Unique Biosynthetic Pathway in Bloom-Forming Cyanobacterial Genus Microcystis Jointly Assembles Cytotoxic Aeruginoguanidines and Microguanidines

International audienceThe 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. M icrocystis is a dominant bloom-forming cyanobacterium occurring in temperate freshwater ecosystems. 1 The genus is infamous for the production of the well-known hepatotoxin microcystin. 2 Both blooms and toxins cause ecosystem disturbance and public health threats and constitute a growing concern in the frame of freshwater eutrophication and global warming. Microcystis has also been described as a producer of a multitude of bioactive natural products, some of interest for biotechnological and pharmaceutical application. 3−5 Cytotoxic aeruginoguanidines (AGDs) represent one of the most remarkable families of compounds described for Microcystis. 6 The three AGD congeners reported for strain Microcystis aeruginosa NIES-98 feature highly unprecedented characteristics such as a 1-(4-hydroxy-3-hydroxymethyl)-phenyl-1-hydroxy-2-propylamine-4′,3′,1-triO -sulfate (Hphpa trisulfate) moiety, along with geranylation and prenylation of arginines (Figure 1A). While bloom-forming Microcystis belong to the most intensively studied cyanobacteria, AGDs were reported only twice from a bloom in Czech Republic and an isolate in Brazil, 7,8 never from any other cyanobacteria. Their intricate features confine AGDs into a unique compound family. 3 Our recent genomic analysis of ten Microcystis strains revealed that the different genotypes share a highly similar core genome, while their biosynthetic gene clusters (BGCs) involved in natural product (NP) formation show a sporadic distribution. Moreover, we uncovered three cryptic BGCs no