title: Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening.
abstract: When produced biologically, especially by photosynthetic organisms, hydrogen gas (H2) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H2-producing enzymes is the absence of methods for the high-throughput screening of H2production in biological systems. Here, we re-engineered the natural H2sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H2. A lacZ gene was placed under the control of the hupA H2-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 H2production levels was found for all automatically selected strains. One of the selected H2-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 H2production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.
authors.affiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, 28223-Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Emma
authors.initials: E
authors.lastname: Barahona
authors.affiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, 28223-Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Emilio
authors.initials: E
authors.lastname: Jiménez-Vicente
authors.affiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, 28223-Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Luis M
authors.initials: LM
authors.lastname: Rubio
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D056804:Biofuels; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods; D057166:High-Throughput Screening Assays; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000235:genetics* / Q000378:metabolism; D007763:Lac Operon; D008958:Models, Molecular; D016296:Mutagenesis; D009591:Nitrogenase / Q000235:genetics* / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010788:Photosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D016354:Rhodobacter capsulatus / Q000201:enzymology / Q000235:genetics*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D056804:Biofuels; D002352:Carrier Proteins; C099188:HupA protein, bacteria; D006859:Hydrogen; D010088:Oxidoreductases; D006864:Hydrogenase; D009591:Nitrogenase; C029130:nitrogenase reductase