title: Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae.
abstract: Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle's high O2consumption and the presence of bacterial-type iron-sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.
authors.affiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) , Campus Montegancedo UPM, 28223, Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Stefan
authors.initials: S
authors.lastname: Burén
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Young
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Sweeny
authors.affiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) , Campus Montegancedo UPM, 28223, Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Gema
authors.initials: G
authors.lastname: Lopez-Torrejón
authors.affiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) , Campus Montegancedo UPM, 28223, Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Marcel
authors.initials: M
authors.lastname: Veldhuizen
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Voigt
authors.affiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) , Campus Montegancedo UPM, 28223, Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Luis M
authors.identifier: 0000-0003-1596-2475
authors.initials: LM
authors.lastname: Rubio
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D009586:Nitrogen Fixation; D009591:Nitrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D009591:Nitrogenase
keywords: Azotobacter vinelandii; NifDK; mitochondria; nif genes; nitrogen fixation; yeast