Sustained photobiological hydrogen production in the presence of N2 by nitrogenase mutants of the heterocyst-forming cyanobacterium Anabaena

• Sustained H2 production by the Anabaena ΔHup cells can be attained under low N2 concentration in the headspace gases.
• Two double-crossover variants were constructed with amino acid substitutions in nitrogenase.
• The variants continuously produced H2 over 21 days under N2-rich atmosphere.
• Extremely low N2-fixing activity of the variant nitrogenases allowed cells to obtain combined N to sustain their activity.
• The dc-Q193S variant produced ethane at low but significant rates along with greater rates of ethylene production.

Heterocyst-forming cells of the cyanobacterium Anabaena sp. strain PCC 7120 ΔHup, lacking an uptake hydrogenase, photobiologically produce H2 by nitrogenase. Under N2-rich atmosphere, the nitrogenase activity declines in a rather short time due to the sufficiency of combined nitrogen. From the parental ΔHup strain, site-directed double-crossover variants, dc-Q193S and dc-R284H, were created with amino acid substitutions presumed to be located in the vicinity of the FeMo-cofactor of nitrogenase. Unlike the case for the ΔHup strain, H2 production activities of the variants were not decreased by the presence of high concentrations of N2 and they continuously produced H2 over 21 days with occasional headspace gas replacement. This property of N2 insensitivity is a potentially useful strategy for reducing the cost of the culture gas in future practical applications of sustainable biofuel production. This Anabaena strain has only the Mo-containing nitrogenase which reduces acetylene to ethylene, but the dc-Q193S variant also produced ethane at low but measurable rates along with greater rates of ethylene production.