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Modular Electron-Transport Chains from Eukaryotic Organelles Function to Support Nitrogenase Activity

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Modular Electron-Transport Chains from Eukaryotic Organelles Function to Support Nitrogenase Activity Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity Jianguo Yanga,1, Xiaqing Xiea,1, Mingxuan Yanga, Ray Dixonb,2, and Yi-Ping Wanga,2 aState Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China; and bDepartment of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, United Kingdom Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved January 12, 2017 (received for review December 7, 2016) A large number of genes are necessary for the biosynthesis reduced by a variety of oxidoreductase systems, depending on the and activity of the enzyme nitrogenase to carry out the process physiology of the host diazotroph (13–17). of biological nitrogen fixation (BNF), which requires large amounts A number of studies have suggested chloroplasts, root plastids, or of ATP and reducing power. The multiplicity of the genes involved, mitochondria as suitable locations for expression of nitrogenase in the oxygen sensitivity of nitrogenase, plus the demand for energy eukaryotes (18–20). These energy-conversion organelles can po- and reducing power, are thought to be major obstacles to tentially provide reducing power and ATP required for the nitro- engineering BNF into cereal crops. Genes required for nitrogen gen-fixation process. Diverse reduction reactions carried out in fixation can be considered as three functional modules encoding these organelles rely on different electron-transport chains (21). electron-transport components (ETCs), proteins required for metal Multiple gene copies of ferredoxins have been identified in all cluster biosynthesis, and the “core” nitrogenase apoenzyme, re- plants, including photosynthetic or nonphotosynthetic ferredoxins spectively. Among these modules, the ETC is important for the mainly expressed in chloroplasts or root plastids, respectively; and supply of reducing power. In this work, we have used Escherichia ferredoxin-like adrenodoxins located in the mitochondria (21, 22). coli as a chassis to study the compatibility between molybdenum The major function of the photosynthetic ferredoxins is to transfer and the iron-only nitrogenases with ETC modules from target electrons from photosystem I to NADPH, catalyzed by leaf-type plant organelles, including chloroplasts, root plastids, and mito- ferredoxin–NADPH oxidoreductase (LFNR) (23). In addition, chondria. We have replaced an ETC module present in diazotrophic photosynthetic ferredoxins work to distribute reducing power de- – bacteria with genes encoding ferredoxin NADPH oxidoreductases rived from the photosynthetic process to several ferredoxin- (FNRs) and their cognate ferredoxin counterparts from plant or- dependent enzymes for nitrogen and sulfur assimilation (24). – ganelles. We observe that the FNR ferredoxin module from chlo- Electron transfer between root-type ferredoxin–NADPH oxidore- roplasts and root plastids can support the activities of both types ductase (RFNR) and ferredoxin in the root plastid is reversed, with of nitrogenase. In contrast, an analogous ETC module from mito- NADPH generated in the oxidative pentose-phosphate pathway chondria could not function in electron transfer to nitrogenase. being used to reduce RFNR and, in turn, ferredoxin (25). In mi- However, this incompatibility could be overcome with hybrid tochondria, adrenodoxin serves to transfer electrons from NADPH- modules comprising mitochondrial NADPH-dependent adreno- Anabaena dependent adrenodoxin oxidoreductase (MFDR) to the cysteine doxin oxidoreductase and the ferredoxins FdxH or desulfurase Nfs1 to participate in the biosynthesis of the biotin (26). FdxB. We pinpoint endogenous ETCs from plant organelles as Recently, we successfully reassembled the Klebsiella oxytoca power supplies to support nitrogenase for future engineering of (Ko) MoFe (27) and the “minimal” Azotobacter vinelandii (Av) diazotrophy in cereal crops. FeFe (28) nitrogenase systems in Escherichia coli (Fig. 1). From the synthetic biology point of view, these two nitrogenase systems nitrogen fixation | electron transport | plant organelles | nitrogenase can be divided into three functional modules: the electron- engineering transport component (ETC) module, the metal cluster biosynthesis itrogen is one of the primary nutrients limiting plant pro- Significance Nductivity in agriculture (1). Industrial nitrogen fertilizers are used to circumvent this limitation, but have resulted in environ- Engineering nitrogenase into cereal crops requires detailed under- mental pollution and expensive economic costs, especially in de- standing of the components required for efficient nitrogen fixation. veloping countries (2, 3). These factors have potentiated a renewed We have used a synthetic biology modular approach to evaluate focus toward engineering biological nitrogen fixation (BNF) in ce- components from chloroplast, root plastids, and mitochondria that real crops. BNF, the process that converts gaseous nitrogen to function as electron donors to both conventional Mo nitrogenase ammonia by nitrogenase enzymes, contributes >60% of the total and the alternative Fe nitrogenase systems. The knowledge atmospheric N2 fixed in the biogeochemical nitrogen cycle (4). obtained in this study not only identifies electron-transfer compo- Nitrogenases are a family of metalloenzymes that consist of two nents from plant organelles that can be used to support nitroge- separable components, dinitrogenase reductase (Fe protein) and nase activity, but also is likely to enable reduction of the number of dinitrogenase (XFe protein, where X is equivalent to Mo, V, or Fe, target genes required to engineer nitrogen fixation in plants. depending on the heterometal composition of the active site co- Author contributions: J.Y., R.D., and Y.-P.W. designed research; J.Y., X.X., and M.Y. performed factor) (Fig. 1 and refs. 5 and 6). All three nitrogenases catalyze the research; J.Y., R.D., and Y.-P.W. analyzed data; and J.Y., X.X., R.D., and Y.-P.W. wrote the paper. biological reduction of N2 according to the following equation: N2 + + − The authors declare no conflict of interest. (6 + 2n)H + (6 + 2n)e → 2NH + nH (n ≥ 1) (7–9). In this 3 2 This article is a PNAS Direct Submission. process, electrons are first transferred to the Fe protein, which, in Freely available online through the PNAS open access option. turn, donates electrons to the XFe protein with hydrolysis of two See Commentary on page 3009. ATP molecules per electron (Fig. 1) (10–12). Although Fe protein 1J.Y. and X.X. contributed equally to this work. is the obligate electron donor for XFe protein in all characterized 2To whom correspondence may be addressed. Email: [email protected] or wangyp@pku. nitrogenase systems, the in vivo electron donor for Fe protein is less edu.cn. stringently conserved (9). Direct electron donors to Fe protein are This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. either reduced flavodoxin or reduced ferredoxin, which, in turn, are 1073/pnas.1620058114/-/DCSupplemental. E2460–E2465 | PNAS | Published online February 13, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1620058114 Downloaded by guest on September 29, 2021 (Zm; ZmFDI and ZmFDIII), Oryza sativa (Os; OsFD1 and PNAS PLUS OsFD4), and Triticum aestivum (Ta; TaFD) were selected for fur- ther study. These ferredoxin-encoding genes were codon-optimized for E. coli (Dataset S1), and expressed from the inducible PLteto-1 promoter (Fig. 2A;detailsareprovidedinSI Materials and Methods). The fdxH gene from As was also introduced as a control to verify effectiveness of the inducible system. To assay electron transport by plant ferredoxins, the fla- vodoxin 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 SEE COMMENTARY 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, in- dicating that reducing power is provided by the pyruvate oxido- reductase 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 Fig. 1. (Upper) Modular arrangement of genes required for MoFe and the minimal FeFe nitrogenase (28) may prefer ferredoxin, rather than minimal FeFe nitrogenase systems. Letters within the arrows represent the flavodoxin, as an electron donor. All of the chloroplast ferredoxins corresponding nif genes or the anfHDGK structural genes encoding FeFe ni- could restore ∼100% activity for the FeFe nitrogenase system, trogenase. (Lower) Schematic pathways for electron donation to nitrogenase with the exception of the NifJ–AtFD2 hybrid module, which and electron transfer within nitrogenase. Structures of representative proteins ∼ B – are shown. PFOR (NifJ), pyruvate–ferredoxin (flavodoxin) oxidoreductase showed 76% activity (Fig. 2 ). In contrast, only the NifJ [Protein Data Bank (PDB) ID code 1B0P]; Rnf complex, NADH–ferredoxin oxi- CrPETF and NifJ–TaFD
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