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A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation

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 dep...

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Autores principales: Payá-Tormo, Lucía, Coroian, Diana, Martín-Muñoz, Silvia, Badalyan, Artavazd, Green, Robert T., Veldhuizen, Marcel, Jiang, Xi, López-Torrejón, Gema, Balk, Janneke, Seefeldt, Lance C., Burén, Stefan, Rubio, Luis M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9209457/
https://www.ncbi.nlm.nih.gov/pubmed/35725884
http://dx.doi.org/10.1038/s41598-022-14453-x
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author Payá-Tormo, Lucía
Coroian, Diana
Martín-Muñoz, Silvia
Badalyan, Artavazd
Green, Robert T.
Veldhuizen, Marcel
Jiang, Xi
López-Torrejón, Gema
Balk, Janneke
Seefeldt, Lance C.
Burén, Stefan
Rubio, Luis M.
author_facet Payá-Tormo, Lucía
Coroian, Diana
Martín-Muñoz, Silvia
Badalyan, Artavazd
Green, Robert T.
Veldhuizen, Marcel
Jiang, Xi
López-Torrejón, Gema
Balk, Janneke
Seefeldt, Lance C.
Burén, Stefan
Rubio, Luis M.
author_sort Payá-Tormo, Lucía
collection PubMed
description 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.
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spelling pubmed-92094572022-06-22 A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation Payá-Tormo, Lucía Coroian, Diana Martín-Muñoz, Silvia Badalyan, Artavazd Green, Robert T. Veldhuizen, Marcel Jiang, Xi López-Torrejón, Gema Balk, Janneke Seefeldt, Lance C. Burén, Stefan Rubio, Luis M. Sci Rep Article 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. Nature Publishing Group UK 2022-06-20 /pmc/articles/PMC9209457/ /pubmed/35725884 http://dx.doi.org/10.1038/s41598-022-14453-x Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Payá-Tormo, Lucía
Coroian, Diana
Martín-Muñoz, Silvia
Badalyan, Artavazd
Green, Robert T.
Veldhuizen, Marcel
Jiang, Xi
López-Torrejón, Gema
Balk, Janneke
Seefeldt, Lance C.
Burén, Stefan
Rubio, Luis M.
A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation
title A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation
title_full A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation
title_fullStr A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation
title_full_unstemmed A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation
title_short A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation
title_sort colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9209457/
https://www.ncbi.nlm.nih.gov/pubmed/35725884
http://dx.doi.org/10.1038/s41598-022-14453-x
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