Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana
ABSTRACT Engineering plants to synthesize nitrogenase and assimilate atmospheric N2 will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a cri...
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American Society for Microbiology
2022-06-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.00268-22 |
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| author | Xi Jiang Diana Coroian Emma Barahona Carlos Echavarri-Erasun Rocío Castellanos-Rueda Álvaro Eseverri Jose A. Aznar-Moreno Stefan Burén Luis M. Rubio |
| author_facet | Xi Jiang Diana Coroian Emma Barahona Carlos Echavarri-Erasun Rocío Castellanos-Rueda Álvaro Eseverri Jose A. Aznar-Moreno Stefan Burén Luis M. Rubio |
| author_sort | Xi Jiang |
| collection | DOAJ |
| description | ABSTRACT Engineering plants to synthesize nitrogenase and assimilate atmospheric N2 will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors. Here, we used a library of 30 distinct nifB sequences originating from different phyla and ecological niches to restore diazotrophic growth of an Azotobacter vinelandii nifB mutant. Twenty of these variants rescued the nifB mutant phenotype despite their phylogenetic distance to A. vinelandii. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, nifB functional modules composed of a nifB variant, together with the A. vinelandii NifS and NifU proteins (for biosynthesis of NifB [Fe4S4] clusters) and the FdxN ferredoxin (for NifB function), were expressed in Nicotiana benthamiana chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts (Methanosarcina acetivorans and Methanocaldococcus infernus) or mitochondria (M. infernus and Methanothermobacter thermautotrophicus). These NifB proteins were shown to accept [Fe4S4] clusters from NifU and were functional in FeMo-co synthesis in vitro. The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants. IMPORTANCE Biological nitrogen fixation is the conversion of inert atmospheric dinitrogen gas into nitrogen-reactive ammonia, a reaction catalyzed by the nitrogenase enzyme of diazotrophic bacteria and archaea. Because plants cannot fix their own nitrogen, introducing functional nitrogenase in cereals and other crop plants would reduce our strong dependency on N fertilizers. NifB is required for the biosynthesis of the active site cofactors of all nitrogenases, which arguably makes it the most important protein in global nitrogen fixation. NifB functionality is therefore a requisite to engineer a plant nitrogenase. The expression of nifB genes from a wide range of prokaryotes into the model diazotroph Azotobacter vinelandii shows a surprising level of genetic complementation suggestive of plasticity in the nitrogenase biosynthetic pathway. In addition, we obtained NifB proteins from both mitochondria and chloroplasts of tobacco that are functional in vitro after reconstitution by providing [Fe4S4] clusters from NifU, paving the way to nitrogenase cofactor biosynthesis in plants. |
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| spelling | doaj.art-0d6af2f64bb541d7842f270802fbbc3e2022-12-22T02:33:41ZengAmerican Society for MicrobiologymBio2150-75112022-06-0113310.1128/mbio.00268-22Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamianaXi Jiang0Diana Coroian1Emma Barahona2Carlos Echavarri-Erasun3Rocío Castellanos-Rueda4Álvaro Eseverri5Jose A. Aznar-Moreno6Stefan Burén7Luis M. Rubio8Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Pozuelo de Alarcón, Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Pozuelo de Alarcón, Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Pozuelo de Alarcón, Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Pozuelo de Alarcón, Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Pozuelo de Alarcón, Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Pozuelo de Alarcón, Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Pozuelo de Alarcón, Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Pozuelo de Alarcón, Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Pozuelo de Alarcón, Madrid, SpainABSTRACT Engineering plants to synthesize nitrogenase and assimilate atmospheric N2 will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors. Here, we used a library of 30 distinct nifB sequences originating from different phyla and ecological niches to restore diazotrophic growth of an Azotobacter vinelandii nifB mutant. Twenty of these variants rescued the nifB mutant phenotype despite their phylogenetic distance to A. vinelandii. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, nifB functional modules composed of a nifB variant, together with the A. vinelandii NifS and NifU proteins (for biosynthesis of NifB [Fe4S4] clusters) and the FdxN ferredoxin (for NifB function), were expressed in Nicotiana benthamiana chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts (Methanosarcina acetivorans and Methanocaldococcus infernus) or mitochondria (M. infernus and Methanothermobacter thermautotrophicus). These NifB proteins were shown to accept [Fe4S4] clusters from NifU and were functional in FeMo-co synthesis in vitro. The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants. IMPORTANCE Biological nitrogen fixation is the conversion of inert atmospheric dinitrogen gas into nitrogen-reactive ammonia, a reaction catalyzed by the nitrogenase enzyme of diazotrophic bacteria and archaea. Because plants cannot fix their own nitrogen, introducing functional nitrogenase in cereals and other crop plants would reduce our strong dependency on N fertilizers. NifB is required for the biosynthesis of the active site cofactors of all nitrogenases, which arguably makes it the most important protein in global nitrogen fixation. NifB functionality is therefore a requisite to engineer a plant nitrogenase. The expression of nifB genes from a wide range of prokaryotes into the model diazotroph Azotobacter vinelandii shows a surprising level of genetic complementation suggestive of plasticity in the nitrogenase biosynthetic pathway. In addition, we obtained NifB proteins from both mitochondria and chloroplasts of tobacco that are functional in vitro after reconstitution by providing [Fe4S4] clusters from NifU, paving the way to nitrogenase cofactor biosynthesis in plants.https://journals.asm.org/doi/10.1128/mbio.00268-22FeMo-cofactormetalloproteinsnifnitrogen fixationsynthetic biology |
| spellingShingle | Xi Jiang Diana Coroian Emma Barahona Carlos Echavarri-Erasun Rocío Castellanos-Rueda Álvaro Eseverri Jose A. Aznar-Moreno Stefan Burén Luis M. Rubio Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana mBio FeMo-cofactor metalloproteins nif nitrogen fixation synthetic biology |
| title | Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana |
| title_full | Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana |
| title_fullStr | Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana |
| title_full_unstemmed | Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana |
| title_short | Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana |
| title_sort | functional nitrogenase cofactor maturase nifb in mitochondria and chloroplasts of nicotiana benthamiana |
| topic | FeMo-cofactor metalloproteins nif nitrogen fixation synthetic biology |
| url | https://journals.asm.org/doi/10.1128/mbio.00268-22 |
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