Site-Directed Mutagenesis of the Anabaena sp. Strain PCC 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

Abstract

ABSTRACT 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 Å 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.