ELECTRON TRANSPORT CHAIN MODULE FROM EUKARYOTIC ORGANELLE AND APPLICATION THEREOF

Abstract

Provided are an electron transport chain module from a eukaryotic organelle and an application thereof in biological nitrogen fixation. The electron transport chain (ETC) module is composed of the NifJ protein from Klebsiella oxytoca and a ferredoxin from plant chloroplasts or leucoplasts; plant-type ferredoxin-NADPH reductase (FNR) and the FdxH or FdxB protein from Anabaena; or an FNR and a Ferredoxin protein from plant organelles.

Claims

1. An electron transport chain (ETC) module for a biological nitrogen fixation system, comprising an NifJ protein and an NifF protein.

2. The ETC module of claim 1, wherein the nitrogen fixation system is a MoFe nitrogenase system and an FeFe nitrogenase system.

3. The ETC module of claim 1, wherein the NifJ protein and the NifF protein are substituted individually or substituted simultaneously by corresponding proteins from eukaryotic organelles, thereby forming hybrid or intact ETC modules.

4. The ETC module of claim 3, wherein the eukaryotic organism is a plant, and the organelle is a plastid or mitochondria.

5. The ETC module of claim 3, wherein the hybrid ETC module is formed by replacing the NifF protein in the ETC module consisting of the NifJ and the NifF with a ferredoxin from a plant plastid.

6. The ETC module of claim 5, wherein the plastid is a chloroplast or a root-plastid, preferably a chloroplast.

7. The ETC module of claim 3, wherein the hybrid ETC module is formed by replacing the NifJ protein in the ETC module consisting of the NifJ and the NifF with a plant-type Ferredoxin-NADPH reductase (FNR) from a plant plastid and mitochondria.

8. The ETC module of claim 7, wherein the plastid is a chloroplast or a root-plastid.

9. The ETC module of claim 3, wherein the hybrid ETC module is composed of an NADPH-dependent adrenodoxin oxidoreductase (MFDR) from a plant mitochondria and an Anabaena FdxB.

10. The ETC module of claim 3, wherein the intact ETC module is composed of an FNR from a target plant organelle and Ferredoxin proteins.

11. The ETC module of claim 10, wherein the target plant organelle is a chloroplast or a root-plastid.

12. Use of the ETC module of claim 1 in biological nitrogen fixation.

13. Use of the ETC module of claim 6 in biological nitrogen fixation.

14. Use of the ETC module of claim 7 in biological nitrogen fixation.

Description

DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1. Upper: Modular arrangement of the recombined MoFe and the minimal FeFe nitrogenase system. Letters in the diagram represent the corresponding nif or anfHDGK genes, e.g. J represents nifJ gene. Bottom: Schematic diagram of electron transport within nitrogenase in a nitrogenase system, with the representative proteins in figure shown with crystal structures, wherein the abbreviation PFO (NifJ) represents pyruvate-ferredoxin (Flavodoxin) oxidoreductase; the abbreviation FNR represents ferredoxin-NADPH oxidoreductase; the abbreviation NifF represents flavodoxin; the abbreviation FdxN represents 2[4Fe-4S]-type ferredoxin; the abbreviation FdxH represents [2Fe-2S]-type ferredoxin; the abbreviation Fe protein represents dinitrogenase reductase; X=Mo, V or Fe in XFe protein. The cofactors of the Fe proteins and XFe proteins are shown in ball-and-stick model. Atom colors are Fe in rust, S in yellow, C in gray, 0 in red and X atom (Mo, V or Fe atom) in purple. Additionally, gene arrangement in this figure does not represent the real gene arrangement for the recombined MoFe and the minimal FeFe nitrogenase system.

[0024] FIG. 2. (A) Sequence alignment of the AsFdxH protein with ferredoxins from plastids. (B) Sequence alignment of the AsFdxH protein with ferredoxins from mitochondria. In the figure, sequences in green shadow in (A) or crimson shadow in (B) are leading peptides for the plant-type ferredoxins; cysteine residues for binding [2Fe-2S] are highlighted with yellow shadow. As represents Anabaena sp. PCC 7120; Cr represents Chlamydomonas reinhardtii; At represents Arabidopsis thaliana; Zm represents Zea mays; Os represents Oryza sativa; Ta represents Triticum aestivum.

[0025] FIG. 3. Plasmid maps of the main vectors used in the invention. The rrnB T1 is a Escherichia coli terminator (BBa_B0010); the T.sub.L is the E. coli thrL gene terminator; the T.sub.0 is a phage lambda t.sub.0 terminator; the T.sub.a is an artificial terminator L3S2P21 reported by chen et al.sup.[1].

[0026] FIG. 4. Gradient induction assays of the controllable expression of the CrPETF or CrFNR. (A) and (B) Gradient induction of the expression of the CrPETF controlled by the P.sub.LtetO-1 promoter with the anhydrotetracycline (aTc). (C) and (D) Gradient induction of the expression of the CrFNR controlled by the Ptac promoter with the isopropyl--d-thiogalactoside (IPTG). In this experiment, the expression of CrPETF was first induced with 200 ng/mL of aTc, and then the expression of CrFNR was induced with gradient concentration of IPTG. The acetylene reduction activities shown in the figure were relative activities obtained under the condition that the activities obtained from FeFe or MoFe nitrogenase system carrying NifJ-NifF module are defined as 100% activities. FeFe represents the minimal FeFe nitrogenase system; MoFe represents the recombined MoFe nitrogenase system. Error bars indicate the standard deviation observed from at least three independent experiments.

[0027] FIG. 5. Substitution effect of the hybrid ETC modules consisting of the NifJ protein and ferredoxin from plastid was assayed by acetylene reduction. (A) Schematic picture of electron transport pathways between the hybrid ETC modules and the core enzyme module. NifJ-NifF module was replaced by the hybrid ETC modules comprising the NifJ with chloroplast ferredoxins (B and C); NifJ with root-plastid ferredoxins (D and E); NifJ with mitochondria ferredoxins (F and G). The activities obtained from FeFe or MoFe nitrogenase system carrying NifJ-NifF module without adding inducers are defined as 100%. FeFe, represents the minimal FeFe nitrogenase system; MoFe, represents the recombined MoFe nitrogenase system. As represents Anabaena sp. PCC 7120; Cr represents Chlamydomonas reinhardtii; At represents Arabidopsis thaliana; Zm represents Zea mays; Os represents Oryza sativa; Ta represents Triticum aestivum. Error bars indicate the standard deviation observed from at least three independent experiments.

[0028] FIG. 6. MoFe or FeFe nitrogenase activities after a high-copy plasmid carrying the GroESL-encoding genes was cotransformed with NifJ-AtMFD1 or NifJ-AtMFD2 hybrid modules were assayed by acetylene reduction. The activities obtained from FeFe or MoFe nitrogenase system carrying NifJ-NifF module without adding inducers are defined as 100%. FeFe, represents the minimal FeFe nitrogenase system; MoFe, represents the recombined MoFe nitrogenase system. At represents Arabidopsis thaliana. Error bars indicate the standard deviation observed from at least three independent experiments.

[0029] FIG. 7. Substitution effect of the hybrid ETC modules consisting of the KoNifF or AsFdxB with FNRs from different plant organelles was assayed by acetylene reduction. The NifJ-NifF module was replaced by the hybrid modules consisting of plant-type FNRs and the KoNifF (A and B) or AsFdxB (C and D). The acetylene reduction activities obtained from FeFe or MoFe nitrogenase system carrying NifJ-NifF module without adding inducers are defined as 100%. FeFe, represents the minimal FeFe nitrogenase system; MoFe, represents the recombined MoFe nitrogenase system. Ko represents Klebsiella oxytoca; As represents Anabaena sp. PCC 7120; Cr represents Chlamydomonas reinhardtii; At represents Arabidopsis thaliana; Zm represents Zea mays. Error bars indicate the standard deviation observed from at least three independent experiments.

[0030] FIG. 8. Substitution effect of the hybrid ETC modules consisting of the plant FNRs and AsFdxH was assayed by acetylene reduction. (A) Schematic picture of electron transport pathways between the hybrid ETC modules and the core enzyme module. The NifJ-NifF module was replaced by the hybrid modules consisting of the plant FNRs and AsFdxH (B and C). The activities obtained from FeFe or MoFe nitrogenase system carrying NifJ-NifF module without adding inducers are defined as 100%. FeFe, represents the minimal FeFe nitrogenase system; MoFe, represents the recombined MoFe nitrogenase system. As, Anabaena sp. PCC 7120; Cr, Chlamydomonas reinhardtii; At, Arabidopsis thaliana; Zm, Zea mays. Error bars indicate the standard deviation observed from at least three independent experiments.

[0031] FIG. 9. Substitution effect of the intact ETC modules of chloroplast and root-plastid was assayed by acetylene reduction and .sup.15N assimilation assays (D and E). (A) Schematic picture shows electron transport pathways between the intact ETC modules from plant organelles and the core enzyme module. The activities obtained from FeFe or MoFe nitrogenase system carrying NifJ-NifF module without adding inducers are represented by 100%. FeFe, represents the minimal FeFe nitrogenase system; MoFe, represents the recombined MoFe nitrogenase system. As, Anabaena sp. PCC 7120; Cr, Chlamydomonas reinhardtii; At, Arabidopsis thaliana; Zm, Zea mays. Error bars for the acetylene reduction assay indicate the standard deviation observed from at least three independent experiments. Error bars for the .sup.15N assimilation assay indicate the standard deviation observed from at least two independent experiments.

[0032] FIG. 10. Single component of the intact ETC module is not enough to support nitrogenase activity for the FeFe (A) or MoFe (B) nitrogenase system. The acetylene reduction activities obtained from FeFe or MoFe nitrogenase system carrying NifJ-NifF module without adding inducers are defined as 100%. FeFe, represents the minimal FeFe nitrogenase system; MoFe, represents the recombined MoFe nitrogenase system. As, Anabaena sp. PCC 7120; Cr, Chlamydomonas reinhardtii; At, Arabidopsis thaliana; Zm, Zea mays. Error bars indicate the standard deviation observed from at least three independent experiments.

EXAMPLES

[0033] Materials and Methods

[0034] Bacterial Strains and Plasmids Used in the Examples are Shown in Table 1

TABLE-US-00001 TABLE 1 Bacterial Strains and plasmids used in the Examples Strain or Source or plasmid Relevant feature reference E. coli Strains Top 10 F.sup. mcrA (mrr-hsdRMS-mcrBC) 80lacZM15 Invitrogen lacX74 nupG recA1 araD139 (ara-leu)7697 galE15 galK16 rpsL(Str.sup.R) endA1 .sup. JM109 recA endA1 gyrA96 hsdR17 supE44 relA1 Takara (lac-proAB)/F[traD36 proAB.sup.+ lacI.sup.q lacZM15] Bio Plasmids pBR322M pBR322 derivative, Amp.sup.R, labeled as nifJ/nifF in (8) the text pBR322M-P.sub.LtetO-1 pBR322M derivative carrying P.sub.LtetO-1 inducible Examples expression cassette pBR322M-P.sub.tac pBR322M derivative carrying P.sub.tac inducible Examples expression cassette pKU7815 pACYC184 derivative carrying the minimal FeFe (8) nitrogenase system, Cm.sup.R pKU7017 pACYC184 derivative carrying the recombined (9) MoFe nitrogenase system, Cm.sup.R pKU7830 pKU7815 derivative, nifJ/nifF Examples pKU7831 pKU7017 derivative, nifJ/nifF Examples pKU7832 pBR322M derivative carrying KonifJ/nifF genes, Examples labeled as nifJ/nifF in the text pKU7833 pKU7830 derivative with nifF gene replaced by Examples P.sub.LtetO-1 inducible expression cassette, labeled as nifJ/nifF in the text pKU7834 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-AsfdxH.sub.ori Examples pKU7835 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-CrPETF.sub.syn Examples pKU7836 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-AtFD2.sub.syn Examples pKU7837 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-ZmFDI.sub.syn Examples pKU7838 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-OsFD1.sub.syn Examples pKU7839 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-TaFD.sub.syn Examples pKU7840 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-AtFD3.sub.syn Examples pKU7841 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-ZmFDIII.sub.syn Examples pKU7842 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-OsFD4.sub.syn Examples pKU7843 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-AtMFD1.sub.syn Examples pKU7844 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-AtMFD2.sub.syn Examples pKU7845 pEASY-Blunt derivative carrying EcgroES operon Examples with its original promoter pKU7846 pKU7832 derivative with nifJ gene replaced by P.sub.tac Examples inducible expression cassette, labeled as nifJ/nifF in the text pKU7847 pKU7846 derivative carrying P.sub.tac-AspetH.sub.ori/nifF Examples pKU7848 pKU7846 derivative carrying P.sub.tac-CrFNR.sub.syn/nifF Examples pKU7849 pKU7846 derivative carrying P.sub.tac-ZmLFNR.sub.syn/nifF Examples pKU7850 pKU7846 derivative carrying P.sub.tac-ZmRFNR.sub.syn/nifF Examples pKU7851 pKU7846 derivative carrying P.sub.tac-AtMFDR.sub.syn/nifF Examples pKU7852 pKU7834 derivative with nifJ gene replaced by P.sub.tac Examples inducible expression cassette, labeled as nifJ/ P.sub.LtetO-1-AsfdxH.sub.ori in the text pKU7853 pKU7847 derivative carrying P.sub.tac-AspetH.sub.ori/ Examples P.sub.LtetO-1-AsfdxH.sub.ori pKU7854 pKU7848 derivative carrying P.sub.tac-CrFNR.sub.syn/ Examples P.sub.LtetO-1-AsfdxH.sub.ori pKU7855 pKU7849 derivative carrying P.sub.tac-ZmLFNR.sub.syn/ Examples P.sub.LtetO-1-AsfdxH.sub.ori pKU7856 pKU7850 derivative carrying P.sub.tac-ZmRFNR.sub.syn/ Examples P.sub.LtetO-1-AsfdxH.sub.ori pKU7857 pKU7851 derivative carrying P.sub.tac-AtMFDR.sub.syn/ Examples P.sub.LtetO-1-AsfdxH.sub.ori pKU7858 pKU7833 derivative carrying nifJ/P.sub.Lteto-1-AsfdxB.sub.ori Examples pKU7859 pKU7847 derivative carrying P.sub.tac-AspetH.sub.ori/ Examples P.sub.LtetO-1-AsfdxB.sub.ori pKU7858 pKU7848 derivative carrying P.sub.tac-CrFNR.sub.syn/ Examples P.sub.LtetO-1-AsfdxB.sub.ori pKU7859 pKU7849 derivative carrying P.sub.tac-ZmLFNR.sub.syn/ Examples P.sub.LtetO-1-AsfdxB.sub.ori pKU7860 pKU7850 derivative carrying P.sub.tac-ZmRFNR.sub.syn/ Examples P.sub.LtetO-1-AsfdxB.sub.ori pKU7861 pKU7851 derivative carrying P.sub.tac-AtMFDR.sub.syn/ Examples P.sub.LtetO-1-AsfdxB.sub.ori pKU7862 pKU7848 derivative carrying P.sub.tac-CrFNR.sub.Syn/ Examples P.sub.LtetO-1-CrPETF.sub.syn pKU7863 pKU7849 derivative carrying P.sub.tac-ZmLFNR.sub.syn/ Examples P.sub.LtetO-1-ZmFDI.sub.syn pKU7864 pKU7850 derivative carrying P.sub.tac-ZmRFNR.sub.syn/ Examples P.sub.LtetO-1-ZmFDIII.sub.syn pKU7865 pKU7851 derivative carrying P.sub.tac-AtMFDR.sub.syn/ Examples P.sub.LtetO-1-AtMFD.sub.syn pKU7866 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-AspetF.sub.ori Examples pKU7867 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-CrFDX2.sub.syn Examples pKU7868 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-AtFD1.sub.syn Examples pKU7869 pKU7833 derivative carrying nifJ/P.sub.LtetO-1-ZmFDII.sub.syn Examples

[0035] Bacterial Strains and Growth Medium

[0036] 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 Na.sub.2HPO.sub.4, 3.4 g/L of KH.sub.2PO.sub.4, 26 mg/L of CaCl.sub.2. 2H.sub.2O, 30 mg/L of MgSO.sub.4, 0.3 mg/L of MnSO.sub.4, 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 g/mL for ampicillin, 25 g/mL for chloramphenicol.

[0037] Construction of Recombinant Plasmids

[0038] Plasmid pKU7815 and pKU7017 are pACYC184 derivatives carrying the minimal FeFe.sup.[28] or the recombined MoFe.sup.[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-P.sub.LtetO-1 plasmid was obtained by direct reassembly of the tetR expression cassette and the P.sub.LtetO-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-P.sub.tac plasmid was obtained by direct reassembly of the lad expression cassette and the P.sub.tac promoter carrying a weak RBS with pBR322M as backbone. To lower the leakage expression of the plant-type FNRs, a Lad mutant LacI.sup.WF[31] with higher affinity for the lac operator was used for construction of pBR322M-P.sub.tac plasmid. The nifF gene in the pKU7832 was replaced with the SwaI restricted fragment [tetR-P.sub.LtetO-1] fragment from plasmid pBR322M P.sub.LtetO-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 P.sub.LtetO-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 FIG. 2. The nifJ gene in the pKU7832 was replaced with the ScaI restricted fragment [lacI-P.sub.tac] fragment from plasmid pBR322M-P.sub.tac, resulting in plasmid pKU7846. To construct the plasmid expressing the hybrid modules consisting of the plant-type FNRs and NifF, original FNR gene sequences or chemically synthesized FNR gene sequences were cloned downstream of the P.sub.tac promoter of the pKU7846 plasmid by using NdellSpeI restriction sites. The pKU7853 plasmid, carrying P.sub.tac-AspetH.sub.ori/P.sub.LtetO-1-AsfdxH.sub.ori, was constructed by replacing the nifF gene in plasmid pKU7847 with ScaI restricted fragment [P.sub.LtetO-1-AsfdxH] fragment from pKU7834. Similar methods were used to construct the pKU7854-pKU7857 and pKU7859-pKU7865. The PCR product carrying the groES with its original promoter, flanking the XbaI/SpeI restriction sites, was PCR amplified from the genome of E. coli and directly ligated to the pEASY-Blunt vector (TransGene, CB101) to generate pKU7845. Plasmids maps are provided in FIG. 3. Each of the above constructed plasmids was confirmed by DNA sequencing before any further experiments.

[0039] Acetylene Reduction Assay

[0040] The C.sub.2H.sub.2 reduction method was used to assay the nitrogenase activity as described in the literature.sup.[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 OD.sub.600 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 M of isopropyl--d-thiogalactoside (IPTG) was added to induce the expression of the ferredoxins or FNRs respectively as indicated by results shown in FIG. 4. Air in the tube was repeatedly evacuated and flushed with argon three times. After static culture at 30 C. for 68 h, 2 mL C.sub.2H.sub.2 was added. The activity was detected 16 h later with a Shimadzu GC-2014 gas chromatograph. Data presented are mean values based on at least three replicates.

[0041] .sup.15N.sub.2 Assimilation Assay

[0042] To detect the .sup.15N.sub.2 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 .sup.15N.sub.2 gas (99%.sup.+, 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 .sup.15N whose values are a linear transform of the isotope ratios, .sup.15N/.sup.14N representing the per mille difference between the isotope ratios in a sample and in atmospheric N.sub.2.sup.[33]. Data presented are mean values based on at least two replicates.

Example 1: Hybrid ETC Modules Consisting of the NifJ Protein and Plastid Ferredoxins can Functionally Support Nitrogenase Activity

[0043] Most plants are known to have multiple copies of ferredoxins in different organelles.sup.[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.sup.[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 P.sub.Lteto-1 promoter. The fdxH gene from As was used as a control to verify effectiveness of the inducible system.

[0044] 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.sup.[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 FIG. 5 and Table 2). This result suggests that the AvAnfH protein in the hybrid minimal FeFe nitrogenase.sup.[28] may prefer ferredoxin, rather than flavodoxin, as electron donor. All the chloroplast ferredoxins derived hybrid modules could restore about 100% activities for the FeFe nitrogenase system, except the NifJ-AtFD2 hybrid module, which showed 76% activity (FIG. 5B). The NifJ-CrPETF and NifJ-TaFD hybrid modules could restore >90% activities for the MoFe nitrogenase system. The NifJ-AtFD2, NifJ-ZmFDI and NifJ-OsFD1 hybrid modules showed restored activity to <70% (FIG. 5C). All root-plastid ferredoxin derived hybrid modules showed lower nitrogenase activities when compared with the chloroplast ferredoxin derived modules from the same organism (FIG. 5B-5E).

TABLE-US-00002 TABLE 2 The activities of the FeFe or MoFe nitrogenase system after NifF was replaced by ferredoxins from different plant plastids Relative nitrogenase activity, Redox % acetylene reduction.sup.a Organism Location Genes potential FeFe MoFe Ko nifF 412 mV(2) 100 6 100 15 nifF 31 6 10 3 As Heterocyst fdxH 351 mV(3) 153 21 100 6 Vegetative petF 384 mV(3) 110 14 81 11 Cr Chloroplast PETF 398 mV(4) 105 11 90 5 Chloroplast FDX2 321 mV(4) 92 4 87 4 Zm Chloroplast FDI 423 mV(5) 96 9 56 6 Chloroplast FDII 406 mV(5) 75 7 50 7 Root plastid FDIII 321 mV(6) 82 9 51 7 At Chloroplast FD1 425 mV(7) 59 6 36 6 Chloroplast FD2 433 mV(7) 76 11 50 11 Root plastid FD3 337 mV(7) 68 4 34 8 .sup.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.

[0045] 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 (FIGS. 5F and 5G). Further, in order to exclude the effect of GroESL proteins on proper and efficient folding of the mitochondrial ferredoxins in E. coli, as indicated by Picciocchi et al..sup.[26], a high-copy plasmid carrying the GroESL encoding genes was co-transformed with a MoFe or FeFe nitrogenase system carrying either the NifJ-AtMFD1 or NifJ-AtMFD2 hybrid modules. Similar negative results were finally obtained (FIG. 6). Simultaneously, phylogenetic analysis also showed that the mitochondrial ferredoxins do not have similar evolutionary relationships with any electron donors of nitrogenase. Overall, these results suggest that the mitochondria ferredoxins cannot couple with the NifJ protein to form functional ETC modules capable of providing electrons for the nitrogenase systems.

Example 2: Study of Electron Supply of Hybrid ETC Modules Consisting of Plant-Type FNR and KoNifF, AsFdxH and AsFdxB, Respectively, to Nitrogenase System

[0046] 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.sup.[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 (FIG. 7); all hybrid modules formed with the plant-type FNRs and AsFdxH could restore nitrogenase activity for both of the MoFe and FeFe nitrogenase systems (FIG. 8).

Example 3: The Intact ETC Modules from the Chloroplast and Root-Plastid Support Nitrogenase Activity

[0047] 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 P.sub.tac controlled FNRs with P.sub.LtetO-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.

[0048] 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 .sup.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 .sup.15N assimilation activities for both MoFe and FeFe nitrogenases (FIG. 9); no restoration of activity was observed, when either of the two components from each of the modules was expressed individually (FIG. S6), indicating that for the functionality of each plant intact ETC module, both components have to be present.

[0049] For the FeFe nitrogenase system, the chloroplast modules, CrFNR-PETF and ZmLFNR-FDI, showed almost equal amount of restored acetylene reduction activities (45%) and .sup.15N assimilation activities (>30%) as that with the AsPetH/FdxH (FIGS. 5B and D). Similar results were obtained for the MoFe nitrogenase system, chloroplast modules CrFNR-PETF and ZmLFNR-FDI, and AsPetH/FdxH module each restored 46% of .sup.15N assimilation activities (FIG. 9E). The ZmRFNR-FDIII module from the root-plastid only restored lower activities for both MoFe and FeFe nitrogenases, compared with chloroplast module from the same organism (FIGS. 9B, C, D and E). Weakly increased activities were observed from the the MoFe nitrogenase system carrying AtMFDR-MFD module (11% .sup.15N assimilation activity) when compared with the NifJ-NifF deficient negative control (6% .sup.15N assimilation activity) in the MoFe nitrogenase system (FIG. 9E). But this phenotype was not observed in the FeFe nitrogenase system (FIG. 9D). As multiple-copies of ferredoxins exist in E. coli, we believe that the enhanced activity of MoFe nitrogenase system carrying the AtMFDR-MFD module may be contributed by hybrid modules formed with AtMFDR and the ferredoxins from E. coli. For the FeFe nitrogenase system, such effect may be masked by high background activities due to its high background activities (FIG. 9D).

[0050] 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.

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