Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening.

When produced biologically, especially by photosynthetic organisms, hydrogen gas (H) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H-producing enzymes is the absence of methods for the high-throughput screening of H production in biological systems. Here, we re-engineered the natural H sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H. A lacZ gene was placed under the control of the hupA H-inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H production levels was found for all automatically selected strains. One of the selected H-overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.