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Biological nitrogen fixation (BNF) is the reduction of N 2 into NH 3 in a group of prokaryotes by an extremely O 2 -sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S 2 V red ) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S 2 V red to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O 2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O 2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O 2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops., (© 2022. The Author(s).)

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.