Besides anthropogenic pollutants, proliferations of phytoplanktonic cyanobacteria in freshwater ecosystems represent a source of growing concern worldwide due to the ecological and economical disturbances they create, as well as the significant impact of their toxins on animal and human health. One of the most prevalent and notorious bloom-forming genus of freshwater cyanobacteria is very likely Microcystis. However, the toxicological impairs of Microcystis spp., yet remain incompletely investigated. Although microcystins (MCs), a family of hepatic cyanotoxins initially described from Microcystis blooms, have been extensively characterized, it comprises now more than 250 known variants with variable toxicological ranges. In addition to the MC risk, recent concerns have highlighted that Microcystis blooms may be of even greater concern for environmental and human health, due to their production of various other deleterious bioactive compounds, the toxicological characterization of which is sorely lacking. We hypothesize that some emerging Microcystis metabolites (mostly cyanopeptides) are of (eco)toxicological concern and should be included in future environmental monitoring and sanitary regulations. Four main questions will be specifically investigated: 1) which emerging cyanobacterial metabolites are the most frequently produced by the different Microcystis genotypes? 2) What are the biosynthetic gene clusters (BGC) associated with the production of these compounds, and when they are unknown, can we characterize them? 3) What is the chemical structure of these different metabolites and when unknown, can we determine them? 4) Are these different compounds potentially toxic and at which level compared to regulated microcystins (MCs)

- WP1: Description of the chemical diversity of Microcystis genotypes retrieved by genome mining and metabolomics from mono-clonal strains - coordinated by PCC & MCAM Deliverable: Determination of most occurrent Microcystis metabolite families and identification of 6 most representative genotypes, that will be selected for further metabolite purification, structural characterization (WP2), and toxicological analyses (WP4), together with the selection of novel BGCs for gene editing (WP3). - WP2: Purification and characterization of most potentially toxic Microcystis metabolites (except MCs) - coordinated by MCAM & PCC Deliverable: purification and structural characterization of potentially toxic purified Microcystis metabolites to serve as analytical standards useable for absolute quantification and for further toxicity assessment (WP4). - WP3: genome editing of Microcystis strains - coordinated by TACGENE & PCC Deliverable: mutant Microcystis strain(s) presenting specific inactivation of targeted BGCs and comparison of its chemical phenotype with those of the wild strains for BGC production characterization. - WP4: Toxicological assays - coordinated by SEBIO & MCAM Deliverable: evaluation multi-model of toxicities of most abundant Microcystis metabolites, comparison with MC.

WP4: Toxicological assays on carp and trout cell lines. The 6 different Microcystis strains selected from WP1 and their 12-15 respective purified metabolites (WP2) will be investigated for their toxicological properties according to various assays involving different organism and cell line models: A/ medaka fish embryo assays (MCAM); B/ trout and carp cell-line assays (RT-HEP, CLC and CCB – SEBIO); C/ vertebrate cell-line assays (PCBM, HaCat and RAW264.7 – MCAM, subcontracting PCBIS25). Taken together, these results will be integrated within a weight-of-evidence approach (WoE), in order to determine the most noxious Microcystis strains and metabolites, according to molecular absolute quantification.