Dataset Information

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 N₂ fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle's high O₂ consumption 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.

SUBMITTER: Buren S

PROVIDER: S-EPMC5477005 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae.

Burén Stefan S Young Eric M EM Sweeny Elizabeth A EA Lopez-Torrejón Gema G Veldhuizen Marcel M Voigt Christopher A CA Rubio Luis M LM

ACS synthetic biology 20170303 6

Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N<sub>2</sub> fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle's high O<sub>2</sub> consumption 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 Azo ...[more]

PMID: 28221768

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Project description:Biological nitrogen fixation, the conversion of inert N2 to metabolically tractable NH3, is only performed by certain microorganisms called diazotrophs and is catalyzed by the nitrogenases. A [7Fe-9S-C-Mo-R-homocitrate]-cofactor, designated FeMo-co, provides the catalytic site for N2 reduction in the Mo-dependent nitrogenase. Thus, achieving FeMo-co formation in model eukaryotic organisms, such as Saccharomyces cerevisiae, represents an important milestone toward endowing them with a capacity for Mo-dependent biological nitrogen fixation. A central player in FeMo-co assembly is the scaffold protein NifEN upon which processing of NifB-co, an [8Fe-9S-C] precursor produced by NifB, occurs. Prior work established that NifB-co can be produced in S. cerevisiae mitochondria. In the present work, a library of nifEN genes from diverse diazotrophs was expressed in S. cerevisiae, targeted to mitochondria, and surveyed for their ability to produce soluble NifEN protein complexes. Many such NifEN variants supported FeMo-co formation when heterologously produced in the diazotroph A. vinelandii. However, only three of them accumulated in soluble forms in mitochondria of aerobically cultured S. cerevisiae. Of these, two variants were active in the in vitro FeMo-co synthesis assay. NifEN, NifB, and NifH proteins from different species, all of them produced in and purified from S. cerevisiae mitochondria, were combined to establish successful FeMo-co biosynthetic pathways. These findings demonstrate that combining diverse interspecies nitrogenase FeMo-co assembly components could be an effective and, perhaps, the only approach to achieve and optimize nitrogen fixation in a eukaryotic organism.IMPORTANCEBiological nitrogen fixation, the conversion of inert N2 to metabolically usable NH3, is a process exclusive to diazotrophic microorganisms and relies on the activity of nitrogenases. The assembly of the nitrogenase [7Fe-9S-C-Mo-R-homocitrate]-cofactor (FeMo-co) in a eukaryotic cell is a pivotal milestone that will pave the way to engineer cereals with nitrogen fixing capabilities and therefore independent of nitrogen fertilizers. In this study, we identified NifEN protein complexes that were functional in the model eukaryotic organism Saccharomyces cerevisiae. NifEN is an essential component of the FeMo-co biosynthesis pathway. Furthermore, the FeMo-co biosynthetic pathway was recapitulated in vitro using only proteins expressed in S. cerevisiae. FeMo-co biosynthesis was achieved by combining nitrogenase FeMo-co assembly components from different species, a promising strategy to engineer nitrogen fixation in eukaryotic organisms.

Dataset Information

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae.

Publications

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae.

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