Expression of Active Subunit of Nitrogenase via Integration into Plant Organelle Genome (original) (raw)
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State of the art in eukaryotic nitrogenase engineering
FEMS microbiology letters, 2018
Improving the ability of plants and plant-associated organisms to fix and assimilate atmospheric nitrogen has inspired plant biotechnologists for decades, not only to alleviate negative effects on nature from increased use and availability of reactive nitrogen, but also because of apparent economic benefits and opportunities. The combination of recent advances in synthetic biology and increased knowledge about the biochemistry and biosynthesis of the nitrogenase enzyme has made the seemingly remote and for long unreachable dream more possible. In this review, we will discuss strategies how this could be accomplished using biotechnology, with a special focus on recent progress on engineering plants to express its own nitrogenase.
Analysis of Nitrogenase Fe Protein Activity in Transplastomic Tobacco
Frontiers in Agronomy, 2021
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 ac...
Plant Biotechnology Journal, 2020
SummaryThe generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural ‘green’ revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH, M, U and S, were re‐designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl‐prolyl cis‐trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe‐4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of nifU and nifS co‐expression. These results support th...
Functional expression of the nitrogenase Fe protein in transgenic rice
Communications Biology
Engineering cereals to express functional nitrogenase is a long-term goal of plant biotechnology and would permit partial or total replacement of synthetic N fertilizers by metabolization of atmospheric N2. Developing this technology is hindered by the genetic and biochemical complexity of nitrogenase biosynthesis. Nitrogenase and many of the accessory proteins involved in its assembly and function are O2 sensitive and only sparingly soluble in non-native hosts. We generated transgenic rice plants expressing the nitrogenase structural component, Fe protein (NifH), which carries a [4Fe-4S] cluster in its active form. NifH from Hydrogenobacter thermophilus was targeted to mitochondria together with the putative peptidyl prolyl cis‐trans isomerase NifM from Azotobacter vinelandii to assist in NifH polypeptide folding. The isolated NifH was partially active in electron transfer to the MoFe protein nitrogenase component (NifDK) and in the biosynthesis of the nitrogenase iron-molybdenum c...
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...
Transcriptional regulators of nitrate metabolism: Key players in improving nitrogen use in crops
Journal of Biotechnology, 2020
Green revolution has boosted crop yields by the development of varieties which rely on high fertilizer application. Since then, higher productivity has largely witnessed excessive nitrogen (N) fertilizer application resulting in many environmentally and agronomically unsustainable consequences. One possible solution to this problem is to develop varieties with efficient N use endowed with genetically superior N metabolizing machinery, thereby significantly reducing N loss in soil and facilitating gainful yield performance at lower N conditions. Nitrate (NO 3 −) is the major form of N acquired by plants in aerobic soils. Hence, its efficient acquisition, transport, assimilation into complex organic compounds, and overall homeostasis is crucial to ensure productivity under optimal and suboptimal N conditions. Transcription factors are prime regulators of these processes, and insights into their mechanism of action and the resultant effect on N metabolism are crucial to generating crops with efficient and durable nitrogen use efficiency. The present review, therefore, presents a comprehensive updated account of major N responsive transcription factor families, their cross-talk with other growth factors, and explores existing and potential areas of their biotechnological application to maximize crop yields.
International Journal of Molecular Sciences, 2021
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 C...
PLoS ONE, 2013
Biological nitrogen fixation is a complex process requiring multiple genes working in concert. To date, the Klebsiella pneumoniae nif gene cluster, divided into seven operons, is one of the most studied systems. Its nitrogen fixation capacity is subject to complex cascade regulation and physiological limitations. In this report, the entire K. pneumoniae nif gene cluster was reassembled as operon-based BioBrick parts in Escherichia coli. It provided ,100% activity of native K. pneumoniae system. Based on the expression levels of these BioBrick parts, a T7 RNA polymerase-LacI expression system was used to replace the s 54-dependent promoters located upstream of nif operons. Expression patterns of nif operons were critical for the maximum activity of the recombinant system. By mimicking these expression levels with variable-strength T7dependent promoters, ,42% of the nitrogenase activity of the s 54-dependent nif system was achieved in E. coli. When the newly constructed T7-dependent nif system was challenged with different genetic and physiological conditions, it bypassed the original complex regulatory circuits, with minor physiological limitations. Therefore, we have successfully replaced the nif regulatory elements with a simple expression system that may provide the first step for further research of introducing nif genes into eukaryotic organelles, which has considerable potentials in agro-biotechnology.
Journal of Experimental Botany
Plants depend upon beneficial interactions between roots and root-associated microorganisms for growth promotion, disease suppression, and nutrient availability. This includes the ability of free-living diazotrophic bacteria to supply nitrogen, an ecological role that has been long underappreciated in modern agriculture for efficient crop production systems. Long-term ecological studies in legume–rhizobia interactions have shown that elevated nitrogen inputs can lead to the evolution of less cooperative nitrogen-fixing mutualists. Here we describe how reprogramming the genetic regulation of nitrogen fixation and assimilation in a novel root-associated diazotroph can restore ammonium production in the presence of exogenous nitrogen inputs. We isolated a strain of the plant-associated proteobacterium Kosakonia sacchari from corn roots, characterized its nitrogen regulatory network, and targeted key nodes for gene editing to optimize nitrogen fixation in corn. While the wild-type strai...
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...