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

Este 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.

Detalles Bibliográficos
Autores: Barahona, Emma, Jímenez-Vicente, Emilio, Rubio, Luis Manuel
Tipo de recurso: artículo
Fecha de publicación:2016
País:España
Institución:Universidad Rey Juan Carlos
Repositorio:BURJC-Digital. Repositorio Institucional de la Universidad Rey Juan Carlos
OAI Identifier:oai:burjcdigital.urjc.es:10115/27914
Acceso en línea:https://hdl.handle.net/10115/27914
Access Level:acceso abierto
Palabra clave:fluorescence-activated cell sorting flow cytometry
high-throughput screening
directed evolution of nitrogenase
photosynthetic organisms
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spelling Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screeningBarahona, EmmaJímenez-Vicente, EmilioRubio, Luis Manuelfluorescence-activated cell sorting flow cytometryhigh-throughput screeningdirected evolution of nitrogenasephotosynthetic organismsEste 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.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 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.Nature Research202320232016info:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/10115/27914reponame:BURJC-Digital. Repositorio Institucional de la Universidad Rey Juan Carlosinstname:Universidad Rey Juan CarlosInglésAtribución 4.0 Internacionalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:burjcdigital.urjc.es:10115/279142026-06-24T12:48:17Z
dc.title.none.fl_str_mv Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening
title Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening
spellingShingle Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening
Barahona, Emma
fluorescence-activated cell sorting flow cytometry
high-throughput screening
directed evolution of nitrogenase
photosynthetic organisms
title_short Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening
title_full Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening
title_fullStr Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening
title_full_unstemmed Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening
title_sort Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening
dc.creator.none.fl_str_mv Barahona, Emma
Jímenez-Vicente, Emilio
Rubio, Luis Manuel
author Barahona, Emma
author_facet Barahona, Emma
Jímenez-Vicente, Emilio
Rubio, Luis Manuel
author_role author
author2 Jímenez-Vicente, Emilio
Rubio, Luis Manuel
author2_role author
author
dc.subject.none.fl_str_mv fluorescence-activated cell sorting flow cytometry
high-throughput screening
directed evolution of nitrogenase
photosynthetic organisms
topic fluorescence-activated cell sorting flow cytometry
high-throughput screening
directed evolution of nitrogenase
photosynthetic organisms
description Este 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.
publishDate 2016
dc.date.none.fl_str_mv 2016
2023
2023
dc.type.none.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/10115/27914
url https://hdl.handle.net/10115/27914
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv Atribución 4.0 Internacional
http://creativecommons.org/licenses/by/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv Atribución 4.0 Internacional
http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Nature Research
publisher.none.fl_str_mv Nature Research
dc.source.none.fl_str_mv reponame:BURJC-Digital. Repositorio Institucional de la Universidad Rey Juan Carlos
instname:Universidad Rey Juan Carlos
instname_str Universidad Rey Juan Carlos
reponame_str BURJC-Digital. Repositorio Institucional de la Universidad Rey Juan Carlos
collection BURJC-Digital. Repositorio Institucional de la Universidad Rey Juan Carlos
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,811543

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