BIOCHEMISTRY OF NITROGEN FIXATION

Group leader: Luis Manuel Rubio Herrero - Senior Researcher CSIC
This email address is being protected from spambots. You need JavaScript enabled to view it.  910679189 (Office 276 )   910679140 (Lab 278-280/S58-S74/ALF3)

 

Personnel:

 

The generation of cereal plants able to express nitrogenase, and thus of assimilating atmospheric nitrogen, has the potential of changing agriculture systems worldwide.

Goals

(1) To engineer direct biological nitrogen fixation (BNF) in cereals by introducing the nitrogenase metabolic pathway into plant cells.
(2) To understand the biosynthesis of the iron-molybdenum cofactor to further establish a basis for rational metabolic engineering of nitrogenase.

 

General Overview

Cereal crop yields are generally increased by addition of chemically synthesized nitrogen fertilizers, which are inaccessible to many smallholder farmers in Sub-Saharan Africa. Our long-term goal is to engineer varieties of cereals that require little or no nitrogen input and deliver higher and more resilient yields. This goal will be achieved by making the plants acquire nitrogen from the atmosphere rather than from synthetic nitrogen fertilizers. The strategy is to transfer to the plant the bacterial genes needed for the biogenesis of nitrogenase, the protein complex that performs biological nitrogen fixation (Fig. 1). The direct transfer of nitrogen fixation genes to the plant has the advantage of developing technology that is in the seeds. We aim to reconstitute the nitrogenase biosynthetic pathway, delivering concepts, knowledge, tools and methodologies for engineering nitrogen fixation in staple cereal crops. Synthetic biology, plant and yeast cell cultures, combinatorial genetic transformation of cereals and model plants, and biochemical complementation assays of resulting nitrogen fixation proteins are the tools being used. In addition, my laboratory tries to understand the unique reactions taking place in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase.




Figure 1. Industrial (left) and biological (center) N2 fixation for nitrogen fertilizer production (right) Generation of self-fertilizing transgenic cereals. Adapted from Burén and Rubio (doi.org/10.1093/femsle/fnx274).

 

 

 

Research Activity

 

Nitrogenase Engineering in Eukaryotes

The most immediate obstacles to engineering BNF in plants are (1) the extreme sensitivity of nitrogenase to O2 and (2) the complexity of the nitrogenase pathway, which requires many genetic parts (modules) to function optimally (Fig. 2). BNF-Cereals focuses on synthetic biology of BNF components in cereals and evaluation of their functionality. We also use model plants (tobacco and Arabidopsis) and yeasts to obtain relevant biochemical and physiological information to guide cereal engineering. Our bioengineering program aims to streamline the transfer of genetic determinants of nitrogen fixation (nif) to cereal plants by using a carefully designed product validation pipeline so that engineering can be carried out at reasonable cost and time.




Figure 2. Minimum genetic requirements to produce a functional recombinant nitrogenase

 

In recent years we have significantly advanced and overcome genetic (heterologous expressions) and physiological (protection from oxygen and utilization of host reducing power) barriers on the way to the nitrogen-fixing eukaryote. Our work has established that it is possible to produce NifH, the most oxygen-sensitive catalytic component of nitrogenase, in yeast mitochondria in active form (Nat Commun 2016). We also produced in vivo the key precursor (called NifB-co) of the nitrogenase catalytic cofactor in yeast mitochondria (PNAS 2019). These findings, together with prior demonstrations that both NifH and NifB (the enzyme responsible for NifB-co biosynthesis) can be expressed in active forms in rice and tobacco (Plant Biotecnol J 2020; Commun Biol 2021; mBio 2022; Commun Biol 2022; ACS Synth Biol 2022), provide evidence that the development of a eukaryotic system capable of fixing nitrogen at detectable levels might be feasible within a medium-term timeframe.

Understanding the biosynthesis of nitrogenase and its cofactors

In addition to the BNF engineering research line, our group aims to understand the unique reactions that take place in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase. FeMo-co, located in the active site of the nitrogenase enzyme, is a complex metal cluster that serves as a paradigm for the biosynthesis of simpler [Fe-S] centers, which are ubiquitous in nature and play basic roles in all life forms. We hypothesize that the complex machinery required for FeMo-co synthesis (Fig. 3) is a limiting factor in nitrogenase activity. We use a multidisciplinary approach to study FeMo-co synthesis. Broadly, our research on this topic has focused on (i) understanding the biosynthesis of the Fe-S-C core FeMo-co by the SAM-radical protein NifB; (ii) understanding the modifications that this core undergoes in the NifEN molecular framework, as well as the exact mechanism of molybdenum incorporation to generate FeMo-co; and (iii) to find simpler metabolic pathways for nitrogenase biosynthesis in the existing microbial diversity and to investigate their peculiarities. We combine genetic, biochemical and spectroscopic analyses with X-ray crystallography, nuclear magnetic resonance and cryogenic electron microscopy, in close collaboration with our international network of collaborators including geneticist Dennis Dean (Virginia Tech), spectroscopists Yisong Guo (Carnegie Mellon University) and Stephen Cramer (University California-Davis), structural biologists Yvain Nicolet (Institut de Biologie Structurale-Grenoble) and David Wemmer (University of California-Davis), and biochemists Nick Le Brun (University of East Anglia) and Mario Piccioli (Universitá di Firenze).




Figure 3. Model for the biosynthesis of nitrogenase FeMo-cofactor (Jiménez-Vicente et al. 2015)

 

By understanding the molecular mechanisms underlying the interactions and activities of the enzymes involved in this process, we aim to establish a basis for rational metabolic engineering of nitrogenase and thus accelerate the BNF engineering process. Therefore, this line will also contribute to achieve a more productive and sustainable agriculture.

Most important recent achievements

  1. First expression of an active component of nitrogenase in eukaryotes (Nat Commun 2016). Collected as a milestone in "The nitrogen fix" by E. Stokstad. Science 353: 1225-1227. European patent EP3044312B1.
  2. First attempt to assemble a complete nitrogenase in Saccharomyces cerevisiae (ACS Synth Biol 2017). Chosen by the Royal Society of Biology as one of the "major scientific advances" of 2017 in Biology.
  3. Biosynthesis of the iron-sulfur-carbon intermediate critical for producing the nitrogenase FeMo-co in yeast (PNAS 2019). U.S. Patent 10941428.
  4. Transgenic rice functionally expressing the NifH structural component of nitrogenase and the FeMo-cofactor maturase NifB (Commun Biol 2022; ACS Synth Biol 2022).
  5. Leading the nitrogenase biosynthesis research. Please see our historical-critical analysis of the field in Chemical Reviews 2020.
  6. Study of a novel, simpler, pathway for FeMo-co biosynthesis (PNAS 2024).

 

Representative Publications

Payá Tormo, L., Nguyen, T.-Q., Fyfe, C., Basbous, H., Dobrzyńska, K., Echavarri-Erasun, C., Martin, L., Caserta, G., Legrand, P., Thorn, A., Amara, P., Schoehn, G., Cherrier, M.V. ✉, Rubio, L.M.✉, Nicolet, Y. ✉ 2025. Dynamics driving the precursor in NifEN scaffold during nitrogenase FeMo-cofactor assembly. Nature Chemical Biology 1–9. DOI: 10.1038/s41589-025-02070-4

García-Molina, G., Echavarri-Erasun, C., del Barrio, M., González-García, S., Rubio, L.M., Pita, M., De Lacey, A.L. 2025. Electroenzymatic N2 reduction to ammonia by nitrogenase from Azotobacter vinelandii immobilized on low density graphite electrodes under direct electron transfer regime. Electrochimica Acta 529, 146342. DOI: 10.1016/j.electacta.2025.146342

Salinero-Lanzarote, A., Lian, J., Namkoong, G., Suess, D.L.M., Rubio, L.M., Dean, D.R., Pérez-González, A. 2025. Molecular sorting of nitrogenase catalytic cofactors. Journal of Biological Chemistry 301. DOI: 10.1016/j.jbc.2025.108291

Payá-Tormo, L., Echavarri-Erasun, C., Makarovsky-Saavedra, N., Pérez-González, A., Yang, Z.-Y., Guo, Y., Seefeldt, L.C., Rubio, L.M. 2024. Iron-molybdenum cofactor synthesis by a thermophilic nitrogenase devoid of the scaffold NifEN. Proceedings of the National Academy of Sciences 121, e2406198121. DOI: 10.1073/pnas.2406198121

Barahona, E., Collantes-García, J.A., Rosa-Núñez, E., Xiong, J., Jiang, X., Jiménez-Vicente, E., Echávarri-Erasun, C., Guo, Y., Rubio, L.M., González-Guerrero, M. 2024. Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase. Journal of Biological Chemistry 107900. DOI: 10.1016/j.jbc.2024.107900

Dobrzyńska, K., Pérez-González, A., Echavarri-Erasun, C., Coroian, D., Salinero-Lanzarote, A., Veldhuizen, M., Dean, D.R., Burén, S., Rubio, L.M. 2023. Nitrogenase cofactor biosynthesis using proteins produced in mitochondria of Saccharomyces cerevisiae. mBio e03088-23. DOI: 10.1128/mbio.03088-23

Rosa-Núñez, E., Echavarri-Erasun, C., Armas, A.M., Escudero, V., Poza-Carrión, C., Rubio, L.M., González-Guerrero, M. 2023. Iron Homeostasis in Azotobacter vinelandii. Biology 12, 1423. DOI: 10.3390/biology12111423

González-Guerrero, M., Navarro-Gómez, C., Rosa-Núñez, E., Echávarri-Erasun, C., Imperial, J., Escudero, V. 2023. Forging a symbiosis: transition metal delivery in symbiotic nitrogen fixation. New Phytologist. DOI: 10.1111/nph.19098

Martin del Campo, J.S., Rigsbee, J., Bueno Batista, M., Mus, F., Rubio, L.M., Einsle, O., Peters, J.W., Dixon, R., Dean, D.R., Dos Santos, P.C. 2023. Overview of physiological, biochemical, and regulatory aspects of nitrogen fixation in Azotobacter vinelandii. Critical Reviews in Biochemistry and Molecular Biology 1–47. DOI: 10.1080/10409238.2023.2181309

Barahona, E., Isidro, E.S., Sierra-Heras, L., Álvarez-Melcón, I., Jiménez-Vicente, E., Buesa, J.M., Imperial, J., Rubio, L.M. 2022. A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatus. Frontiers in Microbiology 13. DOI: 10.3389/fmicb.2022.991123

Baysal, C., Burén, S., He, W., Jiang, X., Capell, T., Rubio, L.M., Christou, P. 2022. Functional expression of the nitrogenase Fe protein in transgenic rice. Communications Biology 5, 1–9. DOI: 10.1038/s42003-022-03921-9

He, W., Burén, S., Baysal, C., Jiang, X., Capell, T., Christou, P., Rubio, L.M. 2022. Nitrogenase Cofactor Maturase NifB Isolated from Transgenic Rice is Active in FeMo-co Synthesis. ACS Synthetic Biology. DOI: 10.1021/acssynbio.2c00194

Payá-Tormo, L., Coroian, D., Martín-Muñoz, S., Badalyan, A., Green, R.T., Veldhuizen, M., Jiang, X., López-Torrejón, G., Balk, J., Seefeldt, L.C., Burén, S., Rubio, L.M. 2022. A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation. Scientific Reports 12, 10367. DOI: 10.1038/s41598-022-14453-x

Jiang, X., Coroian, D., Barahona, E., Echavarri-Erasun, C., Castellanos-Rueda, R., Eseverri, Á., Aznar-Moreno, J.A., Burén, S., Rubio, L.M. 2022. Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana. mBio 0, e00268-22. DOI: 10.1128/mbio.00268-22

Pérez-González, A., Jimenez-Vicente, E., Gies-Elterlein, J., Salinero-Lanzarote, A., Yang, Z.-Y., Einsle, O., Seefeldt, L.C., Dean, D.R. 2021. Specificity of NifEN and VnfEN for the Assembly of Nitrogenase Active Site Cofactors in Azotobacter vinelandii. mBio 12, e0156821. DOI: 10.1128/mBio.01568-21

He, W., Burén, S., Baysal, C., Jiang, X., Capell, T., Christou, P., Rubio, L.M. 2022. Nitrogenase Cofactor Maturase NifB Isolated from Transgenic Rice is Active in FeMo-co Synthesis. ACS Synthetic Biology. DOI: 10.1021/acssynbio.2c00194

Pérez-González, A., Jimenez-Vicente, E., Gies-Elterlein, J., Salinero-Lanzarote, A., Yang, Z.-Y., Einsle, O., Seefeldt, L.C., Dean, D.R. 2021. Specificity of NifEN and VnfEN for the Assembly of Nitrogenase Active Site Cofactors in Azotobacter vinelandii. mBio 12, e0156821. DOI: 10.1128/mBio.01568-21

Phillips, A.H., Hernandez, J.A., Payá-Tormo, L., Burén, S., Cuevas-Zuviría, B., Pacios, L.F., Pelton, J.G., Wemmer, D.E., Rubio, L.M. 2021. Environment and coordination of FeMo–co in the nitrogenase metallochaperone NafY. RSC Chemical Biology. DOI: 10.1039/D1CB00086A

Jenner, L.P., Cherrier, M.V., Amara, P., Rubio, L.M., Nicolet, Y. 2021. An unexpected P-cluster like intermediate en route to the nitrogenase FeMo-co. Chemical Science 12, 5269–5274. DOI: 10.1039/D1SC00289A

Eseverri, Á., Baysal, C., Medina, V., Capell, T., Christou, P., Rubio, L.M., Caro, E. 2020. Transit Peptides From Photosynthesis-Related Proteins Mediate Import of a Marker Protein Into Different Plastid Types and Within Different Species. Frontiers in Plant Science 11, 1474. DOI: 10.3389/fpls.2020.560701

López‐Torrejón, G., Burén, S., Veldhuizen, M., Rubio, L.M. 2021. Biosynthesis of cofactor-activatable iron-only nitrogenase in Saccharomyces cerevisiae. Microbial Biotechnology. DOI: https://doi.org/10.1111/1751-7915.13758

Jiang, X., Payá-Tormo, L., Coroian, D., García-Rubio, I., Castellanos-Rueda, R., Eseverri, Á., López-Torrejón, G., Burén, S., Rubio, L.M. 2021. Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N 2 -fixation in plants. Communications Biology 4, 1–11. DOI: 10.1038/s42003-020-01536-6

Fajardo, A.S., Legrand, P., Payá-Tormo, L., Martin, L., Pellicer Martı́nez, M.T., Echavarri-Erasun, C., Vernède, X., Rubio, L.M., Nicolet, Y. 2020. Structural Insights into the Mechanism of the Radical SAM Carbide Synthase NifB, a Key Nitrogenase Cofactor Maturating Enzyme. Journal of the American Chemical Society. DOI: 10.1021/jacs.0c02243

Crowther, M., Grozinger, L., Pocock, M., Taylor, C.P.D., McLaughlin, J.A., Mısırlı, G., Bartley, B.A., Beal, J., Goñi-Moreno, A., Wipat, A. 2020. ShortBOL: A Language for Scripting Designs for Engineered Biological Systems Using Synthetic Biology Open Language (SBOL). ACS Synthetic Biology 9, 962–966. DOI: 10.1021/acssynbio.9b00470

Beal, J., Goñi-Moreno, A., Myers, C., Hecht, A., de Vicente, M. del C., Parco, M., Schmidt, M., Timmis, K., Baldwin, G., Friedrichs, S., Freemont, P., Kiga, D., Ordozgoiti, E., Rennig, M., Rios, L., Tanner, K., de Lorenzo, V., Porcar, M. 2020. The long journey towards standards for engineering biosystems. EMBO reports e50521. DOI: 10.15252/embr.202050521

Eseverri, Á., López‐Torrejón, G., Jiang, X., Burén, S., Rubio, L.M., Caro, E. 2020. Use of synthetic biology tools to optimize the production of active nitrogenase Fe protein in chloroplasts of tobacco leaf cells. Plant Biotechnology Journal. DOI: 10.1111/pbi.13347

Burén, S., Jiménez-Vicente, E., Echavarri-Erasun, C., Rubio, L.M. 2020. Biosynthesis of Nitrogenase Cofactors. Chemical Reviews. DOI: 10.1021/acs.chemrev.9b00489

Burén, S., Pratt, K., Jiang, X., Guo, Y., Jimenez-Vicente, E., Echavarri-Erasun, C., Dean, D.R., Saaem, I., Gordon, D.B., Voigt, C.A., Rubio, L.M. 2019. Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences. DOI: 10.1073/pnas.1904903116

Baysal, C., Pérez-González, A., Eseverri, Á., Jiang, X., Medina, V., Caro, E., Rubio, L., Christou, P., Zhu, C. 2019. Recognition motifs rather than phylogenetic origin influence the ability of targeting peptides to import nuclear-encoded recombinant proteins into rice mitochondria. Transgenic Research. DOI: 10.1007/s11248-019-00176-9

Navarro-Rodríguez, M., Buesa, J.M., Rubio, L.M. 2019. Genetic and biochemical analysis of the Azotobacter vinelandii molybdenum storage protein. Frontiers in Microbiology 10. DOI: 10.3389/fmicb.2019.00579

 

Patent

  1. Luis Manuel Rubio Herrero, Stefan Burén, Xi Jiang, Carlos Echavarri Erasun, Gema López Torrejón. 2018. Reagents and Methods for the Expression of an Active NifB Protein and Uses Thereof. United States Patent and Trademark Office US10941428B2.
  2. Luis Manuel Rubio Herrero, Gema López Torrejón, Emilio Jiménez Vicente, Jose María Buesa Galiano. 2015. Reagents and methods for the expression of oxygen-sensitive proteins. European Patent Office Number: EP3044312 A1.

Funding (selected grants)

  1. Bill & Melinda Gates Foundation INV-067006. BNF Cereals Phase IV (2024-2028), $5,000,000
  2. MICINN PID2021-128802OB-I00. Formation of the iron-sulfur core of nitrogenase catalytic cofactor (2021-2024), 217,800 €.
  3. Bill & Melinda Gates Foundation INV-005889. BNF Cereals Phase III (2020-2023), $6,200,000.
  4. Bill & Melinda Gates Foundation OPP1143172. BNF-Cereals Phase II (2016-2020), $5,000,000.
  5. MINECO BIO20154-59131-R. Biotechnological Applications of Nitrogenase (2015-2017), 242,000 €.
  6. Bill & Melinda Gates Foundation Grant OPP10442444. Engineering nitrogen fixation in mitochondria (2011-2016), $3,127,139.
  7. ERC Starting Grant 205442. Towards optimization of hydrogen production by nitrogenase (2008-2014), 1,968,000 €.