Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

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.


SUPPLEMENTARY TABLES AND FIGURES
Supplementary Table S1  Sequences  Supplementary Table S2 Part assignment Supplementary Table S3  Assembly  Supplementary Table S4 Quality control and results Supplementary Table S5 Mitochondrial signal efficiency Supplementary Table S6 Promoter efficiency Supplementary Table S7 Regression analysis Supplementary Figure S1 Analysis of promoter efficiency in generating detectable NifEN protein Supplementary Figure S2 Migration of NifD and NifK polypeptides Supplementary Figure S3 Mitochondria targeting of Nif proteins Supplementary Figure S4 Expression of Nif proteins in DSN14 Supplementary Figure S5 NifDK tetramer formation in DSN14 Supplementary Figure S6 Detailed TypeIIS hierarchy for cluster assembly Supplementary Figure S7 Level 1 destination vector design Supplementary Figure S8 Spacer design Supporting Figure S1. Analysis of promoter efficiency in generating detectable NifEN protein.
Solid bars indicate % positive clones as detected by Western blotting using antibodies targeting NifE (a) and NifN (b) (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 (right axis, see also Figure 1). Data is ordered according to GFP fluorescence levels. Note that NifE and NifN could not be sufficiently separated by SDS-PAGE, and Western blotting result therefore reflects total NifEN protein. bdl, below detection limit.
Supporting Figure S2. Migration of NifD and NifK polypeptides. (a) 50 ng samples of purified A. vinelandii apo-NifDK protein was loaded on 7% SDS-PAGE gel. Following separation and transfer, membrane was cut in two and probed with NifD and NifK specific antibodies, and finally developed side by side. Dotted line indicate cut of the membrane. (b) Immunoblot of purified A. vinelandii NifDK protein, together with total protein extracts from selected yeast clones, using antibodies recognizing NifDK. NifD (red arrow) and NifK (green arrow) proteins are indicated. Yeast strains expressing NifD or NifDK (DSN14, DSN24 and DOE1), also show presence of faster migrating polypeptide (yellow arrows). (c) Immunoblot analysis of DSN20 developed with antibodies targeting NifD or NifK specifically, or NifDK, as indicated to the right of each panel. NifD (red arrows) and NifK (green arrows) proteins are indicated, together with the faster migrating NifD polypeptide (yellow arrows). (d) Immunoblot analysis of protein extracts from yeast transformed with plasmid encoding mitochondria (SU9) targeted versions of His-tagged NifD and non-tagged NifK. Cells were grown in SD media supplemented with glucose (non-induced, Glc) or galactose (induced, Gal). Antibodies recognizing his-tag detect full-length NifD (red arrows), but not the faster migrating NifD polypeptide (yellow arrow).
Supporting Figure S3. Mitochondria targeting of Nif proteins. Immunoblot analysis of total extracts (TE) and mitochondria isolations (Mito) from three yeast strains with Nif proteins expressed and targeted using SU9, SOD2 or INDH mitochondria leader sequences. Antibodies recognizing cytoplasmic (tubulin) and mitochondria (HSP60) control proteins were used as controls. s.e. and l.e., short and long exposure.
Supporting Figure S4. Expression of Nif proteins in DSN14. Immunoblot analysis to compare migration of Nif proteins from A. vinelandii and yeast strain DSN14.
Supporting Figure S5. NifDK tetramer formation in DSN14. Co-purification of NifK with Histagged NifD. NifK (green arrows) co-migrate with His-tagged NifD (yellow arrows), indicating complex formation of NifK and His-NifD polypeptides. Subclusters were linearized with BsaI and mixed with homology fragments to target the subclusters to the genome. Every assembly step is tabulated in Supporting Table S3.

Supporting
Supporting Figure S7. Level 1 destination vector design. Each Level 1 vector contains the A scar (GTGC) and the D scar (CCTC) for receiving transcription units upon BsaI digestion. Successful ligation of the transcription unit parts into the vector will eliminate the constitutive bacterial RFP expression cassette, causing loss of a red colony color. On either side of the A and D scars, there is a half spacer. See Spacer Design for more details. Outside of the spacers, positional scars are located. These are designated E, F, G, H, I, J, K, L, M, N, and O. When assembling subclusters, these positional scars will dictate the order in which the transcription units assemble. This step will also match the spacers as intended (S1 with S2, S3 with S4, etc.).
Supporting Figure S8. Spacer design. (a) Minimal terminator elements used to design spacers. One efficiency element, four different positioning elements, and two polyadenylation elements used. (b) Spacer with forward and reverse terminator elements. These elements were randomly combined, with random DNA in between the elements, in the orientation shown here.