Genomic insights into the taxonomy, safety and biotechnological potential of Aphanizomenon flos-aquae and related Nostocales.

The Nostocales are filamentous cyanobacteria that include bloom-forming, nitrogen-fixing species with major ecological and biotechnological relevance. Owing to their metabolic diversity, nitrogen-fixing capabilities and nutritional profile, they hold potential for various biotechnological applications including the production of nutraceuticals and biofertilizers. However, the capacity of certain Nostocales species to produce cyanotoxins also raises health concerns. This study provides a comparative bioinformatic analysis of 161 Nostocales genomes from the National Center for Biotechnology Information (NCBI) and three newly sequenced Aphanizomenon flos-aquae (AFA) strains, including Klamath AFA, with a focus on taxonomy, cyanotoxin-associated biosynthetic gene clusters (BGCs), natural competence, restriction-modification systems and vitamin B12 metabolism. Our analysis supports reassessment of several AFA-labelled genomes and refines the boundaries of the core AFA cluster. Cyanotoxin-associated BGCs, specifically for anatoxin-a, saxitoxin, cylindrospermopsin and microcystin/nodularin, were detected in only 7.3% of the genomes. Natural competence genes required for horizontal gene transfer were found in 89.6% of the genomes, although the presence of multiple restriction-modification system-associated proteins in Nostocales species, especially Aphanizomenon, Anabaena, Dolichospermum and Nostoc sp. may limit foreign gene acquisition. Additionally, ~30% of the genomes harbour a shared btu operon arrangement associated with B12 import, observed here across multiple Nostocales genera. B12 content in the AFA strains was measured at ~0.4-0.5 µg g-1 dry biomass and is likely linked to associated B12-producing bacteria. Fluorescent B12 analogue uptake experiments in NIES 4052 and Klamath AFA support a model for the uptake of exogenous B12 in these strains. Together with the detection of cobalamin-producing commensal bacteria, this suggests that B12 in AFA biomass may be linked to cyanobacterium-bacteria interactions. Overall, this study provides a genomic and experimental framework for evaluating the taxonomy, safety, genetic tractability and nutritional potential of AFA and related Nostocales.