Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae (original) (raw)
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Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae
ACS synthetic biology, 2017
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. Ex...
Nature communications, 2016
The extreme sensitivity of nitrogenase towards oxygen stands as a major barrier to engineer biological nitrogen fixation into cereal crops by direct nif gene transfer. Here, we use yeast as a model of eukaryotic cell and show that aerobically grown cells express active nitrogenase Fe protein when the NifH polypeptide is targeted to the mitochondrial matrix together with the NifM maturase. Co-expression of NifH and NifM with Nif-specific Fe-S cluster biosynthetic proteins NifU and NifS is not required for Fe protein activity, demonstrating NifH ability to incorporate endogenous mitochondrial Fe-S clusters. In contrast, expression of active Fe protein in the cytosol requires both anoxic growth conditions and co-expression of NifH and NifM with NifU and NifS. Our results show the convenience of using mitochondria to host nitrogenase components, thus providing instrumental technology for the grand challenge of engineering N2-fixing cereals.
An experimental workflow identifies nitrogenase proteins ready for expression in plant mitochondria
2019
Industrial nitrogen fertilizer is intrinsic to modern agriculture yet expensive and environmentally harmful. We aim to reconstitute bacterial nitrogenase function within plant mitochondria to reduce nitrogen fertilizer usage. Many nitrogen fixation (Nif) proteins are required for biosynthesis and function of the mature nitrogenase enzyme, and these will need to be correctly processed and soluble within mitochondria as a pre-requisite for function. Here we present our workflow that assessed processing, solubility and relative abundance of 16Klebsiella oxytocaNif proteins targeted to the plant mitochondrial matrix using an Arabidopsis mitochondrial targeting peptide (MTP). The functional consequence of the N-terminal modifications required for mitochondrial targeting of Nif proteins was tested using bacterial nitrogenase assays. We found that despite the use of the same constitutive promoter and MTP, MTP::Nif processing and relative abundance in plant leaf varied considerably. Assessm...
Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB
Frontiers in Plant Science
Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O 2-sensitive S-adenosyl methionine (SAM)-radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codonoptimized versions of NifB from the γ-proteobacterium Azotobacter vinelandii and the thermophilic methanogen Methanocaldococcus infernus in Saccharomyces cerevisiae and in Nicotiana benthamiana. NifB proteins were targeted to the mitochondria, where O 2 consumption is high and bacterial-like [Fe-S] cluster assembly operates. In yeast, NifB proteins were co-expressed with NifU, NifS, and FdxN proteins that are involved in NifB [Fe-S] cluster assembly and activity. The synthetic version of thermophilic NifB accumulated in soluble form within the yeast cell, while the A. vinelandii version appeared to form aggregates. Similarly, NifB from M. infernus was expressed at higher levels in leaves of Nicotiana benthamiana and accumulated as a soluble protein while A. vinelandii NifB was mainly associated with the non-soluble cell fraction. Soluble M. infernus NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the in vitro FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening nif genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase.
Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae
Proceedings of the National Academy of Sciences
The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure–Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter ...
Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix
Frontiers in plant science, 2017
The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, a...
Proceedings of the National Academy of Sciences, 2020
Significance Stable expression of each component of the nitrogenase system in an active form is a prerequisite for engineering nitrogen fixation in eukaryotic cells. Mitochondria provide an oxygen-depleted environment for the expression of active nitrogenase in plants, but signal peptides are required to target nuclear encoded Nif proteins to this organelle. We demonstrate that one of the structural subunits of nitrogenase, NifD, is itself susceptible to cleavage by mitochondrial processing peptidases from a variety of plant origins, presenting a major challenge to engineering nitrogen fixation in mitochondria. To overcome this issue, we have engineered NifD variants that are resistant to cleavage and retain high levels of nitrogenase activity, thus providing a potential solution for engineering active MoFe protein in plants.
Communications Biology, 2021
Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. Hydrogenobacter thermophilus (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N2 fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. H. thermophilus NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe4S4] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolat...
Journal of Bacteriology, 1993
The niJBQ transcriptional unit ofAzotobacter vinelandii has been previously shown to be required for activity of the three nitrogenase systems, Mo nitrogenase, V nitrogenase, and Fe nitrogenase, present in this organism. We studied regulation of expression and the role of the nifBQ region by means of translational 13-galactosidase fusions to each of the five open reading frames: nifB, orf2 (fdxN), orJ3 (nifO), nifQ, and orf5. Expression of the first three open reading frames was observed under all three diazotrophic conditions; expression of orfs was never observed. Genes nifB andfdxN were expressed at similar levels. With Mo, expression of nifO and nifQ was-20and =400-fold lower than that offdxN, respectively. Without Mo, expression of nifB dropped threeto fourfold and that of nijQ dropped to the detection limit. However, expression of nifO increased threefold. The products of nijB, fdxN, nifO, and nifQ have been visualized in A. vinelandii as 13-galactosidase fusion proteins with the expected molecular masses. The NifBfusion lacked activity for any of the three nitrogenase systems and showed an iron-molybdenum cofactor-deficient phenotype in the presence of Mo. The FdxNmutation resulted in reduced nitrogenase activities, especially when V was present. Dinitrogenase activity in extracts was similarly affected, suggesting a role of FdxN in iron-molybdenum cofactor synthesis. The NifO-producing mutation did not affect any of the nitrogenases under standard diazotrophic conditions. The NifQ-producing mutation resulted in an increased (-1,000-fold) Mo requirement for Mo nitrogenase activity, a phenotype already observed with Klebsiella pneumoniae. No effect of the NifQ-producing mutation on V or Fe nitrogenase was found; this is consistent with its very low expression under those conditions. Mutations in orfS had no effect on nitrogenase activity.
Plant expression of NifD protein variants resistant to mitochondrial degradation
Proceedings of the National Academy of Sciences, 2020
Significance Engineering nitrogenase in plants may help alleviate economic and environmental issues due to the use of nitrogen fertilizer. Mitochondria have shown promise in supporting the function of nitrogenase, including electron donation and metallocluster assembly. Despite these successes, formation of the catalytic unit, NifDK, has proven difficult. Here, we find that when relocated to plant mitochondria, NifD is subject to errant peptidase-based cleavage and is insoluble. Guided by NifD sequence variation amongst bacteria and structural modeling, we designed NifD variants that avoided cleavage and retained function in bacterial assays. Fusion of NifK to degradation-resistant NifD also improved solubility, and the polyprotein retained function in bacterial assays. This work advances efforts to produce crops less reliant on nitrogen fertilizer.