Patent Application
20190338257
The present invention relates to an electron transport chain (ETC) module from eukaryotic organelles and application of the ETC module in biological nitrogen fixation.
Nitrogen is one of the primary nutrients limiting crop growth and yield in agriculture[1]. The use of industrial nitrogen fertilizer can circumvent this limitation and provide sufficient nitrogen source for crop growth. However, the extensive use of industrial nitrogen fertilizers can lead to environmental problems, and the economic costs of nitrogen fertilizers used are relatively high. These problems are particularly significant in developing countries[2-3]. These factors have led researchers to refocus on reconstructing the biological nitrogenase system in crops by engineering methods to achieve crop self-nitrogen fixation to solve the problem of nitrogen fertilizer use. Biological nitrogen fixation (BNF), a process that converts gaseous nitrogen to ammonia by nitrogenases in diazotrophs, contributes over 60% of the nitrogen in the atmospheric nitrogen cycle[4]. Nitrogenases are a family of metalloenzymes that consist of two separable components, dinitrogenase reductase (Fe protein) and dinitrogenase (XFe protein, where X is Mo, V, or Fe, depending on the metal atom composition of the active site cofactor) (see
A number of studies have suggested chloroplasts, root-plastids or mitochondria can be ideal locations to introduce a nitrogenase system in eukaryotes[18-20]. These organelles responsible for energy conversion can potentially provide reducing power and ATP required for nitrogen fixation process. Diverse reduction reactions carried out in these organelles rely on different electron transport chains (ETCs)[21]. Existing researches have confirmed that multiple copies of ferredoxin are contained in plant cells, including photosynthetic or non-photosynthetic ferredoxins expressed in chloroplasts or root-plastids; and mitochondria ferredoxin-like adrenodoxins (MFD) located in the mitochondria[21-22]. The major function of the photosynthetic ferredoxins expressed in chloroplasts is to mediate the transfer of electrons from photosystem I (PSI) to Ferredoxin-NADPH oxidoreductase (leaf-type FNR, LFNR) to catalyze the production of NADPH[23]. In addition, photosynthetic ferredoxins are also responsible for the distribution of reducing power derived from the photosynthetic process to proteins involved in nitrogen and sulfur assimilation[24]. Electron transfer between root-type FNR (RFNR) and ferredoxin in the root-plastid is opposite to that in the chloroplast, with NADPH generated in the oxidative pentose-phosphate pathway (OxPPP) transferring electrons to ferredoxin via RFNR to reduce the ferredoxin protein[25]. In mitochondria, MFD mediates the transfer of electrons from NADPH-dependent adrenodoxin oxidoreductase (MFDR) to the cysteine desulfurase Nfs1 to participate in the biosynthesis of the biotin[26].
In the previous study, we have successfully constructed recombinant MoFe[27] nitrogenase systems from Klebsiella oxytoca (Ko) and the “minimal” FeFe[28] nitrogenase systems from Azotobacter vinelandii (Av) in Escherichia coli (
Biological nitrogen fixation is a complex system involving many genes, and it is also a process that requires a large amount of ATP and reducing power. Thus, the involvement of excess genes in the nitrogenase system, the energy available to the nitrogenase system in a particular host environment, and the reducing power are major bottlenecks in the reconstitution of nitrogenase systems in crops. In recent studies, attempts have been made to reduce the number of structural genes required for MoFe nitrogenase[29] and FeFe nitrogenase[28] to simplify the nitrogenase system. However, it was found that when the number of structural genes was reduced to 9, the activity of MoFe nitrogenase[29] decreased sharply, and when some genes were replenished into the system, the nitrogenase activity could be restored to a higher level[30]. These results demonstrate the difficulty of simplifying the nitrogenase system without losing the efficiency of nitrogenase.
In the present invention, we first introduced the concept of modularization of synthetic biology and systematic biology, and divided the nitrogenase system into three functionally independent modules: an electron transport chain (ETC) module, a metal cluster synthesis module, and a nitrogenase “core” enzyme module. And it was further investigated that if electron transport components from plant chloroplasts, root-plastids or mitochondria, including ferredoxin or Ferredoxin-NADPH oxidoreductase (FNR), can replace NifF or NifJ proteins in ETC modules, respectively, to provide electrons for the MoFe and FeFe nitrogenase systems. In this study, we used the model organism E. coli as the “chassis” to study the compatibility of the “core” enzyme module of the FeFe/MoFe nitrogenase system with ETC modules from plant organelles such as chloroplasts, root-plastids and mitochondria.
In the present invention, a new ETC module in which the NifJ or/and NifF protein is replaced is recombinantly produced, and whether the plant-derived ETC module can substitute the NifJ or NifF protein in the ETC module to provide electrons for the “core” enzyme module of the FeFe/MoFe nitrogenase system to support the activity of nitrogenases is determined by the acetylene reduction method and the 15N2 assimilation assay. The means of “substitution” described herein include: the NifF protein is replaced by the ferredoxin protein derived from plant organelles, or the NifJ protein is replaced by the FNR protein derived from plant organelles, or both of the above replacements are performed simultaneously. The ETC module formed when one of the NifJ or NifF proteins is replaced, is called a hybrid module, and the ETC module formed when they are simultaneously replaced by the ferredoxin protein and the FNR protein of the plant organelle, is called an intact module. More specifically, a hybrid ETC module described herein is formed by replacing NifF in the NifJ-NifF module with ferredoxin from chloroplasts, root-plastids or mitochondria of various representative plants, or is composed by the hybrid ETC module consisting of plant-type FNR and Anabaena sp. PCC 7120 (As) FdxH or FdxB. The intact ETC module described herein is a plant-derived ETC module formed by replacing the NifJ and NifF proteins present in the ETC module of diazotrophs with encoded ferredoxin-NADPH oxidoreductase (FNR) and ferredoxin from the target plant organelle, respectively.
The results of the present study indicate that all plant-derived ferredoxins except ferredoxins from mitochondria can functionally replace NiFe of FeFe and MoFe nitrogenases, which means that the interaction between these ferredoxins and NifH/AnfH can meet the needs of electron transport; the intact ETC module (FNR-Ferredoxin) from the chloroplasts and root-plastids of various plants can support the activity of nitrogenase, which means that engineering and reconstituting nitrogen fixation system in the plant plastid does not need to additionally carry the ETC module; the hybrid module formed by mitochondrial MFDR and Anabaena FdxH/FdxB can support the nitrogenase activity; and after analyzing the source of the above substitution components, it can be concluded that the chloroplast-derived ETC module can provide the most suitable electron supply for the nitrogenase system in E. coli.
Therefore, based on the technical solution of the above-mentioned replaceable ETC module, it is beneficial for us to use the endogenous ETC derived from plant organelles in the process of biological nitrogen fixation in the future, thereby avoiding the technical obstacles caused by excessive number of nitrogenase structural genes, high energy demand and reducing power in the process of engineering and reconstituting the biological nitrogenase system in plant cells.
More specifically, the present invention specifically relates to the following aspects:
One aspect of the invention relates to an electron transport chain (ETC) module for a nitrogen fixation system, comprising an NifJ protein and an NifF protein.
The ETC module described in the above aspect, wherein the nitrogen fixation system is MoFe nitrogenase system and FeFe nitrogenase system.
The ETC module described in the above aspects, wherein the NifJ and NifF proteins are substituted individually or substituted simultaneously by corresponding proteins from eukaryotic organelles, thereby forming a hybrid or intact ETC module.
The ETC module described in the above aspects, wherein the eukaryotic organism is a plant, and the organelle is a plastid or mitochondria.
The ETC module described in the above aspects, wherein the hybrid ETC module is formed by replacing the NifF protein in the ETC module by ferredoxins from a plant plastid.
The ETC module described in the above aspects, wherein the plastid is a chloroplast or a root-plastid, preferably a chloroplast.
The ETC module described in the above aspects, wherein the hybrid ETC module is formed by replacing an NifJ protein in the ETC module consisting of the NifJ and the NifF by plant-type ferredoxin-NADPH reductase (FNR) from a plant plastid and mitochondria.
The ETC module described in the above aspects, wherein the plastid is a chloroplast or a root-plastid.
The ETC module described in the above aspects, wherein the hybrid ETC module is composed of NADPH-dependent adrenodoxin oxidoreductase (MFDR) from plant mitochondria and Anabaena FdxB.
The ETC module described in the above aspects, wherein the intact ETC module is composed of FNRs from target plant organelles and ferredoxin proteins.
The ETC module described in the above aspects, wherein the target plant organelle is a chloroplast or a root-plastid.
The use of the ETC module of any of the preceding items in biological nitrogen fixation.
Materials and Methods
Bacterial Strains and Plasmids Used in the Examples are Shown in Table 1
E. coli Strains
Bacterial Strains and Growth Medium
Luria-Bertani (LB) broth for E. coli growth contained 10 g/L of Tryptone, 5 g/L of Yeast Extract and 10 g/L of NaCl. KPM minimal media used in this study contained 10.4 g/L of Na2HPO4, 3.4 g/L of KH2PO4, 26 mg/L of CaCl2. 2H2O, 30 mg/L of MgSO4, 0.3 mg/L of MnSO4, 36 mg/L of ferric citrate, 10 mg/L of para-aminobenzoic acid, 5 mg/L of biotin, 1 mg/L Vitamin B1 and 0.8% (w/v) glucose, with 20 mM ammonium salt (KPM-HN) or 10 mM glutamate (KPM-LN) as the nitrogen source. Casamino acids (purchased from BD Biosciences, 223050) at final concentration of 0.05% were also added to the KPM minimal media to ensure the normal growth. Antibiotics were used at the following concentration: 50 μg/mL for ampicillin, 25 μg/mL for chloramphenicol.
Construction of Recombinant Plasmids
Plasmid pKU7815 and pKU7017 are pACYC184 derivatives carrying the “minimal” FeFe[28] or the recombined MoFe[27] nitrogenase system. nifF and nifJ double deletion derived plasmid of pKU7815 and pKU7017, designed as pKU7830 and pKU7831 in the Examples, were constructed by direct removal of the nifF and nifJ operons using the specific restriction sites flanking each operon, SwaI for the nifF and ScaI for the nifJ respectively. The complementary plasmid for the pKU7830 and pKU7831 is a pBR322M derived plasmid (pKU7832) carrying the nifF and nifJ genes. The pBR322M-PLtetO-1 plasmid was obtained by direct reassembly of the tetR expression cassette and the PLtetO-1 promoter region with the pBR322M as backbone using Gibson Assemble kit (NEB, E5520S). The tetR expression cassette comprises a strong constitutive promoter (BBa_J23100, https://parts.igem.org), a medium ribosome binding site (RBS, BBa_B0032, https://parts.igem.org) and thrL terminator from E. coli. Similarly, the pBR322M-Ptac plasmid was obtained by direct reassembly of the lad expression cassette and the Ptac promoter carrying a weak RBS with pBR322M as backbone. To lower the leakage expression of the plant-type FNRs, a Lad mutant LacIWF[31] with higher affinity for the lac operator was used for construction of pBR322M-Ptac plasmid. The nifF gene in the pKU7832 was replaced with the SwaI restricted fragment [tetR-PLtetO-1] fragment from plasmid pBR322M PLtetO-1, resulting in plasmid pKU7833. To construct the plasmid for expression of the hybrid modules consisting of the NifJ and plant-type ferredoxins, original ferredoxin gene sequences or chemically synthesized ferredoxin gene sequences were cloned downstream of the PLtetO-1 promoter of the pKU7833 plasmid by using NdeI/SpeI restriction sites. In order to facilitate detection of the expression level of different ferredoxins, a sequence encoding the Histidine-tag was added to each of the synthesized ferredoxin sequence as shown in gray shadow in
Acetylene Reduction Assay
The C2H2 reduction method was used to assay the nitrogenase activity as described in the literature[32]. To measure nitrogenase activity of the recombined E. coli JM109 strains, cells were initially grown overnight in KPM-HN medium. The cells were then diluted into 2 mL KPM-LN medium in 20 mL sealed tubes to a final OD600 of ˜0.3. In order to maximize the restoring effect for the plant-type ferredoxins and FNRs, 200 ng/mL of anhydrotetracycline (aTc) or 200 μM of isopropyl-β-d-thiogalactoside (IPTG) was added to induce the expression of the ferredoxins or FNRs respectively as indicated by results shown in
15N2 Assimilation Assay
To detect the 15N2 assimilation activity, the recombined E. coli JM109 strains were prepared as described in the acetylene reduction assay. Air in the tube was repeatedly evacuated and flushed with nitrogen three times. 3 mL gas was finally removed and 2 mL 15N2 gas (99%+, Shanghai Engineering Research Center for Stable Isotope) was injected. After 48 h of incubation at 30° C., the cultures were collected, and were freeze dried, ground, weighed and sealed into tin capsules. Isotope ratios are expressed as δ15N whose values are a linear transform of the isotope ratios, 15N/14N representing the per mille difference between the isotope ratios in a sample and in atmospheric N2[33]. Data presented are mean values based on at least two replicates.
Most plants are known to have multiple copies of ferredoxins in different organelles[21]. Through preliminary sequence analysis, we found that ferredoxins from plant chloroplast and root-plastid show high sequence identity with the Anabaena sp. PCC 7120 (As)fdxH gene product, which is the primary electron donor for the nitrogenase in the cyanobacteria[34]. To investigate if hybrid ETC modules formed by the NifJ protein and plastid ferredoxins could support nitrogenase activity in E. coli. Coding sequences of several representative plastid ferredoxins from Chlamydomonas reinhardtii (Cr), Arabidopsis thaliana (At), Zea mays (Zm), Oryza sativa (Os), and Triticum aestivum (Ta) were selected for further study. These selected representative ferredoxin encoding genes were codon optimized according to the codon bias of E. coli, optimized gene sequence shown in Sequence Listing, and expressed from the inducible PLteto-1 promoter. The fdxH gene from As was used as a control to verify effectiveness of the inducible system.
The flavodoxin encoded by NifF from the NifJ-NifF module was replaced by the hybrid modules constructed above and the activity of the recombined MoFe, or FeFe systems were analyzed by the method of acetylene reduction[27-28]. The results show that all hybrid ETC modules could restore nitrogenase activities for both the MoFe and FeFe systems, but with different activities. Values greater than 100% were observed for the FeFe nitrogenase system when NifF was replaced with the ferredoxins from As (FdxH), Cr (PETF), or Os (FD1) respectively (see
aActivities of the FeFe or MoFe nitrogenase systems carrying the NifJ-NifF module are represent as 100%. Data presented are mean values based on at least three independent experiments.
As mitochondria are another potential location for nitrogenase in the plant, the capability of mitochondrial ferredoxins in supporting nitrogen fixation was also investigated in E. coli. The same strategy was used to clone the mitochondria ferredoxin coding genes as described in the former part of this section. When mitochondria ferredoxin derived hybrid ETC modules (NifJ-AtMFD1 or NifJ-AtMFD2) were introduced into E. coli, no restoration of the nitrogenase activities were observed (
In plants, three different types of the FNRs existing in different organelles are identified. All of these FNRs function to mediate electron transfer between the ferredoxins and NADPH[23, 25, 26]. To investigate if hybrid ETC modules consisting of the plant-type FNRs and electron donors (KoNifF, AsFdxH and AsFdxB) for nitrogenase could mediate electron transfer to nitrogenase, coding sequences of FNRs from the chloroplast or root-plastid of Cr, Zm, or MFDR from mitochondria of At were selected for testing. These hybrid modules were transformed into the E. coli, and their activities were assayed with acetylene reduction method. The results showed that none of the hybrid ETC modules consisting of the plant-type FNRs and the NifF could restore acetylene reduction activity for either the MoFe or FeFe nitrogenase systems; AsFdxB can form functional ETC module to support both of MoFe and FeFe nitrogenases activities only when it is coupled with the MFDR from mitochondria (
After verifying the function of the hybrid modules as the electron supplier for the nitrogenase systems, further experiments were carried out to investigate whether the intact ETC modules, consisting of FNRs and their cognate ferredoxins from plant organelles, could support the nitrogenase activity. By combining the Ptac controlled FNRs with PLtetO-1 controlled ferredoxins (details are provided in Materials and Methods), two intact chloroplast ETC modules, CrFNR-PETF and ZmLFNR-FDI; one intact root-plastid ETC module ZmRFNR-FDIII; and one intact mitochondria ETC module AtMFDR-MFD were constructed. As it is known that the AsPetH-FdxH module can support nitrogen fixation in its original host, this module was used as a control.
When these intact ETC modules were used to replace the NifJ-NifF modules of either the MoFe or the “minimal” FeFe nitrogenase system respectively, their ability to support nitrogenase activities were assayed with both the acetylene reduction and 15N assimilation methods. The results showed that with the exception of the AtMFDR-MFD module from mitochondria, all other ETC modules can support acetylene reduction activities and 15N assimilation activities for both MoFe and FeFe nitrogenases (
For the FeFe nitrogenase system, the chloroplast modules, CrFNR-PETF and ZmLFNR-FDI, showed almost equal amount of restored acetylene reduction activities (˜45%) and 15N assimilation activities (>30%) as that with the AsPetH/FdxH (
Taken together, above results demonstrate that the intact ETCs modules from plastids (including chloroplast and root-plastid), but not from mitochondria, are capable of providing the electron and reducing power required to reduce nitrogen for the nitrogenase system.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201710001952.9 | Jan 2017 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2017/117862 | 12/22/2017 | WO | 00 |
