Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity

Jianguo Yang; Xiaqing Xie; Mingxuan Yang; Ray Dixon; Yi-Ping Wang

doi:10.1073/pnas.1620058114

. 2017 Feb 13;114(12):E2460–E2465. doi: 10.1073/pnas.1620058114

Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity

Jianguo Yang ^a,¹, Xiaqing Xie ^a,¹, Mingxuan Yang ^a, Ray Dixon ^b,², Yi-Ping Wang ^a,²

PMCID: PMC5373397 PMID: 28193863

Significance

Engineering nitrogenase into cereal crops requires detailed understanding of the components required for efficient nitrogen fixation. We have used a synthetic biology modular approach to evaluate components from chloroplast, root plastids, and mitochondria that function as electron donors to both conventional Mo nitrogenase and the alternative Fe nitrogenase systems. The knowledge obtained in this study not only identifies electron-transfer components from plant organelles that can be used to support nitrogenase activity, but also is likely to enable reduction of the number of target genes required to engineer nitrogen fixation in plants.

Keywords: nitrogen fixation, electron transport, plant organelles, nitrogenase engineering

Abstract

A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the “core” nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin–NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR–ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops.

Nitrogen is one of the primary nutrients limiting plant productivity in agriculture (1). Industrial nitrogen fertilizers are used to circumvent this limitation, but have resulted in environmental pollution and expensive economic costs, especially in developing countries (2, 3). These factors have potentiated a renewed focus toward engineering biological nitrogen fixation (BNF) in cereal crops. BNF, the process that converts gaseous nitrogen to ammonia by nitrogenase enzymes, contributes >60% of the total atmospheric N₂ fixed in the biogeochemical 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 equivalent to Mo, V, or Fe, depending on the heterometal composition of the active site cofactor) (Fig. 1 and refs. 5 and 6). All three nitrogenases catalyze the biological reduction of N₂ according to the following equation: N₂ + (6 + 2n)H⁺ + (6 + 2n)e⁻ → 2NH₃ + nH₂ (n ≥ 1) (7–9). In this process, electrons are first transferred to the Fe protein, which, in turn, donates electrons to the XFe protein with hydrolysis of two ATP molecules per electron (Fig. 1) (10–12). Although Fe protein is the obligate electron donor for XFe protein in all characterized nitrogenase systems, the in vivo electron donor for Fe protein is less stringently conserved (9). Direct electron donors to Fe protein are either reduced flavodoxin or reduced ferredoxin, which, in turn, are reduced by a variety of oxidoreductase systems, depending on the physiology of the host diazotroph (13–17).

Fig. 1. — (*Upper*) Modular arrangement of genes required for MoFe and the minimal FeFe nitrogenase systems. Letters within the arrows represent the corresponding *nif* genes or the *anfHDGK* structural genes encoding FeFe nitrogenase. (*Lower*) Schematic pathways for electron donation to nitrogenase and electron transfer within nitrogenase. Structures of representative proteins are shown. PFOR (NifJ), pyruvate–ferredoxin (flavodoxin) oxidoreductase [Protein Data Bank (PDB) ID code 1B0P]; Rnf complex, NADH–ferredoxin oxidoreductase [the structure shown in gray is a homology model based on the Nqr complex (Na⁺-translocating NADH–quinone oxidoreductase from *Vibrio* alginolyticus; PDB ID code 4P6V) using the online software from https://swissmodel.expasy.org]; FNR (PDB ID code 1QUE); NifF, flavodoxin (PDB ID code 2WC1); FdxN, 2[4Fe–4S]-type ferredoxin (PDB ID code 2OKF); FdxH, [2Fe–2S]-type ferredoxin (PDB ID code 1FRD); Fe protein, dinitrogenase reductase (PDB ID code 1G5P); and XFe protein (where X refers to Mo, V, or Fe), dinitrogenase (PDB ID code 3K1A; MoFe nitrogenase). The cofactors of the Fe and XFe proteins are shown as ball-and-stick models. Atom colors are Fe in rust, S in yellow, C in gray, O in red, and heterometal X in purple.

A number of studies have suggested chloroplasts, root plastids, or mitochondria as suitable locations for expression of nitrogenase in eukaryotes (18–20). These energy-conversion organelles can potentially provide reducing power and ATP required for the nitrogen-fixation process. Diverse reduction reactions carried out in these organelles rely on different electron-transport chains (21). Multiple gene copies of ferredoxins have been identified in all plants, including photosynthetic or nonphotosynthetic ferredoxins mainly expressed in chloroplasts or root plastids, respectively; and ferredoxin-like adrenodoxins located in the mitochondria (21, 22). The major function of the photosynthetic ferredoxins is to transfer electrons from photosystem I to NADPH, catalyzed by leaf-type ferredoxin–NADPH oxidoreductase (LFNR) (23). In addition, photosynthetic ferredoxins work to distribute reducing power derived from the photosynthetic process to several ferredoxin-dependent enzymes for nitrogen and sulfur assimilation (24). Electron transfer between root-type ferredoxin–NADPH oxidoreductase (RFNR) and ferredoxin in the root plastid is reversed, with NADPH generated in the oxidative pentose-phosphate pathway being used to reduce RFNR and, in turn, ferredoxin (25). In mitochondria, adrenodoxin serves to transfer electrons from NADPH-dependent adrenodoxin oxidoreductase (MFDR) to the cysteine desulfurase Nfs1 to participate in the biosynthesis of the biotin (26).

Recently, we successfully reassembled the Klebsiella oxytoca (Ko) MoFe (27) and the “minimal” Azotobacter vinelandii (Av) FeFe (28) nitrogenase systems in Escherichia coli (Fig. 1). From the synthetic biology point of view, these two nitrogenase systems can be divided into three functional modules: the electron-transport component (ETC) module, the metal cluster biosynthesis module, and the “core” enzyme module (Fig. 1). In the present study, ETC modules from plastids and mitochondria, representing potential locations for nitrogenase in plants, were used to test their capability to support the activity of either MoFe or FeFe nitrogenase in E. coli. Our results indicate that intact ETC modules from the chloroplast and root plastid, or hybrid modules from mitochondria, can functionally support nitrogenase activity. Therefore, our results unravel the requirements for electron-transport components for engineering diazotrophy in different plant organelles.

Results

Hybrid ETC Modules Consisting of the NifJ Protein and Plastid Ferredoxins Can Functionally Support Nitrogenase Activity.

In many diazotrophs, the direct electron donor to nitrogenase is either reduced ferredoxin or reduced flavodoxin, which have been demonstrated to receive electrons from the pyruvate–flavodoxin (ferredoxin) oxidoreductase, encoded by nifJ, in some cases (13–15). Most plants are known to have multiple copies of ferredoxins located in different organelles (21). Through preliminary sequence analysis, we found that chloroplast and root-plastid ferredoxins from plants show high sequence identity with the Anabaena sp. PCC 7120 (As) fdxH gene product (Fig. S1), which is the primary electron donor for nitrogenase in cyanobacteria (29). To investigate whether 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; CrPETF), Arabidopsis thaliana (At; AtFD2 and AtFD3), Zea mays (Zm; ZmFDI and ZmFDIII), Oryza sativa (Os; OsFD1 and OsFD4), and Triticum aestivum (Ta; TaFD) were selected for further study. These ferredoxin-encoding genes were codon-optimized for E. coli (Dataset S1), and expressed from the inducible P_Lteto-1 promoter (Fig. 2A; details are provided in SI Materials and Methods). The fdxH gene from As was also introduced as a control to verify effectiveness of the inducible system.

Fig. S1. — Sequence alignment of the AsFdxH protein with ferredoxins from plastids (A) or mitochondria (B). Sequences shaded in green in A or crimson in B are signal peptides for the plant-type ferredoxins. Cysteine residues for liganding the [2Fe–2S] cluster are highlighted in yellow. Abbreviations are the same as in the main text (Fig. 2).

Fig. 2. — Influence of hybrid ETC modules consisting of the NifJ protein with ferredoxins (FDs) from different plant organelles on nitrogenase activity in *E. coli.* (A) Schematic diagram for electron transport between hybrid ETC modules and nitrogenases. (B–G) In each case, *nifF* was replaced by FDs to form hybrid modules consisting of NifJ with chloroplast FDs (B and C), NifJ with root-plastid FDs (D and E), or NifJ with mitochondrial FDs (F and G). In all cases, cultures were assayed for acetylene reduction either in the absence (filled bars) or presence (open bars) of the appropriate inducer required to express heterologous ETCs as described in *SI Materials and Methods* and Fig. S9. The activity of FeFe or MoFe nitrogenases when expressed in the presence of the NifJ–NifF module from native *nif* promoters represents 100% activity in each case (in the absence of added inducer). FeFe represents the minimal FeFe nitrogenase system, and MoFe represents the reassembled MoFe nitrogenase system. Assembly of these nitrogenases requires both the “metal cluster biosynthesis module” and the “core enzyme” module, respectively, as shown in Fig. 1. As, *Anabaena* sp. PCC 7120; At, *Arabidopsis thaliana*; Cr, *Chlamydomonas reinhardtii*; Os, *Oryza sativa*; Ta, *T. aestivum*; Zm, *Zea mays*. Error bars indicate the SD observed from at least three independent experiments.

To assay electron transport by plant ferredoxins, the flavodoxin encoded by nifF in the Ko NifJ–NifF module was replaced by coexpression of the respective plant gene and the resultant activity of the reassembled MoFe (27) or the minimal FeFe (28) nitrogenase was analyzed by the method of acetylene reduction. Although significant background activity was observed in the absence of the NifJ–NifF module, all hybrid ETC modules stimulated nitrogenase activity by both the MoFe and FeFe systems, to varying extents (Fig. 2 B–E). Stimulation of activity by plastid ferredoxins was dependent on the presence of NifJ, indicating that reducing power is provided by the pyruvate oxidoreductase activity of this electron donor (Fig. S2). Interestingly, values >100% were observed for the FeFe nitrogenase system when NifF was replaced with the ferredoxins from As (FdxH), Cr (PETF), or Os (FD1), respectively (Fig. 2B and Table S1). This phenomenon suggests that the AvAnfH protein in the hybrid minimal FeFe nitrogenase (28) may prefer ferredoxin, rather than flavodoxin, as an electron donor. All of the chloroplast ferredoxins could restore ∼100% activity for the FeFe nitrogenase system, with the exception of the NifJ–AtFD2 hybrid module, which showed ∼76% activity (Fig. 2B). In contrast, only the NifJ–CrPETF and NifJ–TaFD hybrid modules could restore >90% activity to the MoFe nitrogenase system, whereas the NifJ–AtFD2, NifJ–ZmFDI, and NifJ–OsFD1 hybrid modules exhibited <70% activity (Fig. 2C). All root-plastid ferredoxin-derived hybrid modules showed lower nitrogenase activities compared with their chloroplast ferredoxin counterparts derived from the same organism (Fig. 2 B–E). It is possible that the different activities observed on substitution of ferredoxins from various plant origins could result from variations in expression levels. However, we were unable to confirm this possibility because we could not detect ferredoxin proteins by Western blotting using an anti-His monoclonal antibody (details are provided in SI Materials and Methods). Together, these results indicated that hybrid ETC modules formed with the NifJ protein, and plastid ferredoxins can direct the transfer of electrons to nitrogenase.

Fig. S2. — Plant-type ferredoxins are insufficient to support nitrogenase activity for either the FeFe (A) or MoFe (B) nitrogenase in the absence of NifJ. In all cases, cultures were assayed for acetylene reduction either in the absence (filled bars) or presence (open bars) of the appropriate inducer required to express heterologous ETCs as described in *SI Materials and Methods*. The activity of FeFe or MoFe nitrogenases when expressed in the presence of the NifJ–NifF module in the absence of added inducer represents 100% activity in each case. FeFe represents the minimal FeFe nitrogenase system; MoFe represents the reassembled MoFe nitrogenase system. Error bars indicate the SD observed from at least three independent experiments.

Table S1.

The redox potential of ferredoxins is not the primary factor limiting electron transfer to nitrogenase from pyruvate–ferredoxin (flavodoxin) oxidoreductase (NifJ)

Organism^*	Location	Genes	Redox potential^†	Relative nitrogenase activity, % acetylene reduction^‡
Organism^*	Location	Genes	Redox potential^†	FeFe	MoFe
Ko	—	nifF	−412 mV (34)	100 ± 6	100 ± 15
	—	ΔnifF	—	31 ± 6	10 ± 3
As	Heterocyst	fdxH	−351 mV (46)	153 ± 21	100 ± 6
	Vegetative	petF	−384 mV (46)	110 ± 14	81 ± 11
Cr	Chloroplast	PETF	−398 mV (47)	105 ± 11	90 ± 5
	Chloroplast	FDX2	−321 mV (47)	92 ± 4	87 ± 4
Zm	Chloroplast	FDI	−423 mV (48)	96 ± 9	56 ± 6
	Chloroplast	FDII	−406 mV (48)	75 ± 7	50 ± 7
	Root plastid	FDIII	−321 mV (49)	82 ± 9	51 ± 7
At	Chloroplast	FD1	−425 mV (50)	59 ± 6	36 ± 6
	Chloroplast	FD2	−433 mV (50)	76 ± 11	50 ± 11
	Root plastid	FD3	−337 mV (50)	68 ± 4	34 ± 8

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Origin of flavodoxin or ferredoxin genes expressed in E. coli.

^{^†}

Redox potentials reported previously with references in parentheses.

^{^‡}

Nitrogenase activity of E. coli strains expressing NifJ in the presence of the indicated flavodoxin or ferredoxin components. Activities observed from the minimal FeFe or reassembled MoFe nitrogenase systems in the presence of the NifJ–NifF module represent 100% activity, respectively. Data presented are mean values based on at least three independent experiments.

Because mitochondria represent another potential location for nitrogenase in plants, the capability of mitochondrial ferredoxins to support nitrogen fixation was also investigated in E. coli. The same strategy was used to clone the mitochondrial adrenodoxin coding genes from At (AtMFD1 and AtMFD2) as described above. When mitochondrial ferredoxin-derived hybrid ETC modules (NifJ–AtMFD1 or NifJ–AtMFD2) were introduced into E. coli, no restoration of activity was observed compared with the nifF-deficient MoFe or FeFe nitrogenase systems (Fig. 2 F and G). To exclude the possibility that the GroESL proteins are potentially required for proper and efficient folding of mitochondrial ferredoxins in E. coli (26), a high-copy plasmid carrying the GroESL-encoding genes was cotransformed with MoFe or FeFe nitrogenase systems carrying either the NifJ–AtMFD1 or NifJ–AtMFD2 hybrid modules. Similar negative results were obtained (Fig. S3). Interestingly, phylogenetic analysis showed that the mitochondrial ferredoxins are not clustered with any of the well-defined electron donors for nitrogenase (Fig. S4). Overall, these results suggest that the mitochondria ferredoxins cannot couple with the NifJ protein to form functional ETC modules for nitrogenase systems.

Fig. S3. — Acetylene reduction by FeFe (A) or MoFe nitrogenase systems (B) expressed in *E. coli* with hybrid ETC modules consisting of NifJ and mitochondrial ferredoxins from At *(At*MFDs) and cotransformed with a high-copy plasmid carrying the *groES* operon. In all cases, cultures were assayed for acetylene reduction either in the absence (filled bars) or presence (open bars) of the appropriate inducer required to express heterologous ETCs as described in *SI Materials and Methods*. The activity of FeFe or MoFe nitrogenases when expressed in the presence of the NifJ–NifF module in the absence of inducer represents 100% activity in each case. FeFe represents the minimal FeFe nitrogenase system; MoFe represents the reassembled MoFe nitrogenase system. Error bars indicate the SD observed from at least three independent experiments.

Fig. S4. — Phylogenetic analysis of the ferredoxins and flavodoxins from different organisms using the maximum-likelihood method with MAGA software (Version 6.0). A Poisson model was used to analysis the substitution rate of each site. The scale with the solid line represents 0.5 expected substitutions per site, and the scale with the dashed line represents 2.5 expected substitutions per site. Bootstrap values of 500 replicates are indicated in numbers at each junction. FdxH from As is colored in red; chloroplast FDs are colored in green; root-plastid FDs are shown in gray; and mitochondria FDs are represented in brown. FD or Fld sequence assignments are as follows: (As) AsFdxH, WP_010995602.1; AsPetF, WP_010998287.1; (Cr) CrPETF, XP_001692808.1; CrFDX2, XP_001697912.1; (At) AtFD1, NP_172565.1; AtFD2, NP_176291.1; AtFD3, NP_180320.1; AtMFD1, AAL82812.1; AtMFD2, AAL82813.1; (Zm) ZmFDI, P27787.1; ZmFDII, O80429.1; ZmFDIII, NP_001105346.1; (Os) OsFD1, NP_001060779.1; OsFD4, NP_001055675.1; OsMFD1, XP_015612701.1; OsMFD2, XP_015647181.1; (*T. aestivum*) TaFD, P00228.2; (Ko) KoNifF, WP_004138775.1; (Av) AvNifF, ACO76434.1; AvFdxN, WP_012703542.1; AvVnfFd, WP_012698954.1; (*R. capsulatus*) RcNifF, AAC05792.1; RcFdxN, CAA35699.1; (*Rhizobium meliloti*) SmFdxN, NP_435687.1; (*Saccharomyces cerevisiae*) ScYAH1, NP_015071.1; and (*Mus musculus*) MmADX, NP_032022.1.

Influence of Hybrid ETC Modules Consisting of Plant-Type Ferredoxin–NADPH Oxidoreductases and Well-Defined Electron Donors on Nitrogenase Activity.

In plants, three different types of the ferredoxin–NADPH oxidoreductases (FNRs) are identified and exist in different organelles. All of these FNRs function to mediate electron transfer between ferredoxins and NADPH (23, 25, 26). From phylogenetic analysis, we found that the chloroplast FNRs form a subgroup with the FNR proteins from cyanobacteria, whereas the root-plastid FNRs form another subgroup with the FNR proteins from algae (Fig. S5). In contrast, the mitochondrial MFDRs diverged away from these two subgroups during early evolution (Fig. S5).

Fig. S5. — Phylogenetic analysis of FNRs from different organisms using the maximum-likelihood method with MAGA software (Version 6.0). A Poisson model was used to analysis the substitution rate of each site. The scale with the solid line represents 0.2 expected substitutions per site, and the scale with the dashed line represents 0.8 expected substitutions per site. Bootstrap values of 500 replicates are indicated in numbers at each junction. FNR from At is shown in red, chloroplast FNRs in green, root-plastid FNRs in gray, and mitochondrial FNRs in brown. FNR sequence assignments are as follows (As) AsFNR, WP_010998260.1; (*Synechocystis* sp. PCC 6803) SsFNR, WP_010873079.1; (Cr) CrFNR, XP_001697352.1; (*Ostreococcus taurii*) OtFNR, XP_003084170.1; (At) AtLFNR1, AAF79911.1; AtLFNR2, CAB52472.1; AtRFNR1, CAB81081.1; AtRFNR2, NP_174339.1; AtMFR, NP_194962.2; (Zm) ZmLFNR1, NP_001105568.1; ZmLFNR2, NP_001104851.1; ZmLFNR3, NP_001149023.2; ZmRFNR1, NP_001295409.1; ZmRFNR2, ACG35047.1; (Os) OsLFNR1, XP_015640980.1; OsLFNR2, BAS76542.1; OsRFNR1, XP_015629836.1;OsRFNR2, XP_015646844.1; OsMFDR, XP_015626908.1; (*E. coli*) EcFpr, WP_000796332.1; (*S. cerevisiae*) ScARH1, AAC49500.1; and (*M. musculus*) MmFDXR, NP_032023.1.

To investigate whether hybrid ETC modules consisting of the plant-type FNRs and well-defined electron donors (KoNifF, AsFdxH, and AsFdxB) could direct electron transfer to nitrogenase in E. coli, well-characterized chloroplast or root-plastid FNRs from Cr and Zm, plus the At mitochondrial MFDR, were selected to verify this assumption. These hybrid modules were transformed into the E. coli, and their activities were assayed by the acetylene reduction method. None of the hybrid ETC modules consisting of the plant-type FNRs and NifF could stimulate acetylene reduction by either the MoFe or FeFe nitrogenase systems (Fig. S6). In contrast, all hybrid modules formed with the plant-type FNRs and AsFdxH could partially restore nitrogenase activity to both the MoFe and FeFe nitrogenase systems (Fig. 3 B and C). However, only MFDR from mitochondria could support electron transfer to the nitrogenases when coupled to AsFdxB (Fig. S6).

Fig. S6. — Influence of hybrid ETC modules consisting of KoNifF or AsFdxB with FNRs from different plant organelles on nitrogenase activity in *E. coli*. The NifJ–NifF module was replaced either by hybrid modules consisting of KoNifF with plant-type FNRs (A and B) or AsFdxB with plant-type FNRs (C and D). In all cases, cultures were assayed for acetylene reduction either in the absence (filled bars) or presence (open bars) of the appropriate inducer required to express heterologous ETCs as described in *SI Materials and Methods*. The activity of FeFe or MoFe nitrogenases when expressed in the presence of the NifJ–NifF module in the absence of added inducer represents 100% activity in each case. MoFe represents the reassembled MoFe nitrogenase system. Error bars indicate the SD observed from at least three independent experiments.

Fig. 3. — Influence of hybrid ETC modules consisting of the cyanobacterial ferredoxin AsFdxH combined with FNRs from different plant organelles on nitrogenase activity in *E. coli*. (A) Schematic diagram for electron transport between hybrid ETC modules and nitrogenases. (B and C) The NifJ–NifF ETC module was replaced by hybrid modules consisting of plant-type FNRs and AsFdxH. Experimental details and abbreviations are the same as in Fig. 2. Error bars indicate the SD observed from at least three independent experiments.

The fact that ETC modules formed with the plant-type FNRs and NifF could not functionally support nitrogenase activity is not surprising, because FNRs known to reduce flavodoxins are limited to a few bacteria, such as Fpr from E. coli and FNRs from some species of cyanobacteria, which are usually related to the biosynthesis of cytochrome P450 (30). Microbial FNRs showed a distant evolutionary relationship with the chloroplast and root-plastid FNRs (Fig. S5). In addition, no flavodoxins have been identified in plants (31). Therefore, the plant-type FNRs do not appear to have coevolved with the flavodoxins.

Intact ETC Modules from Chloroplasts and Root Plastids Can Functionally Support Nitrogenase Activity.

After evaluating the function of the hybrid modules, further experiments were carried out to investigate whether intact ETC modules, consisting of FNRs and their cognate ferredoxin counterparts from plant organelles, could support nitrogenase activity. By combining the P_tac-controlled FNRs with P_LtetO-1-controlled ferredoxins (details are provided in SI Materials and Methods), two chloroplast ETC modules, CrFNR–PETF and ZmLFNR–FDI; one root-plastid ETC module, ZmRFNR–FDIII; and one mitochondrial ETC module, AtMFDR–MFD1 were constructed. Because it is known that the AsPetH–FdxH module from cyanobacteria can function to support nitrogen fixation in its original host, this module was also constructed and used as a control.

The ability of the intact ETC modules to support nitrogenase activity as replacement for the NifJ–NifF module was assayed by both acetylene reduction and ¹⁵N-assimilation methods. With the exception of the AtMFDR–MFD1 module from mitochondria, all other ETC modules conferred ability to partially restore acetylene reduction and ¹⁵N assimilation to both MoFe and FeFe nitrogenases (Fig. 4 B–E). In contrast, no obvious stimulation of activity could be observed, when either of the two components from each of the modules was expressed individually, demonstrating that the plant-type ferredoxins do not function in electron transport in the absence of their cognate FNR and, conversely, that the plant FNRs cannot donate electrons to nitrogenase in the absence of a coexpressed ferredoxin component (Fig. S7). Thus, both members of the plant ETC pairs are necessary for functionality.

Fig. 4. — Influence of intact ETC modules consisting of FNRs from different plant organelles with their cognate ferredoxins (FDs) on nitrogenase activity in *E. coli*. (A) Schematic representation for electron transport between intact plant ETC modules and nitrogenases. (B–E) Nitrogen fixation by FeFe or MoFe nitrogenases was assayed either by acetylene reduction (B and C; black bars) or ¹⁵N assimilation (D and E; striped bars). Experimental details and abbreviations are the same as in Fig. 2. Error bars for the acetylene reduction assay indicate the SD observed from at least three independent experiments. Error bars for ¹⁵N assimilation indicate the SD observed from at least two independent experiments.

Fig. S7. — Single components of each ETC module are insufficient to support nitrogenase activity for either the FeFe (A) or MoFe (B) nitrogenase. In all cases, cultures were assayed for acetylene reduction either in the absence (filled bars) or presence (open bars) of the appropriate inducer required to express heterologous ETCs as described in *SI Materials and Methods*. The activity of FeFe or MoFe nitrogenases when expressed in the presence of the NifJ–NifF module in the absence of added inducer represents 100% activity in each case. FeFe represents the minimal FeFe nitrogenase system; MoFe represents the reassembled MoFe nitrogenase system. Error bars indicate the SD observed from at least three independent experiments.

Two of the chloroplast modules, CrFNR–PETF and ZmLFNR–FDI, showed comparable amounts of activity to those observed with the AsPetH–FdxH module from cyanobacteria (∼45% acetylene reduction and ∼30% ¹⁵N assimilation) with both FeFe and MoFe nitrogenases (Fig. 4). However, the ZmRFNR–FDIII module from the root plastid was less active than its corresponding chloroplast module from the same plant with respect to both nitrogenases (Fig. 4 B–E). Interestingly, weak complementation was observed with the AtMFDR–MFD1 module (11% ¹⁵N assimilation activity) when combined with MoFe nitrogenase, compared with the NifJ–NifF-deficient negative control (6% ¹⁵N assimilation activity) (Fig. 4E). However, this phenotype was not observed with FeFe nitrogenase. (Fig. 4D). Because multiple copies of ferredoxins exist in E. coli, it is possible that the enhanced activity of MoFe nitrogenase resulted from the contribution of hybrid modules formed between AtMFDR and those from E. coli ferredoxins. This effect may not be observed with the FeFe nitrogenase system because of its higher background activity in the absence of nifF and nifJ (Fig. 4D). Together, these results demonstrate that intact ETC modules from plastids, but not their equivalents from mitochondria, are capable of transferring electrons to nitrogenases.

SI Materials and Methods

Bacterial Strains and Growth Media.

Luria–Bertani broth for E. coli growth contained 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl. KPM minimal medium used in this study contained 10.4 g/L Na₂HPO₄, 3.4 g/L KH₂PO₄, 26 mg/L CaCl₂·2H₂O, 30 mg/L MgSO₄, 0.3 mg/L MnSO₄, 36 mg/L ferric citrate, 10 mg/L para-aminobenzoic acid, 5 mg/L biotin, 1 mg/L vitamin B1, and 0.8% (wt/vol) glucose, with 20 mM ammonia (KPM-HN) or 10 mM glutamate (KPM-LN) as the nitrogen source. Casamino acids (BD Biosciences, catalog no. 223050) at a final concentration of 0.05% was also added to the KPM minimal medium to ensure normal growth. Antibiotics were used at the following concentrations: 50 μg/mL for ampicillin and 25 μg/mL for chloramphenicol.

Construction of Recombinant Plasmids.

Plasmid pKU7815 and pKU7017 are pACYC184 derivatives carrying the minimal FeFe (28) or the reassembled MoFe nitrogenase system (27). nifF and nifJ double deletion derivatives of pKU7815 and pKU7017, designated as pKU7830 and pKU7831 in this study, were constructed by direct removal of the nifF and nifJ operons by using the specific restriction sites flanking each transcription unit: SwaI for nifF and ScaI for nifJ, respectively. The complementary plasmid for pKU7830 and pKU7831 is a pBR322M derivative (pKU7832) carrying both the nifF and nifJ operons. The pBR322M-P_LtetO-1 plasmid was constructed by direct assembly of the tetR expression cassette and the P_LtetO-1 promoter region with the pBR322M as backbone by using the Gibson Assembly Cloning kit (NEB, catalog no. E5520S). The tetR expression cassette consists of a strong constitutive promoter (BBa_J23100; parts.igem.org/Main_Page), a medium strength ribosome-binding site (RBS, BBa_B0032; parts.igem.org/Main_Page), and the thrL terminator from E. coli. Similarly, the pBR322M-P_tac plasmid was constructed by direct assembly of the lacI expression cassette and the P_tac promoter carrying a weak RBS with pBR322M as backbone. To lower the leaky expression of plant-type FNRs, a LacI mutant LacI^WF (43) with higher affinity for the lac operator expressed from P_lacI^q was used for construction of the pBR322M-P_tac plasmid. The pKU7833 plasmid was constructed by replacing the nifF gene in the pKU7832 with the SwaI restricted tetR-P_LtetO-1 fragment from plasmid pBR322M-P_LtetO-1. To construct plasmids with hybrid modules expressing NifJ and plant-type ferredoxins, original gene sequences or chemically synthesized gene sequences (Dataset S1) were cloned downstream of the P_LtetO-1 promoter of the pKU7833 plasmid using NdeI/SpeI restriction sites. To facilitate detection of the expression level of different ferredoxins, a sequence encoding a hexahistidine tag was added to each of the synthesized ferredoxin sequences as shown by the gray shadows in Dataset S1. The pKU7846 plasmid was constructed by replacing the nifJ gene in pKU7832 with the ScaI-restricted lacI-P_tac fragment from plasmid pBR322M-P_tac. For construction of plasmids with hybrid modules expressing plant-type FNRs and NifF, original gene sequences or chemically synthesized gene sequences (Dataset S1) were cloned downstream of the P_tac promoter of the pKU7846 plasmid using NdeI/SpeI restriction sites. The pKU7853 plasmid, carrying P_tac-AspetH_ori/P_LtetO-1-AsfdxH_ori, was constructed by replacing the nifF gene in plasmid pKU7847 with the ScaI P_LtetO-1-AsfdxH fragment from pKU7834. Similar methods were used to construct the pKU7854-pKU7857 and pKU7859-pKU7865 plasmids. The PCR product carrying groES with its original promoter, flanked by XbaI/SpeI restriction sites, was PCR amplified from the genome of E. coli and directly ligated to the pEASY-Blunt vector (TransGene, catalog no. CB101) to generate pKU7845. Plasmids maps are provided in Fig. S8. Each of the above constructs was confirmed by DNA sequencing before any further experiments.

Fig. S8. — Plasmid maps of the main vectors used in this study. rrnB T1 is an *E. coli* terminator (BBa_B0010); T_L is the *E. coli thrL* gene terminator; T₀ is the phage lambda t₀ terminator; and T_a is an artificial terminator L3S2P21 described by Chen et al. (45).

Acetylene Reduction Assay.

To measure the activity of E. coli JM109 derivatives expressing reassembled nitrogenase systems, cells were initially grown overnight in KPM-HN medium. The cells were then diluted into 2 mL of KPM-LN medium in 20-mL sealed tubes to a final OD₆₀₀ of ∼0.3. To optimize expression of the plant components, 200 ng/mL anhydrotetracycline (aTc) or 200 μM isopropyl-β-d-thiogalactoside (IPTG) was added to induce expression of the ferredoxins or FNRs, respectively, as indicated by the results in Fig. S9. Air in the tubes was repeatedly evacuated and replaced with argon. After incubation at 30 °C for 6–8 h, 2 mL of C₂H₂ was injected, and the gas phase was analyzed ∼16 h later with a Shimadzu GC-2014 gas chromatograph. Data presented are mean values based on at least three replicates.

Fig. S9. — Induction profiles of CrPETF or CrFNR expression in relation to nitrogenase activity. (A and B) Induction profile of CrPETF expression, controlled by the P_LtetO-1 promoter in response to aTc concentration. (C and D) Induction profile of CrFNR expression controlled by the P_tac promoter in response to IPTG concentration. Acetylene reduction activity shown on the y axis is relative to the activity obtained from the FeFe or MoFe nitrogenase system expressed in the presence of the NifJ–NifF module. FeFe represents the minimal FeFe nitrogenase system; MoFe represents the recombined MoFe nitrogenase system. Error bars indicate the SD observed from at least three independent experiments.

¹⁵N₂ Assimilation Assay.

To detect ¹⁵N₂ assimilation, E. coli JM109 derivatives expressing reassembled nitrogenase systems were grown as described for the acetylene reduction assay. The tubes were filled with N₂ gas, and then 3 mL of gas was removed, and 2 mL of ¹⁵N₂ gas (99%⁺; Shanghai Engineering Research Center for Stable Isotopes) was injected. After 48 h of incubation at 30 °C, the cultures were collected, freeze -dried, ground, weighed, and then sealed into tin capsules. Isotope ratios are expressed as δ¹⁵N, where 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 (44). Data presented are mean values based on at least two replicates.

Discussion

It is generally accepted that several factors may limit successful engineering of nitrogenase in cereal crops, including provision of the appropriate physiological environment for the enzyme [e.g., availability of energy (ATP), reducing power, and a low-oxygen environment], in addition to the relative large number of nif genes required for biosynthesis of nitrogenase itself. Attempts have been made to reduce the number of genes required for both MoFe (32) and FeFe nitrogenase biosynthesis (28) by using E. coli as a chassis. However, in the case of nif genes originating from Paenibacillus sp., it was observed that nitrogenase activity decreased drastically when the number of genes were decreased to nine for MoFe nitrogenase (32), but activity could be recovered when additional genes were recruited back into the system (33). These findings have highlighted the difficulties in minimizing nif gene sets for synthetic biology.

One approach to reducing the number of genes required for nitrogenase activity is to make use of host-encoded ETCs. Bioinformatic analysis indicated that ETC modules from plant organelles show close evolutionary relationships with nitrogenase ETCs from nitrogen-fixing cyanobacteria (Figs. S4 and S5). Therefore, it is reasonable to question whether these ETCs can replace the NifJ–NifF module and support nitrogen fixation. We have used the modularity concept to analyze compatibility between ETCs from different plant organelles with nitrogenase enzyme modules. Our results indicate that plastid ferredoxins can efficiently participate in electron transfer to nitrogenase from either the nif-specific pyruvate–flavodoxin (ferredoxin) reductase encoded by nifJ or from its FNR counterparts in plants. Overall, we observed little difference in the response of the MoFe or FeFe nitrogenases to the various ETC modules we analyzed, suggesting that plant ETCs can function with either enzyme.

We found that almost all plant-originated ferredoxins used in this study (except adrenodoxins from mitochondria) could functionally substitute for NifF for both FeFe and MoFe nitrogenases (Fig. 2). This finding implies that the interface between these ferredoxins and NifH/AnfH are competent for electron transfer. In many cases, the previously reported redox potentials of these ferredoxins (Table S1) are higher than those of the Fe protein NifH (−412 mV) (34), which disfavors electron transfer to nitrogenase in vitro (35). Furthermore, the redox potential of the NADH/NAD⁺ couple (midpoint potential −380 mV) is, in theory, too high to drive electron donation to nitrogenase by FNRs, although NAD(P)H-dependent electron-transfer chains apparently support nitrogenase activity in vivo in aerobic diazotrophs. To account for this conundrum, it has been proposed that either proton motive force or electron bifurcation drives the reversed electron flow required to overcome these bioenergetic constraints in vivo (36, 37). Because multiple factors, including expression levels, interface compatibility, and protein ratios, may affect electron transfer to nitrogenase, it is not possible to determine whether ferredoxin redox potentials relate to the efficiency of electron transfer in our experimental system.

Overall, our results lay a solid foundation for the use of extant ETC modules from plant organelles to engineer BNF in cereal crops. The observation that ETC modules from both chloroplasts and root plastids can functionally support nitrogenase activity implies that engineering diazotrophy in plastids does not require insertion of an ETC module. For instance, for the FeFe nitrogenase system, of the minimal 10 genes required (28), 2 of these function as the bacterial ETC module and presumably would not be essential for engineering nitrogenase activity in plants. To further reduce the number of nif/anf genes required, plant genes that can substitute for components of the “metal cluster biosynthesis” module (Fig. 1), such as those required for iron–sulfur cluster assembly, could also be considered. Two recent studies have shown that the [Fe–S] cluster biosynthesis components from eukaryotic organelles can substitute for NifU and NifS to incorporate the [4Fe–4S] cluster into the Fe protein of nitrogenase (19, 20).

Accumulated mutational studies indicate that many diazotrophs have multiple pathways that mediate electron flow to nitrogenase. These pathways include multiple electron-transfer proteins (ferredoxins and flavodoxins) and multiple oxidoreductases, including pyruvate–flavodoxin (ferredoxin) oxidoreductase, FNR, and the Rnf complex [a membrane-bound ion-translocating NADH–ferredoxin oxidoreductase, first identified in Rhodobacter capsulatus by Schmehl et al. (16)] (Fig. 1). The putative membrane-associated electron-transferring flavoprotein complex encoded by the fixABCX operon has also been proposed to transfer electrons to nitrogenase (38). Such redundancy has endowed nitrogenase with the ability to cope with heterogenous ETC modules. Conversely, the [2Fe–2S]-type ferredoxin is the most extensively used electron shuttle, which is maintained throughout the tree of life and is involved in a plethora of metabolic, regulatory, dissipative, and developmental processes (31). To satisfy the requirements for electron transfer to diverse proteins, ferredoxins have evolved multiple interfaces to cope with different metabolic partners (39). This interface flexibility may provide another explanation for the compatibility of the nitrogenase system with heterogenous ETC modules from plant chloroplasts and root plastids.

Combining the results obtained from this study, a schematic model for electron transfer to nitrogenase in organelles of engineered plants is proposed (Fig. 5). Our results imply that the endogenous ETC module present in mitochondria is not competent to support nitrogenase activity (Fig. 4), reflecting the requirement for an additional ETC component, such as AsFdxH or AsFdxB, that can function as a hybrid module for direct electron transfer to nitrogenase (Fig. 3). In this scenario, NADPH generated either by glycolysis or by isocitrate dehydrogenase can provide reducing equivalents for electron transfer to nitrogenase through the hybrid MFDR–ferredoxin pathway (Fig. 5). In root plastids, the NADPH generated by degradation of glucose through the oxidative pentose phosphate pathway is used by RFNR to catalyze reduction of ferredoxins. We propose that this electron-transfer pathway can be used to provide reducing power for nitrogenase in the absence of the nif-specific NifJ–NifF ETC module (Fig. 5).

Fig. 5. — Schematic model illustrating potential routes for electron transfer to nitrogenase in engineered plant organelles. The diagram depicts an artificial plant cell in which a chloroplast and root plastid coexists in the same cell. The main components or processes for generation of reducing power are shown. Components within organelles with solid outlines represent existing proteins present in plants, whereas components with dashed outlines represent those required to engineer nitrogenase activity as suggested by our results. The red arrow in the root plastid represents RFNR–ferredoxin-mediated electron transfer from NADPH to nitrogenase, with reducing equivalents being supplied by the oxidative pentose phosphate pathway (OxPPP). In mitochondria, the red arrow indicates the election transfer pathway from MFDR to nitrogenase, which can function if a heterologous ferredoxin such as AsFdxH or AsFdxB is introduced. NADPH can be supplied either through glycolysis or via the oxidative TCA cycle by isocitrate dehydrogenase (ICDH). In chloroplasts, light-activated photosystem II (PSII) extracts electrons from water and transfers them to plastoquinone (PQ), and then through cytochrome b6f (Cytb₆f) to plastocyanin (PC), which then feeds electrons to the light-oxidized photosystem I (PSI). PSI-derived electrons are used to reduce ferredoxin, which then transfers reductant to either LFNR, for NADPH production, or to nitrogenase, for nitrogen fixation. The NADPH generated can also promote reverse electron transfer to nitrogenase via ferredoxin, catalyzed by LFNR. The bottom lighter half of the chloroplast represents the light condition, with the blue arrow representing the photo-coupled “charging” process for accumulating NADPH; the darker half of the chloroplast represents the dark condition, with the yellow arrow representing the LFNR–ferredoxin mediated “discharging” process for transferring electrons from NADPH to nitrogenase (N₂ase). As the major site for ammonia assimilation, ammonia produced in the chloroplast can be immediately assimilated by glutamine synthetase (GS) (42). Glc-6P, glucose-6-P; Rib-5P, ribose-5-P.

Although chloroplasts are potentially problematic for nitrogenase engineering in terms of oxygen evolution, it is possible to consider solutions based on mechanisms of oxygen avoidance used by cyanobacteria. The electron-transfer route to LFNR in chloroplasts involves light activation of photosystems II and I and the subsequent reduction of ferredoxin, which feeds electrons into LFNR. The reaction catalyzed by LFNR is reversible; whereas reduction by ferredoxin favors biosynthesis of NADPH (23), the reverse reaction uses NADPH to reduce oxidized ferredoxin (40). In this case, LFNR can be considered to be analogous to a battery, which accumulates reducing equivalents in the light and discharges this power (as NADPH) in the dark to fuel redox reactions (Fig. 5). Because the chloroplast ETC modules studied in this work function with photosystem I and can support nitrogenase activity in E. coli (Fig. 4), it is possible to envisage a scenario for energetic coupling of photosynthesis with nitrogen fixation, with temporal separation of their activities in the light and dark periods. In nonheterocystous cyanobacteria, nature has been able to reconcile the processes of nitrogen fixation and oxygenic photosynthesis through temporal separation, in which photosynthetic CO₂ fixation occurs in the light and nitrogen fixation is carried out in the dark (41). According to our model, electron transfer from the ferredoxin to LFNR would occur during the light period, resulting in accumulation of excess NADPH. This reductant could be used in the dark to drive the reverse-catalytic reaction of LFNR, enabling reduction of nitrogenase by reduced ferredoxin (Fig. 5).

Materials and Methods

Bacterial Strains and Growth Medium.

Bacterial strains used in this study are listed in Table S2. Media and antibiotics were used as described (28) and are detailed in SI Materials and Methods.

Table S2.

Strains and plasmids used in this study

Strain or plasmid	Relevant feature	Source
Ec strains
Top10	F⁻ mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(Str^R) endA1 λ⁻	Invitrogen
JM109	recA endA1 gyrA96 hsdR17 supE44 relA1 Δ(lac-proAB)/F′[traD36 proAB⁺ lacI^q lacZΔM15]	Takara Bio
Plasmids
pBR322M	pBR322 derivative, Amp^R, labeled as ΔnifJ/ΔnifF in the text	Ref. 28
pBR322M-P_LtetO-1	pBR322M derivative carrying P_LtetO-1 inducible expression cassette	This study
pBR322M-P_tac	pBR322M derivative carrying P_tac inducible expression cassette	This study
pKU7815	pACYC184 derivative carrying the minimal FeFe nitrogenase system, Cm^R	Ref. 28
pKU7017	pACYC184 derivative carrying the reassembled MoFe nitrogenase system, Cm^R	Ref. 27
pKU7830	pKU7815 derivative, ΔnifJ/ΔnifF	This study
pKU7831	pKU7017 derivative, ΔnifJ/ΔnifF	This study
pKU7832	pBR322M derivative carrying KonifJ/nifF genes, labeled as nifJ/nifF in the text	This study
pKU7833	pKU7830 derivative with nifF gene replaced by P_LtetO-1 inducible expression cassette, labeled as nifJ/ΔnifF in the text	This study
pKU7834	pKU7833 derivative carrying nifJ/P_LtetO-1-AsfdxH_ori	This study
pKU7835	pKU7833 derivative carrying nifJ/P_LtetO-1-CrPETF_syn	This study
pKU7836	pKU7833 derivative carrying nifJ/P_LtetO-1-AtFD2_syn	This study
pKU7837	pKU7833 derivative carrying nifJ/P_LtetO-1-ZmFDI_syn	This study
pKU7838	pKU7833 derivative carrying nifJ/P_LtetO-1-OsFD1_syn	This study
pKU7839	pKU7833 derivative carrying nifJ/P_LtetO-1-TaFD_syn	This study
pKU7840	pKU7833 derivative carrying nifJ/P_LtetO-1-AtFD3_syn	This study
pKU7841	pKU7833 derivative carrying nifJ/P_LtetO-1-ZmFDIII_syn	This study
pKU7842	pKU7833 derivative carrying nifJ/P_LtetO-1-OsFD4_syn	This study
pKU7843	pKU7833 derivative carrying nifJ/P_LtetO-1-AtMFD1_syn	This study
pKU7844	pKU7833 derivative carrying nifJ/P_LtetO-1-AtMFD2_syn	This study
pKU7845	pEASY-Blunt derivative carrying EcgroES operon with its original promoter	This study
pKU7846	pKU7832 derivative with nifJ gene replaced by P_tac inducible expression cassette, labeled as ΔnifJ/nifF in the text	This study
pKU7847	pKU7846 derivative carrying P_tac-AspetH_ori/nifF	This study
pKU7848	pKU7846 derivative carrying P_tac-CrFNR_syn/nifF	This study
pKU7849	pKU7846 derivative carrying P_tac-ZmLFNR_syn/nifF	This study
pKU7850	pKU7846 derivative carrying P_tac-ZmRFNR_syn/nifF	This study
pKU7851	pKU7846 derivative carrying P_tac-AtMFDR_syn/nifF	This study
pKU7852	pKU7834 derivative with nifJ gene replaced by P_tac inducible expression cassette, labeled as ΔnifJ/P_LtetO-1-AsfdxH_ori in the text	This study
pKU7853	pKU7847 derivative carrying P_tac-AspetH_ori/P_LtetO-1-AsfdxH_ori	This study
pKU7854	pKU7848 derivative carrying P_tac-CrFNR_syn/P_LtetO-1-AsfdxH_ori	This study
pKU7855	pKU7849 derivative carrying P_tac-ZmLFNR_syn/P_LtetO-1-AsfdxH_ori	This study
pKU7856	pKU7850 derivative carrying P_tac-ZmRFNR_syn/P_LtetO-1-AsfdxH_ori	This study
pKU7857	pKU7851 derivative carrying P_tac-AtMFDR_syn/P_LtetO-1-AsfdxH_ori	This study
pKU7858	pKU7833 derivative carrying nifJ/P_LtetO-1-AsfdxB_ori	This study
pKU7859	pKU7847 derivative carrying P_tac-AspetH_ori/P_LtetO-1-AsfdxB_ori	This study
pKU7858	pKU7848 derivative carrying P_tac-CrFNR_syn/P_LtetO-1-AsfdxB_ori	This study
pKU7859	pKU7849 derivative carrying P_tac-ZmLFNR_syn/P_LtetO-1-AsfdxB_ori	This study
pKU7860	pKU7850 derivative carrying P_tac-ZmRFNR_syn/P_LtetO-1-AsfdxB_ori	This study
pKU7861	pKU7851 derivative carrying P_tac-AtMFDR_syn/P_LtetO-1-AsfdxB_ori	This study
pKU7862	pKU7848 derivative carrying P_tac-CrFNR_syn/P_LtetO-1-CrPETF_syn	This study
pKU7863	pKU7849 derivative carrying P_tac-ZmLFNR_syn/P_LtetO-1-ZmFDI_syn	This study
pKU7864	pKU7850 derivative carrying P_tac-ZmRFNR_syn/P_LtetO-1-ZmFDIII_syn	This study
pKU7865	pKU7851 derivative carrying P_tac-AtMFDR_syn/P_LtetO-1-AtMFD_syn	This study
pKU7866	pKU7833 derivative carrying nifJ/P_LtetO-1-AspetF_ori	This study
pKU7867	pKU7833 derivative carrying nifJ/P_LtetO-1-CrFDX2_syn	This study
pKU7868	pKU7833 derivative carrying nifJ/P_LtetO-1-AtFD1_syn	This study
pKU7869	pKU7833 derivative carrying nifJ/P_LtetO-1-ZmFDII_syn	This study

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Amp, ampicillin; Cm, chloramphenicol; Ec, E. coli; ^R, resistance; Δ, deletion. _syn represents the codon optimized and synthetic gene sequence; _ori represents original gene sequence amplified from the genome of its original host.