Deletion of Flv3A facilitates long-term H₂ photoproduction in diazotrophic Anabaena sp. PCC 7120

Molecular hydrogen (H₂) is a promising energy carrier, and its production by photosynthetic microorganisms holds substantial potential for advancing renewable energy generation. The nitrogenase-mediated H₂ production using heterocyst-forming cyanobacteria represents a promising approach, as the process utilizes light energy and photosynthetic reductants while being naturally protected from O₂-rich environments by its restriction to microoxic heterocyst cells. We investigated the impact of deleting the vegetative cell-specific flavodiiron protein, Flv3A, on the long-term H₂ photoproduction of the model heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. The H₂ photoproduction response was evaluated under varying atmospheric conditions, with or without N₂ and O₂, and compared to the ∆hupL mutant, which is deficient in the large subunit of uptake hydrogenase, and the ∆hupL/flv3A double mutant. Unlike the ΔhupL mutant, H₂ photoproduction in Δflv3A is not enhanced by increased nitrogenase activity or high accumulation of sugars in cells. Our results suggest that the absence of the vegetative cell-localized Flv3A positively affects H₂ photoproduction in heterocysts by simultaneously downregulating hupL expression and enhancing the O₂ tolerance of nitrogenase via a yet unexplored mechanism. These findings advance our understanding of nitrogenase-driven H₂ production and provide a new strategy to address key limitations in long-term photobiological H₂ production.