Expression of Active Subunit of Nitrogenase via Integration into Plant Organelle Genome

Ivleva, Natalia B.; Groat, Jeanna; Staub, Jeffrey M.; Stephens, M. A.

doi:10.1371/journal.pone.0160951

Cited by 89 publications

(73 citation statements)

References 44 publications

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“…In plant chloroplasts ex vivo Fe protein activity required the lowering of ambient oxygen conditions (Ivleva et al, 2016). By contrast, aerobically grown yeast expressing matrix targeted NifH and NifM were capable of producing a functional Fe protein (López-Torrejón et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix

et al. 2017

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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, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

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Section: Discussionmentioning

confidence: 99%

Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix

et al. 2017

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“…1), such as those required for iron-sulfur cluster assembly, could also be considered. Two recent studies have shown that the [Fe-S] cluster biosynthesis components from eukaryotic organelles can substitute for NifU and NifS to incorporate the [4Fe-4S] cluster into the Fe protein of nitrogenase (19,20).…”

Section: Discussionmentioning

confidence: 99%

“…A number of studies have suggested chloroplasts, root plastids, or mitochondria as suitable locations for expression of nitrogenase in eukaryotes (18)(19)(20). These energy-conversion organelles can potentially provide reducing power and ATP required for the nitrogen-fixation process.…”

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confidence: 99%

Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity

Yang

Xie

Yang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the "core" nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin-NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR-ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops.nitrogen fixation | electron transport | plant organelles | nitrogenase engineering

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“…In recent studies it has been shown that an active dinitrogenase reducatase can be produced in eukaryotes, provided it is targeted to either mitochondria in yeast or plastids in tobacco (12,13). These achievements are noteworthy for two reasons.…”

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confidence: 99%

“…However, neither NifU nor NifS is necessary for the production of an active nitrogenase reductase in either yeast or tobacco (12,13), indicating that counterparts involved in assembly of iron-sulfur cofactors in eukaryotic organisms-for example Isu and Nfs1 (15)-can substitute for the nitrogen-fixation-specific assembly components. It should be noted that NifU and NifS also supply the iron-sulfur building blocks necessary for formation of the complex cofactors associated with dinitrogenase (Fig.…”

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confidence: 99%