S141: Production of hydrogen by transgenic strains of the cyanobacterium Anabaena variabilis expressing hydrogenase genes

The cyanobacterium Anabaena variabilis is a filamentous, photosynthetic prokaryote that is capable of growth with nothing more than sunlight, air and water with small amounts of inorganic compounds. Unlike most other photosynthetic organisms, this strain can fix atmospheric dinitrogen to ammonium in a process called nitrogen fixation and it does so under aerobic growth conditions. This is because the oxygen-labile enzyme, nitrogenase, is protected in specialized cells called heterocysts that provide a nearly anaerobic environment for nitrogen fixation. While nitrogenase is capable of hydrogen production itself, it is not an efficient hydrogenase. Therefore, we attempted to express Fe-hydrogenase genes in A. variabilis with the goal of producing large amounts of hydrogen using sunlight as the source of energy. The hydA genes and their maturation factor genes were from Clostridium acetobutylicum and Chlamydomonas reinhardii. The hydA genes, with or without their maturation factor genes, were fused to the ferredoxin-NADP⁺ reductase (FNR) gene. This includes two promoters for overexpressing Fe-hydrogenase in heterocysts as well as vegetative cells, along with the N-terminal CpcD-like domain of FNR. FNR catalyzes electron transfer from reduced ferredoxin to NADP⁺ in the terminal step of the photosynthetic electron transport chain, while the CpcD-like domain targets FNR to the photosynthetic membranes. Fusing the Fe-hydrogenase genes to the CpcD-like domain targets the Fe-hydrogenase to the photosynthetic apparatus, replacing the normal FNR and facilitating the transfer of electrons from ferredoxin directly to the Fe-hydrogenase. We report here the construction of these transgenic strains and their production of hydrogen.