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

When produced biologically, especially by photosynthetic organisms, hydrogen gas (H(2)) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H(2)-producing enzymes is the absence of methods for the high-throughput screening of H(2) production in biological systems. Here, we re-engineered the natural H(2) sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H(2). A lacZ gene was placed under the control of the hupA H(2)-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(2) production levels was found for all automatically selected strains. One of the selected H(2)-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(2) production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.