Improvement of Nitrogenase-Based Photobiological Hydrogen Production by Cyanobacteria by Gene Engineering – Hydrogenases and Homocitrate Synthase

Abstract

We have created several cyanobacterial mutants of hydrogenase and of homocitrate synthase for improvement of nitrogenase-based hydrogen production activity. From Nostoc sp. PCC 7120, mutants were engineered that were disrupted in the uptake hydrogenase gene (ΔhupL), the bidirectional hydrogenase gene (ΔhoxH), and both (ΔhupL/ΔhoxH). Disruption of the Hup structural gene, but not of the Hox structural gene, was very effective in increasing H2 accumulation capacity in this strain. Disruption of hypF, which encodes for one of NiFe-hydrogenase maturation proteins, was also effective. These results indicate that elimination of Hup activity is effective in improving the hydrogen production. The ΔhupL mutant from Nostoc sp. PCC 7422 was able to accumulate hydrogen to 20–30% (v/v) in 3–8 days, and the efficiency of light energy conversion into hydrogen was 3.7% in visible light or about 1.7% in total radiation. The presence of 20% O2 in the initial gas phase inhibited H2 accumulation by less than 20% until day 7. Nostoc PCC 7120 has two homocitrate synthase genes, nifV1 and nifV2, and homocitrate bound to Fe-Mo cofactor of dinitrogenase is considered to be important for efficient nitrogen fixation, but not for hydrogen production. We have created mutants disrupted in one of the two genes and the both of them with ΔhupL as the parent, and found that modulation of homocitrate synthase activity is effective in prolonging the high hydrogen production period.