Group leader: Luis Manuel Rubio Herrero - Research Professor CSIC-INIA
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The generation of cereal plants able to express nitrogenase, and thus of assimilating atmospheric nitrogen, has the potential of changing agriculture systems worldwide.


  1. To engineer active nitrogenase in eukaryotic organisms, with special focus on cereal crops
  2. To understand the biosynthesis of the iron-molybdenum cofactor of nitrogenase
  3. To improve biological hydrogen production by using nitrogenase directed evolution

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. 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. Finally, we contribute knowledge and technology to the other widely recognized biotechnological application of nitrogenase, which is the biological production of H2, a clean energy vector.


Research Activity

Nitrogenase Engineering in Eukaryotes


Only a small group of bacteria and archaea are capable of biological nitrogen fixation (BNF), a process by which the inert N2 is reduced to NH3 and thus is made available all organisms. Our vision is to engineer BNF in plants by direct transfer of bacterial nitrogen fixation (nif) genes in order to express a functional “eukaryotic” nitrogenase. The most immediate obstacles to engineer BNF in plants are the sensitivity of nitrogenase towards O2 and the complexity of nitrogenase, which requires a large number of genetic parts to function optimally. We lead a consortium of researchers that are engineering BNF components in cereals and assessing their functionality. Model plants (tobacco and common bean) and yeast systems are also being used to gain relevant biochemical and physiological information to guide cereal engineering.


BNF-Cereals Phase I (2011-2016) was an early stage proof-of-concept. Its main outcome was the expression and maturation of one functional, O2-labile nitrogenase component in mitochondria of aerobically grown yeast (Figure 1). By breaking through the limitation imposed by O2 in the production of nitrogenase within a eukaryotic cell, we delivered enabling technology that is instrumental to engineer nitrogen-fixing cereals. BNF-Cereals Phase II aims to engineer BNF components in higher plant organelles and to assess their functionality. Our goals in this research line are: (1) to provide concepts, tools and methodologies for engineering BNF in cereal crops; (2) to establish the functionality of all individual components of the nitrogenase biosynthetic pathway in eukaryotic cells.

Figure 1. Expression of active nitrogenase Fe protein in mitochondria of aerobically grown yeast (López-Torrejón et al 2016). Mitochondria respiration protects the Fe protein from O2 damage. Four bacterial genes are involved in producing active Fe protein.

Optimization of hydrogen production by nitrogenase

In the process of reducing N2 to NH3, the nitrogenase enzyme evolves H2. This old observation has encouraged researches to investigate the applications of nitrogenase as catalyst for biological H2 production. However, major barriers to re-engineer and optimize H2 production by nitrogenase became apparent, mostly due to nitrogenase complexity and low catalytic efficiency. We have recently developed an efficient high-throughput screening to select H2 overproducers in libraries containing millions of nitrogenase variants (Figure 2). This system is being used to generate new nitrogenase (and hydrogenase) variants by in vitro protein evolution techniques. Our goal is to provide tools to help overcome these barriers and improve their use as potential catalysts for direct biophotolysis.

Figure 2.Biosensor to select H2-overproducing nitrogenase variants. This tool consists of a library of random nitrogenase variants and a biological H2 sensor, which is based on the sensing hydrogenase of Rhodobacter capsulatus and produces fluorescent signals that are proportional to the amount of H2 produced by each variant. High-throughput selection by coupling this tool to FACS cytometry was achieved. The selection of a nitrogenase variant resulting in 1000% improvement in bacterial H2 production is shown as proof of concept in Barahona et al. 2016.


Understanding the biosynthesis of nitrogenase and its cofactors

Due to its great agronomical and ecological significance, nitrogenase has been subject of extensive biochemical, genetic, and structural analyses. The iron-molybdenum cofactor (FeMo-co) of nitrogenase, located at the active site of the nitrogenase enzyme is ultimately responsible for BNF activity. Understanding the details of FeMo-co biosynthesis and nitrogenase assembly will improve BNF agronomical applications.

FeMo-co is a complex metallocluster that serves as paradigm to understand the biosynthesis of simpler [Fe-S] clusters, which are ubiquitous in nature and perform basic functions in all forms of life. We aim to characterize the enzymes and metal clusters needed for nitrogenase to function efficiently. We hypothesize that the complex machinery required for FeMo-co synthesis could be a limiting factor to nitrogenase activity (Figure 3). We use a multidisciplinary approach to study FeMo-co synthesis. By understanding the molecular mechanisms, underlying interactions, and activities of enzymes involved in this process, we aim to establish a foundation for rational metabolic engineering of nitrogenase. Our specific research lines in this topic are: (1) to understand the biosynthesis of the central Fe-S core of FeMo-co by the SAM-radical protein NifB; and (2) to find and investigate simpler metabolic pathways for nitrogenase biosynthesis.

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

Most important recent achievements

  1. First active NifB purified from a eukaryotic cell
  2. First expression of an active component of nitrogenase in eukaryotes. Highlighted in “The nitrogen fix” by E. Stokstad. Science 353: 1225-1227.
  3. Development of a high-throughput screen for H2-overproducing variants of nitrogenase.
  4. Detailed characterization of NifB and its product NifB-co, essential to all nitrogenases.


Representative Publications

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

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

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

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:

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

Burén, S; López-Torrejón, G; Rubio, LM. 2018. "Extreme bioengineering to meet the nitrogen challenge". Proceedings of the National Academy of Sciences of the United States of America. DOI: 10.1073/pnas.1812247115".

Jimenez-Vicente, E; Yang, ZY; Ray, WK; Echavarri-Erasun, C; Cash, VL; Rubio, LM; Seefeldt, LC; Dean, DR. 2018. "Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation". Journal of Biological Chemistry. DOI: 10.1074/jbc.RA118.002994".

Ortega-Villasante, C; Burén, S; Blázquez-Castro, A; Barón-Sola, Á; Hernández, LE. "Fluorescent in vivo imaging of reactive oxygen species and redox potential in plants". Free Radical Biology and Medicine. DOI: 10.1016/j.freeradbiomed.2018.04.005".

Burén, S; Rubio, LM. 2017. "State of the art in eukaryotic nitrogenase engineering". FEMS Microbiology Letters. DOI: 10.1093/femsle/fnx274".

Arragain, S; Jiménez-Vicente, E; Scandurra, AA; Burén, S; Rubio, LM; Echavarri-Erasun, C. 2017. "Diversity and Functional Analysis of the FeMo-Cofactor Maturase NifB". Frontiers in Plant Science. DOI: 10.3389/fpls.2017.01947".

Pérez-González, A; Kniewel, R; Veldhuizen, M; Verma, HK; Navarro-Rodríguez, M; Rubio, LM; Caro, E. 2017. "Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria". BMC Biotechnology. DOI: 10.1186/s12896-017-0393-y".

Burén, S; Jiang, X; López-Torrejón, G; Echavarri-Erasun, C; Rubio, LM. 2017. "Purification and in vitro activity of mitochondria targeted nitrogenase cofactor maturase NifB". Frontiers in Plant Science. DOI: 10.3389/fpls.2017.01567".

Burén, S; Young, EM; Sweeny, EA; Lopez-Torrejón, G; Veldhuizen, M; Voigt, CA; Rubio, LM. 2017. "Formation of nitrogenase NifDK tetramers in the mitochondria of Saccharomyces cerevisiae". ACS synthetic biology. DOI: 10.1021/acssynbio.6b00371".

Barahona, E; Jimenez-Vicente, E; Rubio, LM. 2016. "Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening". Scientific Reports. DOI: 10.1038/srep38291".

Guo, Y; Echavarri-Erasun, C; Demuez, M; Jiménez-Vicente, E; Bominaar, EL; Rubio, LM. 2016. "The nitrogenase FeMo-cofactor precursor formed by NifB protein: a diamagnetic cluster containing eight iron atoms". Angewandte Chemie International Edition. DOI: 10.1002/anie.201606447".

George, SJ; Hernandez, JA; Jimenez-Vicente, E; Echavarri-Erasun, C; Rubio, LM. 2016. "EXAFS reveals two Mo environments in the nitrogenase iron-molybdenum cofactor biosynthetic protein NifQ". Chemical Communications. DOI: 10.1039/C6CC06370E".

Ortega-Villasante, C; Burén, S; Barón-Sola, Á; Martínez, F; Hernández, LE. 2016. "In vivo ROS and redox potential fluorescent detection in plants: present approaches and future perspectives". Methods. DOI: 10.1016/j.ymeth.2016.07.009".

Wilcoxen, J; Arragain, S; Scandurra, AA; Jimenez-Vicente, E; Echavarri-Erasun, C; Pollmann, S; Britt, RD; Rubio, LM. 2016. "Electron paramagnetic resonance characterization of three iron–sulfur clusters present in the nitrogenase cofactor maturase NifB from Methanocaldococcus infernus". Journal of the American Chemical Society. DOI: 10.1021/jacs.6b03329".

López-Torrejón, G; Jiménez-Vicente, E; Buesa, JM; Hernandez, JA; Verma, HK; Rubio, LM. 2016. "Expression of a functional oxygen-labile nitrogenase component in the mitochondrial matrix of aerobically grown yeast". Nature Communications. DOI: 10.1038/ncomms11426".