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

• Published in: Scientific reports 2016-12, Vol.6 (1), p.38291, Article 38291
• Main Authors: Barahona, Emma, Jiménez-Vicente, Emilio, Rubio, Luis M.
• Format: Article
• Language: English
• Subjects: 42/41; 631/1647/1511; 631/326/2522; 631/61/318; 82/29; 82/80; Amino acid sequence; Amino acids; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Biofuels; Carrier Proteins - genetics; Carrier Proteins - metabolism; Conserved sequence; Directed evolution; Emissions; Enzymes; Ferredoxin hydrogenase; Flow Cytometry; Fluorescence; Gene Expression Regulation, Bacterial; Genes; Genetic Engineering - methods; High-throughput screening; High-Throughput Screening Assays; Humanities and Social Sciences; Hydrogen - metabolism; Hydrogenase - genetics; Hydrogenase - metabolism; Lac Operon; LacZ gene; Models, Molecular; multidisciplinary; Mutagenesis; NifH gene; Nitrogenase; Nitrogenase - genetics; Nitrogenase - metabolism; Oxidoreductases - genetics; Oxidoreductases - metabolism; Photosynthesis - genetics; Promoter Regions, Genetic; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Proteins; Random mutagenesis; Rhodobacter capsulatus - enzymology; Rhodobacter capsulatus - genetics; Science
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep38291

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.