Genomic and chemical characterization of cytotoxic Ahp-cyclodepsipeptides from Phormidium using Thamnocephalus platyurus bioassay

Toxic benthic cyanobacterial mats have been identified globally as a potential health hazard since they can produce potent cyanotoxins. Such mats have been connected to the death of mammals worldwide, with a significant fraction of recent cases associated to benthic cyanobacterial blooms producing anatoxin-a or its derivatives. A recent event involving the death of a dog led to monitoring of mats in the area and to the discovery of the first cyanopeptolin producer among the genus Phormidium. Using metagenomics, the genome of the dominant species in the cyanobacterial mats collected was reconstructed, but no evidence for the production of anatoxin-a or other congeners was obtained. In contrast, the biosynthetic gene cluster for cyanopeptolin was detected in the genome. Further chemical analysis of metabolites extracted from the same mats enabled the isolation and characterization of two novel cyanopeptolins with their truncated products. Toxicity was confirmed using the cytotoxic reporting organism Thamnocephalus platyurus. The untruncated cyanopeptolins had an LC₅₀ as low as 6.4 μM while the truncated products did not exhibit cytotoxicity in the same assay. The presence of truncated analogues lacking cytotoxicity might suggest that other mat-associated microorganisms could degrade those compounds. While Phormidium may not be the direct cause of dog's death, our study provides the first evidence that Phormidium can produce cyanopeptolins. This highlights the underexplored variety of neglected cyanotoxin classes. These findings not only expand the known chemical diversity of cyanotoxins but also raise concerns regarding the potential ecological impact of toxic Ahp-cyclodepsipeptides in freshwater environments.