A Synthetic Biology Workflow Reveals Variation in Processing and Solubility of Nitrogenase Proteins Targeted to Plant Mitochondria, and Differing Tolerance of Targeting Sequences in a Bacterial Nitrogenase Assay

Okada, Shoko; Gregg, Christina M.; Allen, Robert S.; Menon, Amratha; Hussain, Dawar; Gillespie, Vanessa; Johnston, Ema; Byrne, Keren; Colgrave, Michelle L.; Wood, Craig C.

doi:10.3389/fpls.2020.552160

Cited by 14 publications

(24 citation statements)

References 51 publications

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“…3,5,6 Understanding the biosynthesis of nitrogenase metalloclusters is crucial from the practical perspective of endowing certain eukaryotes the capacity to fix nitrogen by transferring the genetic determinants from diazotrophs, as well as for improving the nitrogen fixing capacity of symbiotic nitrogen fixing microbes associated with crop plants. [7][8][9][10] There is some topological similarity between the P-cluster and FeMo-co (Figure 1) as their Fe-S cores can be considered to represent fused or bridged sub-cluster units. Recent refinement of a structural model of NifB, involved in formation of the Fe-S-C core common to all three nitrogenase cofactors, suggested a P-cluster-like intermediate as FeMo-co precursor during its formation.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster

et al. 2022

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster

et al. 2022

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“…This study shows that functional nitrogenase NifH protein can be expressed in tobacco chloroplasts during the dark period, even if plants are grown at standard atmospheric O 2 levels. In contrast to mitochondria-targeted K. oxytoca NifH, which is insoluble even when expressed together with NifM (Okada et al, 2020), the A. vinelandii NifH expressed with NifM inside chloroplasts was soluble overcoming a major obstacle for nitrogenase engineering in plants. Chloroplast metabolism in darkness appears to permit NifH protein maturation, which At least two individual leaves from sixty plants were assayed.…”

Section: Discussionmentioning

confidence: 96%

Analysis of Nitrogenase Fe Protein Activity in Transplastomic Tobacco

et al. 2021

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Integration of prokaryotic nitrogen fixation (nif) genes into the plastid genome for expression of functional nitrogenase components could render plants capable of assimilating atmospheric N2 making their crops less dependent of nitrogen fertilizers. The nitrogenase Fe protein component (NifH) has been used as proxy for expression and targeting of Nif proteins within plant and yeast cells. Here we use tobacco plants with the Azotobacter vinelandii nifH and nifM genes integrated into the plastid genome. NifH and its maturase NifM were constitutively produced in leaves, but not roots, during light and dark periods. Nif protein expression in transplastomic plants was stable throughout development. Chloroplast NifH was soluble, but it only showed in vitro activity when isolated from leaves collected at the end of the dark period. Exposing the plant extracts to elevated temperatures precipitated NifM and apo-NifH protein devoid of [Fe4S4] clusters, dramatically increasing the specific activity of remaining NifH protein. Our data indicate that the chloroplast endogenous [Fe-S] cluster biosynthesis was insufficient for complete NifH maturation, albeit a negative effect on NifH maturation due to excess NifM in the chloroplast cannot be excluded. NifH and NifM constitutive expression in transplastomic plants did not affect any of the following traits: seed size, germination time, germination ratio, seedling growth, emergence of the cotyledon and first leaves, chlorophyll content and plant height throughout development.

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“…This study found that NifF, M, N, S, U, W, X, Y, and Z can be expressed in soluble forms, whereas, NifB, E, H, J, K, Q, and V were insoluble. It was also found that the nitrogenase activity of NifM was reduced to 10% of the control because of the N-terminal processing (MTP) that occurs during mitochondrial transport [74]. Furthermore, the NifD protein itself contains an MTPrecognition sequence, which is subject to processing, thereby making functional expression difficult.…”

Section: Ammonia Production Using Nitrogenasementioning

confidence: 99%

Sustainable Biological Ammonia Production towards a Carbon-Free Society

2021

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A sustainable society was proposed more than 50 years ago. However, it is yet to be realised. For example, the production of ammonia, an important chemical widely used in the agriculture, steel, chemical, textile, and pharmaceutical industries, still depends on fossil fuels. Recently, biological approaches to achieve sustainable ammonia production have been gaining attention. Moreover, unlike chemical methods, biological approaches have a lesser environmental impact because ammonia can be produced under mild conditions of normal temperature and pressure. Therefore, in previous studies, nitrogen fixation by nitrogenase, including enzymatic ammonia production using food waste, has been attempted. Additionally, the production of crops using nitrogen-fixing bacteria has been implemented in the industry as one of the most promising approaches to achieving a sustainable ammonia economy. Thus, in this review, we described previous studies on biological ammonia production and showed the prospects for realising a sustainable society.

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A Synthetic Biology Workflow Reveals Variation in Processing and Solubility of Nitrogenase Proteins Targeted to Plant Mitochondria, and Differing Tolerance of Targeting Sequences in a Bacterial Nitrogenase Assay

Cited by 14 publications

References 51 publications

A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster

A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster

Analysis of Nitrogenase Fe Protein Activity in Transplastomic Tobacco

Sustainable Biological Ammonia Production towards a Carbon-Free Society

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