for cell fitness. In another example, the genes involved in the biosynthesis of a posttranslationally modified novel tricyclic depsipeptide and presumed secondary metabolite microviridin, have been found in all 12 Microcystis genomes sequenced to date and have been identified in diverse other cyanobacteria [80,81]. Adding further evidence that this gene cluster may be of central importance to the ecology of these organisms, microviridin transcripts were among the most highly expressed across all 6 time points collected during a metatranscriptomic analysis of a cyanobacterial bloom sampled over a 24-hour period [82•].…”