Using synthetic biology to increase nitrogenase activity

Background Nitrogen fixation has been established in protokaryotic model Escherichia coli by transferring a minimal nif gene cluster composed of 9 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) from Paenibacillus sp. WLY78. However, the nitrogenase activity in the recombinant E. coli 78-7 is only 10 % of that observed in wild-type Paenibacillus. Thus, it is necessary to increase nitrogenase activity through synthetic biology. Results In order to increase nitrogenase activity in heterologous host, a total of 28 selected genes from Paenibacillus sp. WLY78 and Klebsiella oxytoca were placed under the control of Paenibacillus nif promoter in two different vectors and then they are separately or combinationally transferred to the recombinant E. coli 78-7. Our results demonstrate that Paenibacillus suf operon (Fe–S cluster assembly) and the potential electron transport genes pfoAB, fldA and fer can increase nitrogenase activity. Also, K. oxytocanifSU (Fe–S cluster assembly) and nifFJ (electron transport specific for nitrogenase) can increase nitrogenase activity. Especially, the combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytocanifSU recovers 50.1 % of wild-type (Paenibacillus) activity. However, K. oxytocanifWZM and nifQ can not increase activity. Conclusion The combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytocanifSU recovers 50.1 % of wild-type (Paenibacillus) activity in the recombinant E. coli 78-7. Our results will provide valuable insights for the enhancement of nitrogenase activity in heterogeneous host and will provide guidance for engineering cereal plants with minimal nif genes. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0442-6) contains supplementary material, which is available to authorized users.


Background
Most biological nitrogen fixation is catalyzed by the molybdenum nitrogenase enzyme. The molybdenum nitrogenase is composed of two proteins, MoFe protein (NifDK) and Fe protein (NifH). The MoFe protein is an α 2 β 2 heterotetramer that contains the iron-molybdenum cofactors (FeMo-co) and P clusters. The FeMo-co is a [Mo-7Fe-9S-C-homocitrate] cluster which serves as the active site of nitrogen binding and reduction. The P-cluster is a [8Fe-7S] cluster which shuttles electrons to the FeMo-co. The Fe protein is a γ 2 homodimer bridged by an intersubunit [4Fe-4S] cluster that serves as the obligate electron donor to the MoFe protein [1][2][3][4][5].
Although the biochemical properties and structure of molybdenum nitrogenases are remarkably similar when purified from diverse bacteria and archaea, the organization and numbers of nif genes required for the synthesis and assembly of the enzyme varies greatly among these nitrogen-fixing species [6][7][8]. For example, in K. oxytoca (previously called as K. pneumoniae), 20 nif genes, nifJHDKTYENXUSVWZMFLABQ, organized in 7 transcriptional units are co-located within a 24 kb cluster [4], while Paenibacillus sp. WLY78 possesses a minimal and compact nif gene cluster consisting of 9 genes (nif-BnifHnifDnifKnifEnifNnifXhesAnifV) (Fig. 1) [9,10]. This variability in nif genes content is undoubtedly determined by the environmental lifestyle of each diazotroph on one hand, the minimal nif gene sets are probably complemented by housekeeping counterparts located elsewhere in the genome on the other hand.
Genetic and biochemical studies on the two model diazotrophs K. oxytoca and A. vinelandii revealed that 16 nif genes (nif H,D,K,Y,T,E,N,X,U,S,V,Z,W,M,B,Q) products are probably essential for efficient biosynthesis of nitrogenase [11]. It has been demonstrated that nifH, nifD and nifK genes encodes the structural subunits, the nifE, nifN, nifX, nifB, nifQ, nifV, nifY and nifH contribute to the synthesis and insertion of FeMo-co into nitrogenase, nifU, nifS and nifZ play an important role in synthesis of metalloclusters and nifM is required for proper folder of nitrogenase Fe protein [1][2][3][4][5]. However, mutations in some of these genes (notably nifY, nifT, nifX, nifU, nifS, nifV, nifW, nifM and nifQ) do not completely eliminate nitrogenase activity, and there is evidence that homologues elsewhere on the genome may at least partially substitute for their function [11].
Nitrogen fixation plays an important role in agriculture, and there has been a goal to engineer nitrogen fixation into cereals crops to reduce the use of chemically derived fertilizer. The complex nature of the FeMo-co assembly pathway and the large number of genes required for nitrogenase biosynthesis and maintenance of its activity represent a daunting engineering task, even in the age of systems biology. So far, the nif gene cluster from K. oxytoca and Paenibacillus sp. WLY78 has been successfully transferred into the prokaryotic model E. coli [9,[12][13][14][15]. Initially, the recombinant E. coli carrying a refactored nif cluster composed of a series of synthetic operons containing 16 nif genes of K. oxytoca, resulted in reduced activity (about 10 %) compared with the native system [13]. Excitingly, 57 % of wild-type activity has been recovered through modifying the synthetic nif genes cluster [14].
Paenibacillus sp. WLY78 possesses a minimal and compact nif gene cluster consisting of 9 genes (nifB nifH nifD nifK nifE nifN nifX hesA nifV) [9]. The 9 genes are organized as an operon and possess a σ 70 -dependent promoter located in front of nifB gene. Recently, our lab has transferred the nif gene operon under the control of its own native σ 70 -dependent promoter to E. coli JM109 [9]. The recombinant E. coli 78-7 synthesized catalytically active nitrogenase [8]. However, the specific activity of the enzyme expressed in E. coli was approximately 10 % of that observed in Paenibacillus. The low activity will limit the potential use of the Paenibacillus nif cluster in engineering nitrogen fixation into non-N 2 -fixing organisms. Thus, it is necessary to increase nitrogenase activity through synthetic biology.
In this study, two cloning and expression vectors with Paenibacillus nif promoter and ribosome binding site are constructed for transferring foreign genes to the recombinant E. coli 78-7 which carrying the Paenibacillus nif gene operon. A total of 28 selected genes from Paenibacillus and K. oxytoca were placed under the control of Paenibacillus nif promoter in these vectors and then are transferred to E. coli 78-7. Our results demonstrate that Fe-S cluster assembly system and electron transport system from Paenibacillus or K. oxytoca can increase E. coli nitrogenase activity mediated by the minimal nif gene cluster composed of 9 genes (nifBHDKENXhesAnifV). But K. oxytoca nifWZM and nifQ which are required in synthesis and maturation of nitrogenase in K. oxytoca can not increase any activity. Here is the first time to demonstrate that the potential electron transport genes (pfoAB, fer and fldA) are involved in nitrogen fixation of Paenibacillus. Also, it is the first time to demonstrate that Paenibacillus suf and K. oxytoca nifFJ and nifSU can significantly increase nitrogenase activity in E. coli mediated by the Paenibacillus nif gene operon (nifBHDKENXhe-sAnifV). Our results will provide valuable information for the incoming hot research that engineer nitrogen fixation pathway into cereal crops.

Results
Design of combinatorial assembly of the nif and nif-related genes E. coli 78-7 is a recombinant strain carrying a Paenibacillus nif gene operon (nifBHDKENXhesAnifV) in vector pHY300PLK [9,16] (Fig. 2a). As described in methods, two vectors carrying Paenibacillus nif promoter, ribosome-binding site and the multiple cloning site (MCS) are constructed (Fig. 2b, c) for expressing other nif or nif-related genes in E. coli 78-7. The two vectors can coexist in E. coli cells with plasmid pHY300PLK. Then the two vectors carrying foreign genes from Paenibacillus sp. WLY78 or K. oxytoca were separately or combinationally transferred to E. coli 78-7 (Additional file 1: Table S1, Additional file 2: Table S2).

Paenibacillus Suf system can increase nitrogenase activity of the recombinant E. coli 78-7
Nitrogenase is a complex [Fe-S] enzyme. Many diazotrophs, such as K. oxytoca and A. vinelandii, contain nifU and nifS whose products were involved in the assembly of [Fe-S] clusters of nitrogenase [2][3][4][5]. NifU and NifS separately provide the Fe and S required for nitrogenase maturation. The genome of Paenibacillus sp. WLY78 does not have nifSU, but contains iron-sulfur cluster assembly systems: a complete suf (sufCBSUD) operon and a partial isc system (iscSR). Similarly, there are no nifS and nifU in E. coli, but E. coli has two ironsulfur cluster assembly systems: the sufABCDSE operon and the isc system composed of iscR, iscS, iscU, iscA, hscB, hscA, fdx, and orf3 [17]. The nif gene operon from Paenibacillus sp. WLY78 could enable E. coli to fix nitrogen, suggesting that the assembly of Fe-S clusters for the nitrogenase was provided by E. coli iron-sulfur cluster assembly systems.
In this study, the suf (sufCBSUD) operon and iscSR system from Paenibacillus sp. WLY78 are placed under the control of Paenibacillus nif promoter, respectively, and then are separately transferred into the recombinant E. coli 78-7. As shown in Fig. 3, the suf (sufCBSUD) operon can increase nitrogenase activity of E. coli 78-7 from 10 to 20 %, while iscSR system cannot increase any activity. The data suggest that the suf (sufCBSUD) operon plays an important role in Fe-S cluster assembly in nitrogenase synthesis of Paenibacillus.

Klebsiella oxytoca nifSU can increase nitrogenase activity of the recombinant E. coli 78-7
As described in methods, K. oxytoca nifSU gene cluster was placed under the control of Paenibacillus nif promoter and then was transferred to E. coli 78-7. As shown in Fig. 4, K. oxytoca nifSU can increase activity of E. coli 78-7 from 10 to 19.5 %. Our data that K. oxytoca nifSU or Paenibacillus sufCBSUD can increase nitrogenase activity of E. coli 78-7 is consistent with the fact that nitrogenase is a complex [Fe-S] enzyme which contains 38 Fe atoms, 40 S atoms and 2 Mo atoms.

78-7
Nitrogen fixation is carried out by the enzyme nitrogenase, which transfers electrons originating from low-potential electron carriers, such as flavodoxin or ferredoxin molecules, to molecular N 2 [18]. In K. oxytoca, the physiological electron flow to nitrogenase involves specifically the products of the nifF and nifJ genes [19]. The nifF gene product, a flavodoxin, mediates electron transfer from the nifJ gene product, a pyruvate: flavodoxin oxidoreductase, to the Fe protein of nitrogenase [20][21][22][23].
Unlike K. oxytoca nif gene cluster, Paenibacillus nif gene cluster dose not have nifF and nifJ. Genome sequence analysis revealed that there are several genes encoding ferredoxin, flavodoxin and flavodoxin oxidoreductase in the genome of Paenibacillus sp. WLY78. For example, fer and COG3411 encode ferredoxin, fldA and fldB encode flavodoxin, and fpr encodes ferredoxin-NADP reductase, nfrA encodes NAD(P)H-flavin oxidoreductase, and pfoAB separately encode pyruvate: ferredoxin oxidoreductase gamma subunit and alpha subunit. Of these genes, fldA and fldB shows 30 % identity with K. oxytoca nifF, and pfoAB exhibit 33 % identity with K. oxytoca nifJ, but other genes do not show identity with K. oxytoca nifF or nifJ.
Furthermore, the fer, fldA, fldB and COG3411, the orthlogs of K. oxytoca nifF, were separately transferred  Each experiment was repeated at least three times, and the error bars represent standard error into the recombinant E. coli 78-7. As shown in Fig. 5, each of fer and fldA can increase nitrogenase of E. coli 78-7 from 10 to 20.1 %, while fldB and COG3411 cannot increase any activity. The data suggest that fer (ferredoxin) and fldA (flavodoxin) might be an electron transporter of nitrogenase. Our data are consistent with the previous report that either flavodoxins or ferredoxins are the direct electron donor to nitrogenase in diazotrophic bacteria [21].
Furthermore, nfrA, fpr and pfoAB, the orthlogs of K. oxytoca nifJ, were separately transferred into the recombinant E. coli 78-7. As shown in Fig. 5, pfoAB increase nitrogenase activity from 10 to 15 %, while nfrA and fpr do not increase any activity. The data suggest that pfoAB play a role in nitrogen fixation. Notably, the nitrogenase activity of E. coli 78-7 is increased to 35.1 and 40.1 %, respectively, when pfoAB is combined with Paenibacillus fer gene (Paenibacillus-ferproAB) or Paenibacillus fldA gene (Paenibacillus-fldAproAB). We deduce that in Paenibacillus, pfoAB (pyruvate: ferredoxin oxidoreductase) might be involved in the pyruvate breakdown to yield electrons, and then fldA and fer mediate electron to nitrogenase.

Klebsiella oxytoca nifF and nifJ can increase nitrogenase activity of the recombinant E. coli 78-7
As shown in Fig. 6, K. oxytoca nifF and nifJ, whose products are electron transporters, can increase nitrogenase of E. coli 78-7 from 10 to 20.4 and 12,1 %, respectively. When nifF and nifJ were carried in two different vectors and co-transferred into E. coli 78-7, the activity was increased from 10 to 32 %. However, nitrogenase activity could not be increased when nifF and nifJ were assembled as an operon. Our results are consistent with the report that coordinated and balanced expression of nifF and nifJ genes is important for nitrogenase activity in E. coli carrying K. oxytoca nif clusters [15].

Combination of Fe-S cluster synthesis system and electron transporters can significantly increase nitrogenase activity
As described above, the (potential) electron transporters and iron-sulfur cluster assembly systems from Paenibacillus sp. WLY78 or K. oxytoca can increase activity of E. coli 78-7. Here, combination of the (potential) electron transporters and iron-sulfur cluster assembly system was transferred to E. coli 78-7. Considering that K. oxytoca nifSU genes are much shorter and easier to operate

Klebsiella oxytoca nifWZM and nifQ can not increase nitrogenase activity
It was reported that the nifW and nifZ genes seem to be involved in MoFe protein maturation, while nifM is required for proper folding of nitrogenase Fe protein [1,3]. nifM mutants of K.oxytoca and A. vinelandii were unable to synthesise action Fe protein [24][25][26].Unlike in K. oxytoca, Paenibacillus has not the nifWZM genes. And E. coli has also not the nifWZM genes. In this study, the K. oxytoca nifWZM genes were transferred to E. coli 78-7, but the nitrogenase activity was not enhanced by these genes. The data suggest that the requirement of nifWZM genes on maturation of nitrogenase vary greatly among diazotrophs.
NifQ has been implicated in the processing of molybdenum specifically for the biosynthesis of FeMo-co [1,2]. Unlike in K. oxytoca, Paenibacillus dose not have nifQ gene. In this study, the K. oxytoca nifQ gene was transferred to E. coli 78-7, but the nitrogenase activity was not enhanced by nifQ gene. The result indicates that K. oxytoca nifQ is not involved in the processing of molybdenum specifically for the biosynthesis of FeMo-co of nitrogenase encoded by Paenibacillus nif genes (Fig. 8).

Discussion
Our recent studies have revealed that the genome of Paenibacillus sp. WLY78 contains a minimal nif gene cluster composed of nine genes nifBHDKENXhesAnifV and the nif operon under the control of its own nif promoter enabled E. coli to synthesize the active nitrogenase [9]. However, the specific activity of the enzyme expressed in E. coli was approximately 10 % of that observed in Paenibacillus. In this study, synthetic biology was used to determine whether 28 selected genes from Paenibacillus sp. WLY78 and K. oxytoca can increase nitrogenase activity of the recombinant E. coli 78-7.
Compared with K. oxytoca nif clusters, one of the notable absences in the minimal Paenibacillus nif gene cluster is the two genes nifS and nifU, which provide the nitrogen fixation-specific iron-sulfur cluster assembly. The genome of Paenibacillus sp. WLY78 does not have nifSU, but contains iron-sulfur cluster assembly systems: a complete suf (sufCBSUD) operon and a partial isc system (iscSR). In this study, we demonstrate that Paenibacillus suf (sufCBSUD) operon can increase the nitrogenase activity of the recombinant E. coli 78-7, and K. oxytoca nifSU also can increase activity. The results reveal that iron-sulfur cluster assembly system specific for Fe-S cluster of nitrogenase is very important to nitrogen fixation. The results also imply that although E. coli ironsulfur cluster assembly system can support the synthesis of active nitrogenase, it cannot fully support the requirement for synthesis of Fe-S cluster.
It was reported that pyruvate is a major source of electrons in diazotrophic Clostridium pasteurianum and Bacillus polymyxa (now called as Paenibacillus polymyxa) [22]. In K. oxytoca, the pyruvate oxidoreductase (nifJ gene product) was responsible for the pyruvate breakdown to yield electrons, and then the flavodoxin [pBluescriptII SK (+) and pCAMBIA1301] was used as a control. Each experiment was repeated at least three times, and the error bars represent standard error (the nifF gene product) mediates electron transfer to the Fe protein of nitrogenase [23].The Paenibacillus sp. WLY78 nif gene operon does not contain homologs of nifF (encoding a flavodoxin) and nifJ (pyruvate: flavodoxin oxidoreductase) which provide the electron transport chain to nitrogenase in some diazotrophs [22,23]. In this study, we search and find that the fer (ferredoxin), COG3411 (ferredoxin), fldA (flavodoxin), fldB (flavodoxin), fpr (ferredoxin-NADP reductase), nfrA (NAD(P)H-flavin oxidoreductase) and pfoAB (pyruvate: ferredoxin oxidoreductase) are scared on Paenibacillus genomic regions outside of nif genes cluster. When each of these genes is separately transferred to E. coli 78-7, only fer, fldA and pfoAB can increase activity. Combinational assembly of fer or fldA with pfoAB can significantly increase activity. We deduce that pfoAB gene product (pyruvate: ferredoxin oxidoreductase) might be involved in the pyruvate breakdown to yield electrons, and then fldA and fer mediate electron to nitrogenase.
Here is the first time to reveal that pfoAB, fer and fldA genes are involved in nitrogen fixation mediated by Paenibacillus nif genes. Notably, PfoAB shows 33 % identity with K. oxytoca NifJ. But the K. oxytoca nifJ gene product is a single subunit, while Paenibacillus pfoAB gene products are two subunits. Here, we show that both fldA and fer can enhance nitrogenase activity, suggesting that the both genes can transfer electron to Fe protein. fldA also exists in E. coli and K. oxytoca [27]. Whether fldA is involved in transferring electron to Fe protein of nitrogenase in K. oxytoca is not known. In E. coli, FldA and Fpr (the NADPH-dependent flavin adenine dinucleotide (FAD) containing flavodoxin/ferredoxin reductase) are required for the activation of key enzymes in the synthesis of methionine, biotin, pyruvate and deoxyribonucleotides [28][29][30]. Remarkably, the Fpr-FldA redox system can effectively deliver electrons to non-physiological partners, which include a variety of P450 enzymes [31]. Thus, we deduce that the Fpr-FldA redox system might be responsible for electron transport to nitrogenase in E. coli.
Also, we demonstrate that each of K. oxytoca nifJ and nifF genes can increase nitrogenase activity of E. coli 78-7. The higher activity is obtained when K. oxytoca nifJ and nifF genes were carried in different vectors. However, nitrogenase could not be increased when nifF and nifJ were assembled as an operon. Our results are consistent with the report that coordinated and balanced expression of nifF and nifJ genes is important for nitrogenase activity in E. coli carrying K. oxytoca nif clusters.
Furthermore, in order to increase nitrogenase activity of E. coli 78-7, we design to assemble electron transport genes from Paenibacillus or K. oxytoca with Fe-S cluster synthesis genes from Paenibacillus or K. oxytoca.
Considering K. oxytoca nifSU are much shorter and easier to operate in gene cloning than Paenibacillus suf operon, K. oxytoca nifSU are used in the combinational assembly with electron transport genes. The combinational assembly of Paenibacillus fer-pfoAB with K. oxytoca nifSU, Paenibacillus fldA-pfoAB with K. oxytoca nifSU, K. oxytoca nifF and nifJ with nifSU was constructed. Our results demonstrated that these combinational assemblies can significantly increase activity. Especially, Paenibacillus fldA-pfoAB with K. oxytoca nifSU can recover 50 % activity of wild-type Paenibacillus. Our results provide valuable information for engineering nitrogen fixation pathway into cereal crops.
The nifW and nifZ genes seem to be involved in MoFe protein maturation [1,3], while nifM is required for proper folding of nitrogenase Fe protein in K. oxytoca [24][25][26]. The nifWZM genes are not only absent in the Paenibacillus nif cluster, but also in the nif clusters of the Gram-positive Clostridium, Heliobacterium chlorum and archaeal Methanococcus maripaludis [32]. Also, nifM is absent in Rhizobia, such as Azorhizobium caulinodans, Sinorhizobium meliloti and Rhizobium leguminosarum [7]. Our current studies demonstrate that K. oxytoca nifWZM can not increase nitrogenase activity of E. coli 78-7.These data support that the nifWZM genes are not required for nitrogen fixation in Paenibacillus sp. WLY78. Whether the functions of the nifWZM genes are replaced by other components scared in the genome of Paenibacillus sp. WLY78 and E. coli is not known.
It was reported that nifM encodes a cis-trans peptidyl prolyl isomerase and are involved in proper folding of nitrogenase Fe protein in Azotobacter vinelandii [26]. When the conserved Pro258 located in the C-terminal region of Fe protein (NifH) of A. vinelandii, which wraps around the other subunit in the NifH dimer, is replaced by serine, the correct folding of Fe protein (NifH) can acquire NifM independence [26,33]. We compare the Paenibacillus NifH sequence with other NifH sequences and find that Paenibacillus contains the conserved proline residues identified in other NifH sequences that are considered to be potential substrates for NifM (Additional file 3: Figure S1). It is possible that other amino acid substitutions in NifH may enable assembly of Fe protein in the absence of NifM.
It has been demonstrated that nifQ is required for nitrogen fixation in K. oxytoca and A. vinelandii. Recent results show that NifQ is an iron-sulfur protein with a redox-responsive [Fe-S] cluster and NifQ is also a molybdoprotein that serves as a direct molybdenum donor for FeMo-co synthesis, replacing molybdate in the in vitro FeMo-co synthesis assay [34]. Electron paramagnetic resonance (EPR) spectroscopic studies indicated that NifQ carries a [Mo-Fe3-S4] cluster, and that the presence of this metal cluster in NifQ correlates with its ability to support in vitro FeMo-co synthesis [1]. However, there is no nifQ in diazotrophic Paenibacillus, Clostridium, cyanobacteria and Frankia [35,36]. This study demonstrates that K. oxytoca nifQ did not enhance the activity of E. coli 78-7. Interestingly, there is a hesA gene located within the nif clusters of diazotrophic Paenibacillus, cyanobacteria and Frankia [35,36]. Our deletion analysis demonstrates that hesA is important for nitrogenase activity, but the function of hesA in nitrogen fixation has not so far been determined. HesA belongs to the ThiF-MoeB-HesA family which engages in an ATP-dependent process that activates the C-terminus of partner ubiquitin-like proteins by forming an acyladenylate complex that facilitates sulfur transfer [37,38]. It is to speculate that HesA may perform a role in metallocluster biosynthesis. These data suggest that synthesis and maturation of nitrogenase exhibit some different features between nif gene clusters of Gram-negative K. oxytoca/A. vinelandii and Gram-positive Paenibacillus.

Conclusion
A total of 28 selected genes from Paenibacillus sp. WLY78 and K. oxytoca are separately or combinationally transferred into the recombinant E. coli 78-7. Of these 28 genes, 8 genes (pfoAB, fldA, fldB, fer, fpr, nfrA and COG3411) encoding the potential electron transport and 2 gene clusters (suf and isc) encoding Fe-S cluster synthesis are from Paenibacillus sp. WLY78, and 8 genes (nifF, nifJ, nifSU, nifWZM and nifQ) specific for electron transport, Fe-S cluster synthesis and maturation of nitrogenase are from K. oxytoca. Our results demonstrate that Paenibacillus suf operon and the potential electron transporter genes (pfoAB, fldA and fer) can increase nitrogenase activity. Also, K. oxytoca nifSU and nifFJ can increase nitrogenase activity. Especially, combined assembly of the potential electron transporter genes (pfoABfldA) with K. oxytoca nifSU recovers 50.1 % of wild-type activity. Also, we demonstrate that nifWZM and nifQ can not increase activity, suggest ing that the requirement of nifWZM and nifQ genes on maturation of nitrogenase vary greatly among diazotrophs. This study will provide valuable insights for the enhancement of nitrogenase activity in heterogeneous host and will provide guidance for engineering cereal plants with minimal nif genes.

Strains and medium
Paenibacillus sp. WLY78, a nitrogen-fixer, was isolated by our lab [9]. The recombinant E. coli 78-7 which carries an 11 kb nif genes cluster from Paenibacillus sp. WLY78 was constructed by our lab [9]. Paenibacillus sp. WLY78 and E. coli strains were routinely grown in LB or LD medium (per liter contains: 2.5 g NaCl, 5 g yeast and 10 g tryptone) at 30℃ with shaking. When appropriate, antibiotics were added in the following concentrations: 50 m g/ml kanamycine, 100 m g/ml ampiciline, and 12.5 m g/ml tetracycline for maintenance of plasmids. Nitrogen-free and nitrogen-deficient media were used for assay of nitrogenase activity. Nitrogen-free medium contained (per liter) 10 O, 10 μg p-aminobenzoic acid, 5 μg biotin and 4 g glucose as carbon source. Nitrogendeficient medium contained 2 mM glutamate as nitrogen source in nitrogen-free medium [9].

Nitrogenase activity assays
For nitrogenase activity assays, Paenibacillus sp.WLY78 and the recombinant E. coli strains were grown in 5 ml of LD media (supplemented with antibiotics) in 50 ml flasks shaken at 250 rpm for 16 h at 30 °C. The cultures were collected by centrifugation, washed three times with sterilized water and then resuspended in nitrogen-deficient medium containing 2 mM glutamate as nitrogen source (supplemented with antibiotics for the engineered E. coli strains when necessary) to a final OD600 of 0.2-0.4. Then, 1 ml of the culture was transferred to a 25-ml test tube and the test tube was sealed with robber stopper. The headspace in the tube was then evacuated and replaced with argon gas [14]. After incubating the cultures for 6-8 h at 30℃ with shaking at 250 rpm, C 2 H 2 (10 % of the headspace volume) was injected into the test tubes. After incubating the cultures for a further 3 h, 100 ml of culture headspace was withdrawn through the rubber stopper with a gas tight syringe and manually injected into a HP6890 gas chromatograph to quantify ethylene production. All treatments were in three replicates and all the experiments were repeated three or more times.

Construction of cloning and expression vectors
Since E. coli 78-7 carries a Paenibacillus nif gene operon in vector pHY300PLK which is a shuttle vector with two replication origins:one is p15A which can be reproduced in E. coli and the other is a pAMα1 replicon from a plasmid pAMα1 of Streptococcus faecalis which can be reproduced in Gram-positive Bacillus [16]. The p15A replicon allows itself to coexist in E. coli cells with plasmids of the ColE1 compatibility group (e.g., pBR322, pUC19, pBluescript II SK (+)). Thus, two cloning and expression vectors carrying a Paenibacillus nif promoter and a ribosome binding site are here constructed in order to express foreign genes in the recombinant E. coli 78-7. The first vector (here called pBC) contains the backbone derived from pBluescript II SK (+), including the E. coli origin ColE1, ampiciline resistance marker amp and the multiple cloning sites (MCS). A 307 bp Paenibacillus nif promoter region (carrying XhoI and HindIII restriction sites at both ends) from the genomic DNA of Paenibacillus sp. WLY78 and a 1.2 kb chloramphenicol resistance gene fragment (carrying KpnI restriction sites at both ends) from the plasmid pPR9TT were PCR amplified and then ligated to the ampicillin-resistant plasmid pBluescript II SK (+), resulting vector pBC. The second vector (here named as pCK) contains the E. coli origin ColE1, kanamycine resistance marker kan and the multiple cloning site (MCS) from plasmid pCAMBIA1301 and a 307 bp nif promoter region (carrying XhoI and HindIII restriction sites at both ends) from the genomic DNA of Paenibacillus sp. WLY78.