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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...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2022
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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. |
format | Online Article Text |
id | pubmed-9209457 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
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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