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

• Published in: Scientific reports 2022-06, Vol.12 (1)
• Main Authors: 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.
• Format: Article
• Language: English
• Subjects: Science & Technology - Other Topics
• ISSN: 2045-2322; 2045-2322

Abstract Biological nitrogen fixation (BNF) is the reduction of N2into NH3in a group of prokaryotes by an extremely O2-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 (S2Vred) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S2Vredto 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 O2resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH fromDehalococcoides ethenogenesdid not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH fromGeobacter sulfurreducenswas more resistant to O2exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O2tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.