Hydrogenases catalyze the reversible reaction 2H+ + 2e-↔H2 with an equilibrium constant that is dependent on the reducing potential of electrons carried by their redox partner. To examine the possibility of increasing the photobiological production of hydrogen within cyanobacterial cultures, we expressed the [FeFe] hydrogenase, HydA, from Clostridium acetobutylicum in the non-nitrogen-fixing cyanobacterium Synechococcus elongatus sp. 7942. We demonstrate that the heterologously expressed hydrogenase is functional in vitro and in vivo, and that the in vivo hydrogenase activity is connected to the light-dependent reactions of the electron transport chain. Under anoxic conditions, HydA activity is capable of supporting light-dependent hydrogen evolution at a rate > 500-fold greater than that supported by the endogenous [NiFe] hydrogenase. Furthermore, HydA can support limited growth solely using H2 and light as the source of reducing equivalents under conditions where Photosystem II is inactivated. Finally, we demonstrate that the addition of exogenous ferredoxins can modulate redox flux in the hydrogenase-expressing strain, allowing for greater hydrogen yields and for dark fermentation of internal energy stores into hydrogen gas.
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