Site-Directed Mutagenesis Of The Anabaena Sp. Strain Pcc 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

Cyanobacteria use sunlight and water to produce hydrogen gas (H-2), which is potentially useful as a clean and renewable biofuel. Photobiological H-2 arises primarily as an inevitable by-product of N-2 fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H-2 production by this strain, six nitrogenase amino acid residues predicted to reside within 5 angstrom of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N-2. Most of the 49 variants examined were deficient in N-2-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N-2 atmosphere significantly increased their in vivo rates of H-2 production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H-2 compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H-2 in an aerobic, nitrogen-containing environment.