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. 2017 Jun 16;6(6):1043-1055.
doi: 10.1021/acssynbio.6b00371. Epub 2017 Mar 3.

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

Stefan Burén  1 , Eric M Young  2 , Elizabeth A Sweeny  2 , Gema Lopez-Torrejón  1 , Marcel Veldhuizen  1 , Christopher A Voigt  2 , Luis M Rubio  1
Affiliations

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

Stefan Burén et al. ACS Synth Biol. .

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.

Keywords: Azotobacter vinelandii; NifDK; mitochondria; nif genes; nitrogen fixation; yeast.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Measured strengths of transcription units. The geometric mean and standard deviation of biological replicates is plotted in arbitrary units for each promoter-terminator combination. (a) The four expression levels for each of the nine genes in the designed set. (b) The two expression levels for each of the nine genes in the factorial set. The targeted ratios, and how close each promoter-terminator pair approximates that ratio with GFP is shown in Table S2.
Figure 2
Figure 2
Hierarchal assembly. (a) Assembly strategy for transcription units, subclusters and full clusters inserted by homologous recombination in the genome of S. cerevisiae. The standard KanMX selection cassette, abbreviated as “Sel,” was used to select for G418 resistance as a result of successful integration. (b and c) Assembly tree of the designed (b) and factorial (c) set, where each line represents an assembly step (see Methods section and Supporting Information for further details). Note the variants of the tags on each gene in the designed set (b), while the tags are unchanged for each gene in the factorial set (c). (d) Scheme of nif gene organization in DSN14.
Figure 3
Figure 3
Analysis of promoters/terminators and mitochondria targeting signals. Efficiency of each mitochondria targeting signal (a–c) and promoter (d–j) in generating detectable Nif proteins. As no NifH protein was detected when tagged with INDH (a–c), INDH-NifH was excluded in the promoter analysis (d–j). Solid bars indicate % positive clones as detected by Western blotting using antibodies targeting Nif proteins (a–j, left axis). Striped bars indicate observed fluorescence when GFP was expressed by corresponding transcription unit, with the geometric mean and standard deviation of biological replicates plotted in arbitrary units for each promoter–terminator combination (d–j, right axis, see also Figure 1). Data in (d–j) is ordered according to GFP fluorescence levels. bdl, below detection limit.
Figure 4
Figure 4
Nif protein expression in 20 selected yeast clones. Immunoblot analysis of 20 transformed yeast clones, showing expression and migration of NifDK, NifH, NifU, NifS, NifB, NifM, and NifEN proteins, as well as tubulin (loading control). Theoretical masses of corresponding A. vinelandii Nif proteins are indicated. Wild-type (WT) yeast clone is included as control of Nif antibodies specificity. Red squares highlight migration at the location of corresponding A. vinelandii proteins.
Figure 5
Figure 5
Mitochondria targeting of Nif proteins in DSN14. Immunoblot analysis of total extracts (TE) and mitochondria isolations (Mito) from wild-type yeast (WT) and DSN14, where Nif proteins are expressed and targeted to mitochondria using SU9 leader sequences. Analysis using antibodies recognizing cytoplasmic (tubulin) and mitochondria (HSP60) control proteins is included. s.e. and l.e., short and long exposure.
Figure 6
Figure 6
NifDK tetramer formation. (a) Ponceau and immunoblot analysis of yeast DSN14 protein extract, as well as purified A. vinelandii holo- and apo-NifDK proteins (red and blue squares, respectively), separated on anoxic native gels. Polypeptides cross-reacting with NifD, NifK, and NifDK antibodies are highlighted with black arrows. (b) Co-purification of NifK with His-tagged NifD. NifK (green arrows) comigrates with His-tagged NifD (yellow arrows), indication complex formation of NifK and His-NifD polypeptides. In addition, the faster migrating species, presumably N-terminally processed form of NifD (red arrows) is copurifying with His-tagged NifD, suggesting that NifDK tetramer is formed. (c) Ponceau and immunoblot analysis of yeast DSN14 protein extract, as well as purified A. vinelandii holo- and apo-NifDK proteins (red and blue squares, respectively), separated on anoxic native gels in the absence or presence of NafY. Co-migration of NafY protein with apo-NifDK is indicated by green arrow, as compared to unbound NafY (brown arrow). Faster migrating, presumably FeMo-co bound NafY, is indicated by gray arrow. A population of NafY protein comigrating with yeast NifDK is indicated by yellow arrow. s.e., short exposure.
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