High photobiological hydrogen production activity of a Nostoc sp PCC 7422 uptake hydrogenase-deficient mutant with high nitrogenase activity

We describe a strategy to establish cyanobacterial strains with high levels of H-2 production that involves the identification of promising wild-type strains followed by optimization of the selected strains using genetic engineering. Nostoc sp. PCC 7422 was chosen from 12 other heterocystous strains, because it has the highest nitrogenase activity. We sequenced the uptake hydrogenase (Hup) gene cluster as well as the bidirectional hydrogenase gene cluster from the strain, and constructed a mutant (Delta hupL) by insertional disruption of the hupL gene. The Delta hupL mutant produced H-2 at 100 mu moles mg chlorophyll a(-1) h(-1), a rate three times that of the wild-type. The Delta hupL cells could accumulate H-2 to about 29% (v/v) accompanied by O-2 evolution in 6 days, under a starting gas phase of Ar + 5% CO2. The presence of 20% O-2 in the initial gas phase inhibited H-2 accumulation of the Delta hupL cells by less than 20% until day 7.