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

dc.contributor.authorBarahona, Emma
dc.contributor.authorJímenez-Vicente, Emilio
dc.contributor.authorRubio, Luis Manuel
dc.date.accessioned2023-12-26T20:54:37Z
dc.date.available2023-12-26T20:54:37Z
dc.date.issued2016-12-02
dc.identifier.issn2045-2322
dc.identifier.urihttps://hdl.handle.net/10115/27914
dc.descriptionEste trabajo fue respaldado por la Beca de Inicio del Consejo Europeo de Investigación con el número 205442 y por la Beca MINECO BIO2014-59131-R.es
dc.description.abstractWhen 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 H2 production in biological systems. Here, we re-engineered the natural H2 sensing 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 H2 production 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 H2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.es
dc.language.isoenges
dc.publisherNature Researches
dc.rightsAtribución 4.0 Internacional*
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/*
dc.subjectfluorescence-activated cell sorting flow cytometryes
dc.subjecthigh-throughput screeninges
dc.subjectdirected evolution of nitrogenasees
dc.subjectphotosynthetic organismses
dc.titleHydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screeninges
dc.typeinfo:eu-repo/semantics/articlees
dc.identifier.doi10.1038/srep38291es
dc.rights.accessRightsinfo:eu-repo/semantics/openAccesses


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Atribución 4.0 InternacionalExcept where otherwise noted, this item's license is described as Atribución 4.0 Internacional