Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants

Jiang, Xi; Payá-Tormo, Lucía; Coroian, Diana; García-Rubio, Inés; Castellanos-Rueda, Rocío; Eseverri, Álvaro; López‐Torrejón, Gema; Burén, Stefan; Rubio, Luis M.

doi:10.1038/s42003-020-01536-6

Cited by 35 publications

(65 citation statements)

References 49 publications

(50 reference statements)

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“…Expression of active NifH (the Fe protein component of the Mo nitrogenase) and NifB (a FeMo-co maturating protein) in mitochondria of S. cerevisiae has proven that these extremely O 2 -sensitive nitrogenase proteins can accumulate in a eukaryotic cell, representing the first steps towards engineering nitrogenase in eukaryotes (L opez-Torrej on et al, 2016;Bur en et al, 2017). Recent studies have also shown that both mitochondria and chloroplasts of tobacco can harbour active NifH at the end of the dark period (Eseverri et al, 2020;Jiang et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Biosynthesis of cofactor‐activatable iron‐only nitrogenase in Saccharomyces cerevisiae

López‐Torrejón

Burén

Veldhuizen

et al. 2021

Microbial Biotechnology

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Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe-only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show the expression of stable Fe-only nitrogenase component proteins in the low-oxygen mitochondria matrix of S. cerevisiae. As-isolated Fe protein (AnfH) was active in electron donation to NifDK to reduce acetylene into ethylene. Ancillary proteins NifU, NifS and NifM were not required for Fe protein function. The FeFe protein existed as apo-AnfDK complex with the AnfG subunit either loosely bound or completely unable to interact with it. Apo-AnfDK could be activated for acetylene reduction by the simple addition of FeMo-co in vitro, indicating preexistence of the Pclusters even in the absence of coexpressed NifU and NifS. This work reinforces the use of Fe-only nitrogenase as simple model to engineer nitrogen fixation in yeast and plant mitochondria.

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Section: Introductionmentioning

confidence: 99%

Biosynthesis of cofactor‐activatable iron‐only nitrogenase in Saccharomyces cerevisiae

López‐Torrejón

Burén

Veldhuizen

et al. 2021

Microbial Biotechnology

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“…Strep-tagged NifU purified from E. coli was reconstituted in vitro as described (31) with slight modifications. 20 μM of NifU dimer was prepared in 100 mM MOPS (pH 7.5) buffer containing 8 mM 1,4-dithiothreitol (DTT) and incubated at 37 °C for 30 min.…”

Section: Discussionmentioning

confidence: 99%

Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase

Barahona

Jiang

Jiménez-Vicente

et al. 2021

Preprint

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Azotobacter vinelandii molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe3S4] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] cluster. Protein-protein interaction studies using apo-NifQ and as-isolated NifU demonstrated the interaction between both proteins which is only effective when NifQ is unoccupied by its [Fe-S] cluster. The apo-NifQ iron content increased after the incubation with as-isolated NifU, reaching similar levels to holo-NifQ after the interaction between apo-NifQ and NifU with reconstituted transient [Fe4-S4] groups. These results also indicate the necessity of co-expressing NifU together with NifQ in the pathway to provide molybdenum for the biosynthesis of nitrogenase in engineered nitrogen-fixing plants.

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“…Currently, there are no examples of the other nitrogenase components expressed in C. reinhardtii, although their accumulation has been achieved in plant and yeast mitochondria [19][20][21]24,63,67]. Again, plastid accumulation of the Anf structural subunits (Figure 2C) should be achievable with the correct choice of regulatory elements and environmental conditions (e.g., anaerobiosis) to optimise protein expression.…”

Section: Expression Of the Anf /Nif Genes In The Chloroplast Of C Reinhardtiimentioning

confidence: 99%

The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants

Larrea-Álvarez

Purton

2021

IJMS

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Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N2) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N2 by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme’s sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle’s genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an ‘Fe-only’ nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.

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Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants

Cited by 35 publications

References 49 publications

Biosynthesis of cofactor‐activatable iron‐only nitrogenase in Saccharomyces cerevisiae

Biosynthesis of cofactor‐activatable iron‐only nitrogenase in Saccharomyces cerevisiae

Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase

The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants

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