Towards photosynthetic hydrogen production

The need to develop a sustainable energy is crucial and one of the foremost global challenges facing humanity, driven by a continuing global population expansion and anthropogenic climate change. Hydrogen (H2) is a clean burning fuel which, however production relies on fossil fuels that is both unsustainable and environmentally unfriendly. The current global market is currently estimated at $115.25 Billion and expected to rise to $154.74 Billion by 2022. Microbes such as cyanobacteria offer a sustainable method of H2 production platform with minimal environmental impact, making them an extremely attractive proposition. Cyanobacteria can possess two functionally distinct [NiFe] hydrogenases: an uptake enzyme, only capable of hydrogen oxidation and bidirectional enzyme capable of reducing protons to evolve hydrogen. Synechocystis sp. PCC 6803 (PCC 6803) and Synechococcus sp. PCC 7002 (PCC 7002) encode one bidirectional [NiFe] which is a heteropentameric enzyme composed of a hydrogenase module (HoxH & HoxY), forming the catalytic core and the diaphorase module (HoxE, HoxF & HoxU). Previously it has been demonstrated that the hydrogenase (Hox) of PCC 6803 is thylakoid associated forming two distinct Hox populations, one dispersed throughout the thylakoid and the other forming distinct puncta which correlate with H2 evolution (Burroughs et al., 2014). An understanding of the interaction partner of each population is key in engineering strains for photobiological hydrogen production. We show that a proportion of the Hox population interacts with the type 1 NAD(P)H dehydrogenase (NDH-1) of the electron transport chain under conditions that support hydrogen evolution suggesting a role of the hydrogenase in modulating redox balance in the plastoquinol pool. In order to assess the Hox interaction partner at the thylakoid in Synechocystis sp. PCC 6803, HoxE and HoxY subunits were tagged with GFP in order to carryout pull downs and assess distribution of Hox. The hoxYGFP and hoxEGFP strains showed a cytoplasm localisation under photoautotrophic conditions and would localise to the thylakoid under anoxic dark adapted conditions. Pull downs of the hoxEGFP strain showed proteins co-eluting with HoxEGFP with similar and unique bands being observed between photoautotrophic growth and anoxic dark adapted conditions. Hox in Synechococcus sp. PCC 7002 is also thylakoid localised, forming similar populations as described in Synechocystis sp. PCC 6803. We demonstrate that the hydrogenase puncta of Synechococcus sp. PCC 7002 directly interact with the NDH-1 complex which increase upon the addition of glycerol. The metabolome of Synechocystis sp. PCC 6803 strains were compared in a variety of NDH-1 and Hox mutants in order to assess pathways that are implicated in H2 metabolism. Glutamic acid and lactic acid were shown to be key metabolites that were effect in each mutant relative to wild type, especially under anoxic dark adaptation