Elucidating the Biosynthesis of a Model Ladder-Frame Polyether Toxin

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Project Summary/Abstract The societal damage from Harmful Algal Blooms, or HABs, continues to increase globally, with resulting impacts such as fish kills in the wild and in aquaculture, death of marine mammals, and even direct public health concerns though contamination of drinking water supplies or bioaccumulation of HAB toxins in otherwise edible shellfish. DNA based monitoring of HAB toxin biosynthetic genes currently provides a reliable and species-agnostic method to predict the development of a toxic HAB versus an innocuous algal bloom, but the biosynthetic genes for a number of highly impactful HAB compounds are unknown, preventing this monitoring approach across all toxin classes. The large ladder-frame polyether toxins represent one such long standing biosynthetic question, where their long recognized biosynthetic source, namely being polyketide derived natural products, has not yet led to the identification of the causal genes responsible for toxin biosynthesis in any eukaryotic algal producer, such as the “red-tide” dinoflagellates that cause annual toxic “red tide” events in Florida. The lack of clarity regarding dinoflagellate ladder-frame polyether biosynthesis is possibly due to their remarkably intractable genetics. Toxic dinoflagellate species have very large genomes in the 100s of gigabases, genes arrayed in tandem repeats, and a lack of transcriptional regulation, making the causal determinations of gene-chemotype links experimentally difficult. Here, I propose that an alternative toxic microalgae, the haptophyte Prymnesium parvum, producer of the ‘prymnesin’ ladder-frame polyether toxins, and an impactful HAB organism in its own right, is an ideal model system to elucidate the full biosynthetic pathway of a ladder-frame polyether. This proposal aims to identify and characterize the genes and enzymes responsible for prymnesin biosynthesis in Prymnesium parvum. I propose to use computational genomic, transcriptomic, and metabolomic approaches combined with experimental genetic and biochemical approaches to elucidate the biosynthetic pathway for the toxic ‘prymnesin’ ladder-frame polyethers from haploid strains of the experimentally well-suited haptophyte Prymnesium parvum. First, a substrain of P. parvum will be cloned and reference datasets produced and candidate biosynthetic genes cataloged. Second, activity guided fractionation will be used to identify those enzymes in prymnesin biosynthesis using substrates that can be tractably isolated from Prymnesium cultures. Lastly, a functional CRISPR/Cas9 screen will be used to establish causal links to prymnesin biosynthesis for those genes intractable to heterologous reconstitution and in vitro biochemistry. The research would result in a biosynthetic model of prymnesin production, which could be used to develop biosynthetic gene-based monitoring approaches for toxic polyethers in marine and freshwater ecosystems.