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

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.

Keywords: electron transport; nitrogen fixation; nitrogenase engineering; plant organelles.

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.

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.

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. 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.

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 (AtMFDs) 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.

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.

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.

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. 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.

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.

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.

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).