KR101132839B1 - Host cells transformed with hydrogenase or nitrogenase of Rhodobacter sphaeroide and method for preparing hydrogen using the same - Google Patents

Abstract

The present invention (1) transformed into a host cell of Escherichia sp. Transformed with a hydrogenase gene of Rhodobacter sphaeroide and a nitrogenase gene of Rhodobacter spheroid Preparing a transformed Escherichia spp. Host cell; And (2) co-culturing the host cell to produce hydrogen.

According to the present invention, by co-culturing a host cell transformed with a hydrogenase gene and a host cell transformed with a nitrogenase gene, the host cell transformed with a hydrogenase gene is significantly improved. Hydrogen can be produced in yield.

Rhodobacter spheroid, hupS, hupL, hydrogenase, nifK. nifD, nifH, nitrogenase, host cell of the genus Escherichia

Description

Host cells transformed with hydrogenase or nitrogenase of Rhodobacter sphaeroide and method for preparing hydrogen using the same} of Rhodobacter spheroid hydrogenase or nitrogenase

The present invention comprises the steps of (1) preparing Escherichia spp. Host cells transformed with the Rhobacter spheroid hydrogenase gene and Escherichia spp. Host cells transformed with the Rhodobacter spheroid nitrogenase gene; And (2) co-culturing the host cell to produce hydrogen.

Today we are facing two crises: energy shortages and environmental pollution. The world's energy demand is largely dependent on fossil fuels, which are causing a number of political, economic and social problems due to the ubiquity and exhaustion of fossil fuels. In addition, the emissions emitted by the use of fossil fuels cause global warming and environmental pollution problems. In this reality, many countries are engaged in research to develop alternative energy to solve this crisis. As one solution to this problem, interest in hydrogen energy is increasing. Hydrogen energy is a secondary energy that can be obtained by converting primary energy such as sunlight, solar heat, coal, petroleum, etc., and has a very high energy content per unit mass and generates water mainly as a combustion byproduct. It is more environmentally friendly clean energy. In addition, the need for hydrogen energy is further increased due to the development of fuel cells, and is attracting attention not only for military / space development but also for civil / industrial use.

At present, more than 96% of hydrogen is produced from fossil fuels, but this is not the ultimate method due to the limited reserves and the emission of environmental pollutants. Therefore, a method for producing hydrogen using more clean technologies such as solar, hydro, wind and microorganisms that are more environmentally friendly is being studied. In particular, there is an increasing interest in a biological hydrogen production method for producing hydrogen from water or organic waste resources using microorganisms. Biological hydrogen production methods are less energy intensive because they operate at room temperature and atmospheric pressure than chemical engineering methods, and theoretically because hydrogen is produced from renewable fuels such as water, biomass, and organic waste resources. Infinite hydrogen production is possible. In addition, biological hydrogen production method is a technology that has two advantages, combined with environmental treatment technologies such as organic waste resource treatment, carbon dioxide reduction, reduction of environmental pollution and alternative energy production.

Research into hydrogen production by microorganisms began in the 1800s, but it was limited to basic research, and research into hydrogen energy began in the 1970s. In other words, in the age of energy crisis, he began to study hydrogen energy as an alternative. Biological hydrogen production is mainly a method using a microorganism, and can be classified into two types according to the characteristics of the microorganism. One is hydrogen production by photosynthesis microorganisms using light, and the other is anaerobic fermentation by microorganisms that do not use light. Microorganisms involved in hydrogen production method using photosynthesis can be classified into green algae, cyanobacteria and photosynthetic bacteria, and microorganisms involved in anaerobic fermentation are fermentative bacteria. have. These microorganisms produce hydrogen using their own enzymes, and enzymes involved in hydrogen production, such as hydrogenase, nitrogenase, and formate hydrogen-lyase (FHL) are known.

However, traditional hydrogen producing microorganisms exhibit relatively low energy conversion efficiency and low hydrogen production rate. In addition, due to various inhibitors, the instability inherent in production from these individuals over time has been questioned.

In order to solve the above problems, the present inventors have developed E. coli transformed with the hydrogenase gene of Rhodobacter spheroid, and have disclosed that E. coli produces hydrogen at a significantly higher yield than Rhodobacter spheroid. .

The present inventors co-cultured the E. coli transformed with the Rhodobacter spheroid hydrogenase gene and the E. coli transformed with the Rhodobacter spheroid nitrogenase gene while studying to further increase the hydrogen production yield. It was confirmed that hydrogen can be produced in a significantly improved yield than when culturing E. coli transformed alone, the present invention was completed.

An object of the present invention is to (1) transform into a host cell of Escherichia genus transformed with a hydrogenase gene of Rhodobacter sphaeroide and a nitrogenase gene of Rhodobacter spheroid Preparing an isolated Escherichia spp host cell; And (2) co-culturing the host cell to produce hydrogen.

In the present invention, 'Rhodobacter sphaeroide' is a representative hydrogen-producing photosynthetic bacterium, which is capable of producing hydrogen because it expresses a hydrogenase and a nitrogenase protein. to be. The hydrogenase protein is encoded by the hupS and hupL genes, and the nitrogenase protein is encoded by the nifK, nifD and nifH genes.

In the present invention, the 'hupS gene' can be obtained by performing PCR using genomic DNA of Rhodobacter spheroid as a template and primers shown in SEQ ID NO: 3 and primers shown in SEQ ID NO: 4. . The hupS gene of the present invention is preferably composed of the nucleotide sequence shown in SEQ ID NO: 1.

In the present invention, the 'hupL gene' may be obtained by performing PCR using genomic DNA of Rhodobacter spheroid as a template and primers shown in SEQ ID NO: 5 and primers shown in SEQ ID NO: 6. . The hupL gene of the present invention is preferably composed of the nucleotide sequence shown in SEQ ID NO: 2.

In the present invention, the 'nifgen gene (nitrogenase molybdenum-iron protein beta chain) gene' is an embodiment, using the genomic DNA of Rhodobacter spheroid as a template, and using the primer shown in SEQ ID NO: 9 and the primer shown in SEQ ID NO: 10. Can be obtained by performing PCR. The nifK gene of the present invention is preferably composed of the nucleotide sequence shown in SEQ ID NO: 7.

In the present invention, the 'nifD gene (nitrogenase iron-molybdenum protein, alpha chain) and nifH gene (nitrogenase reductase)' is an embodiment, and the primer and sequence shown in SEQ ID NO: 11 are based on genomic DNA of Rhodobacter spheroid. Two genes can be obtained simultaneously by performing PCR using the primers shown in No. 12. The nifDH gene of the present invention preferably consists of the nucleotide sequence shown in SEQ ID NO: 8. The nifD gene of the present invention is composed of the first base to the 876th base of the nucleotide sequence shown in SEQ ID NO: 8, the nifH gene is composed of 938 base to the 1482 base of the base sequence shown in SEQ ID NO: 8 desirable.

The Escherichia spp host cell transformed with the hydrogenase gene of Rhodobacter spheroid and the Escherichia spp host cell transformed with the nitrogenase gene of Rhodobacter spheroid according to the present invention each contain a corresponding gene. After constructing a recombinant expression vector, the recombinant expression vector can be prepared by transforming a host cell of the genus Escherichia.

In the present invention, the term 'vector' means a nucleic acid molecule capable of transporting another nucleic acid linked thereto. Vectors can perform spontaneous replication or can be integrated into host DNA. The vector includes a restriction enzyme recognition site for insertion of recombinant DNA and may include one or more selectable markers. The vector may be a nucleic acid in the form of a plasmid, bacteriophage or cosmid.

The recombinant expression vector of the present invention preferably comprises a 'regulatory sequence'. In the present invention, regulatory sequence means a DNA or RNA element capable of controlling gene expression. Examples of regulatory sequences include promoters, enhancers, silencers, Shine-Dalgarno sequences, TATA-boxes, internal ribosomal entry sites, IRES attachment sites, transcription terminators ( transcriptional terminators, polyadenylation sites, and the like.

In the present invention, 'promoter' means a nucleotide sequence of DNA or RNA to which RNA polymerase binds to initiate transcription. Promoters can be inducible or constitutive. Preferably, the promoter is a T7, T3, lac, tac, trc promoter. More preferably, the promoter is a T7 promoter or T3 promoter known to function in bacteria, for example E. coli.

In the recombinant expression vectors of the invention, the hupS, hupL, nifK, nifDH genes must be operably linked to regulatory sequences. Operationally linked refers to regulatory sequences or combinations thereof in combination with a coding sequence that are in a linking relationship such that they can direct the expression of a functional relationship, for example, the coding sequence.

In the present invention, a bacterial expression vector containing a T7 promoter system is preferred, and a pET expression vector is particularly preferred.

In the present invention, the recombinant expression vector is transformed into a host cell of Escherichia. The host cell is preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).

Transformation and transfection as used in the present invention means various techniques for introducing foreign nucleic acids known in the art to the host strain. Transformation of a suitable host cell by the recombinant expression vector of the present invention is accomplished by methods known in the art to which the present invention pertains and is typically dependent on the type of vector and host cell. Such techniques include, but are not limited to, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, liposome mediated transfection, chemoporation or electroporation. Do not.

In the present invention, the Escherichia genus host cells transformed with the hydrogenase gene of Rhodobacter sphaeroide thus prepared and the nitrogenase gene of Rhodobacter spheroid Hydrogen is produced by co-culture of host cells of the genus Kerikia.

The transformed host cell of the present invention is cultured according to a host individual, according to methods known in the art. Usually, host cells typically contain a carbon source in the form of sugar, an organic nitrogen source, typically a yeast extract, or a nitrogen source, in the form of a salt, such as ammonium sulfate, trace elements, such as iron, manganese and magnesium, and vitamins as needed. Cultured in the medium.

In the present invention, it is preferable to culture under anaerobic conditions using the hydrogen production titer medium shown in Table 2 at the time of co-culture. It is also desirable to shake at 140-150 rpm at a temperature of 29-31 ° C., because more hydrogenase can maintain soluble form under these conditions. In addition, during co-culture, host cells transformed with nitrogenase gene are preferably immobilized. This is because the expression of the nitrogenase gene can be maximized while inhibiting the production of the host cell. Nitrogenase produces the largest amount of electrons and hydrogenase receives the electrons and converts them into hydrogen, thus increasing hydrogen production.

According to the present invention, a host cell transformed with a hydrogenase gene of Rhodobacter spheroids and a host cell transformed with a nitrogenase gene of Rhodobacter spheroids is co-cultured to a host transformed with a hydrogenase gene. Hydrogen can be produced at 10 times improved efficiency than when cells are cultured alone.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Construction of a Recombinant Vector Comprising a Rhodobacter Spheroid Hydrogenase Gene

In this example, the Rhodobacter sphaeroides KCTC 1434 strain, an anaerobic photosynthetic strain, was used and cloned using a pET-21a (Novagen, Madison, WI, USA) vector known as a protein expression vector. The DNA sequences of hupS and hupL , which are genes encoding hardenase from the genome data of Rhodobacter spheroid, were obtained from NCBI's genebank (www.ncbi.nlm.nih.gov), which corresponds to SEQ ID NO: 1. DNA fragment 1 (F1) and DNA fragment 2 (F2) corresponding to SEQ ID NO: 2, respectively having SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 Primer was prepared. Next, genomic DNA was extracted from the Rhodobacter spheroid strain, and the hupS gene was subjected to PCR (Polymerase chain reaction) using a primer pair having a base sequence of SEQ ID NO: 3 and SEQ ID NO: 4 as a template. The nucleotide sequence (SEQ ID NO: 1) of fragment 1 (F1) of was amplified. In addition, PCR was performed using primers having the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 to amplify the nucleotide sequence (SEQ ID NO: 2) of fragment 2 (F2) of the hupL gene. After electricity from the agarose gel (agarose gel) electrophoretic PCR amplification products hupS got a DNA fragment of 1.11 kb, hupL has identified the gene with a fragment of 1.79 kb. The genes were all purified after GeneAll PCR DNA purification kit.

Primer Sequences for Hydrogenase Gene Identification Primer sequence Remarks SEQ ID NO: hupS-F 5'- ACC CAT ATG CCC CAG ATC GAA ACC TTC -3 ' NdeI 3 hupS-R 5'- TGT GCT AGC GGC CTC CGT CTT TTC GTC T -3 ' NheI 4 hupL-F 5'- ACA GCT AGC ATG GTC GCG ACA CCG AAC -3 ' NheI 5 hupL-R 5 '-TGT AAG CTT GCG GAC CTT GAC GGT GGT GA -3' HindIII 6

Example 2: Preparation of Recombinant Expression Vector (pEMBTL-HJ2)

In this example, pEMBTL-HJ2 (see FIG. 1), which is a hydrogenase expression vector, was prepared using E. coli-protein expression vector pET-21a (Novagen, Madison, WI, USA) as a base vector. As follows.

The hupS DNA fragments amplified in Example 1 were cut into NdeI and NheI and introduced into the pET-21a vector cut the same restriction site, which was named 'pEMBTL-HJ1' (see FIG. 2). Herein, the hupL gene was cleaved with NheI and HindIII, and then introduced into the pET-21a vector which cut the same restriction site and named it 'pEMBTL-HJ2' (see FIG. 2).

The vector pEMBTL-HJ2 prepared in this Example was transformed into E. coli Escherichia coli BL21 (DE3) by electroporation, and colonies obtained from LB solid plate medium containing ampicillin were transferred to a new medium to obtain a single colony. Got it. The DNA sequencing method accurately identified the DNA sequences of hupS and hupL inserted into pEMBTL-HJ2. The recombinant microorganism containing pEMBTL-HJ2 was named 'MBTL-HJ-001'.

Example 3: Overexpression and Solubilization of Hydrogenase Protein in Recombinant Microorganisms

In this example, LTL liquid medium (10 g / L tryptone, 5 g / L yeast extract and 5) containing 50 μg / mL of ampicillin (MBTL-HJ-001), the recombinant microorganism prepared in Example 2, was used. 5 mL of g / L NaCl) was inoculated and shaken at 37 ° C. and 160 rpm for 12 hours. 100 μl of the obtained culture solution was added to 10 mL of LB liquid medium containing ampicillin, followed by main culture at 160 rpm for 2 hours at 37 ° C., and then IPTG (Isopropyl β-D-1-thiogalactopyranoside), a protein expression inducer, was added to 1 mM. After addition, protein overexpression was induced for 2 hours under the same conditions. After 1mL of each induction reaction culture was collected, the cells were crushed by an ultrasonicator, and the protein was quantified by the Bradford method by dividing into soluble form and insoluble form. SDS-PAGE (SDS-discontinuous polyacrylamide gel electrophoresis) analysis confirmed that the insoluble form was expressed more than the soluble form at the 100 kDa position.

In order to solubilize the protein, SDS-PAGE analysis was performed by extracting the protein from the recombinant E. coli grown by lowering the incubation temperature and stirring speed to 30 ° C and 150 rpm and increasing the incubation time and the protein expression induction reaction time to 5 hours and 8 hours, respectively. As a result, the soluble form increased by more than 40% (see Figure 3).

Example 4 Comparison of Hydrogen Productivity of Recombinant Microorganisms

In this example, the recombinant microorganism MBTL-HJ1 prepared in Example 2 was used a hydrogen production titer (see Table 2 below) and was cultured in a vial to confirm the improvement in hydrogen productivity.

* Adjust the pH to 7 using KOH and sterilize by adding 2 g of casamino acid (casamino acid).

In Example 4, the recombinant microorganism was inoculated in 5 mL of LB liquid medium containing 50 μg / ml of ampicillin and shaken at 37 ° C. for 12 hours. 400 μl of the culture solution obtained above and 40 mL of hydrogen production titer containing Ampicillin 50 μg / mL and 5 mM IPTG (Table 4) were placed in a 50 mL vial and purged with nitrogen for 10 minutes to form anaerobic conditions, followed by 30 ° C. Incubated at 150 rpm for 66 hours (see FIG. 4). Cell growth measurement of the strain was measured at 600 nm using a spectrophotometer (Spectrophotometer). The hydrogen content was collected by 500 μl of the gas in the vial head space with a syringe and analyzed by gas chromatography, and the results are shown in FIG. 5 and Table 3 below.

Comparison of Hydrogen Productivity between Rhodobacter Spheroids and Recombinant Microorganisms (MBTL-HJ-001) 0 6 18 24 66 Rhodobacter spheroids (μl / 500 μl) 0 0.065 0.036 0.042 0.039 MBTL-HJ-001 0 0.74 0.3 1.64 9.84

As shown in Table 3, the recombinant microorganism MBTL-HJ-001 produced hydrogen, and it was confirmed that the yield was significantly increased compared to the control Rhodobacter spheroid KCTC1434.

Example 5: Construction of a Recombinant Vector Comprising a Rhodobacter Spheroid Nitrogenase Gene

In this example, the Rhodobacter sphaeroides KCTC 1434 strain, which is usually an anaerobic photosynthetic strain, was used and cloned using a pET-28b vector known as a protein expression vector. DNA sequences of nifK, nifD, and nifH, which are genes encoding nitrogenase from the genome data of Rhodobacter spheroid, were obtained from NCBI genebank (www.ncbi.nlm.nih.gov), and corresponded to SEQ ID NO: 7. DNA fragment 7 (F7) and SEQ ID NO: 8 (nifD gene is 1 ~ 876bp, nifH gene is 938 ~ 1482bp) DNA fragment 8 (F8) corresponding to each can be specifically amplified SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 were prepared. Next, genomic DNA is extracted from the Rhodobacter spheroid strain, and PCR (Polymerase chain reaction) is performed using a primer pair having the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 10 as a template. The base sequence (SEQ ID NO: 7) of fragment 7 (F7) of the nifK gene was amplified. In addition, PCR was performed using primers having the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 12 to amplify the nucleotide sequence (SEQ ID NO: 8) of fragment 8 (F8) of the nifD and nifH genes. After electricity from the agarose gel (agarose gel) electrophoretic PCR amplification products nifK got a 1.5 kb DNA fragment, and nifD nifH gene was confirmed that a gene with a fragment of 7.8 kb. The genes were all purified after GeneAll PCR DNA purification kit.

Primer Sequences for Nitrogenase Gene Identification Primer sequence Remarks SEQ ID NO: nifK-F 5'- ATA GCT AGC ATG CCG CAG TCG GC -3 ' NheI 9 nifK-R 5'- ACT AAG CTT TCA GCG GGT CAG GT -3 ' HindIII 10 nifDH-F  5'- TTC CAT ATG GGA AAA CTC CGG CA -3 ' NdeI 11 nifDH-R  5'- TAA GCT AGC AAC CTT TTC GGC CG -3 ' NheI 12

Example 6: Preparation of Recombinant Vector (pEMBTL-HJ4)

In this embodiment, pEMBTL-HJ4 (see FIG. 6), which is a vector for nitrogenase expression, was produced using the E. coli-protein expression vector pET-28b as a base vector.

The nifK DNA fragment amplified in Example 5 was cut into NheI and HindIII and introduced into the pET-28b vector which cut the same restriction site, which was named 'pEMBTL-HJ3'. Here, the nifDH gene was cleaved with NdeI and NheI, and the same restriction site was introduced into the cleaved vector, which was named 'pEMBTL-HJ4'.

The vector pEMBTL-HJ4 prepared in this Example was transformed into E. coli Escherichia coli BL21 (DE3) by electroporation, and colonies obtained from LB solid plate medium containing kanamycin were transferred to a new medium to obtain a single colony. Got it. DNA sequencing method accurately identified the DNA sequences of nifK and nifDH inserted into pEMBTL-HJ4, and named the recombinant microorganism containing pEMBTL-HJ4 as 'MBTL-HJ-002'.

Example 7: Hydrogen Production by Co-Cultivation of Two Recombinant Microorganisms

In this embodiment, the co-culture of MBTL-HJ-001, a recombinant microorganism prepared in Example 2, and MBTL-HJ-002, a recombinant microorganism prepared in Example 6, was confirmed to improve hydrogen productivity.

In Example 7, the hydrogen production titer containing 0.5% agar at the bottom of the vial (Table 2) was laid down about 0.5 cm and cooled down, and then cooled and mixed with MBTL-HJ-002 before hardening. Liquid) was added and MBTL-HJ-001 was added and incubated together. At this time, MBTL-HJ-002 was inoculated into 5 mL of LB liquid medium containing kanamycin 25 μg / ml, shaken at 160 rpm for 12 hours at 37 ° C., and then cultured to obtain the culture medium, which was used before the logarithmic growth stage (FIG. 7). Reference). Here, as a control, when bacteria were not added to the lower solid medium, that is, the coli enters the mock vector into the lower solid medium and cultured together. Here, the culture conditions were set in the same manner as in Example 4. As a result of the experiment, when the MBTL-HJ-001 and MBTL-HJ-002 were co-cultured, it was confirmed that the hydrogen production yield was improved by 10 times or more compared with the case of MBTL-HJ-001 alone culture (see FIG. 8).

Figure 1 shows a recombinant expression vector comprising the hydrogenase gene of Rhodobacter spheroids.

Figure 2 shows the process of making pEMBTL-HJ2.

Figure 3 shows the overexpression of MBTL-HJ-001 at 30 ℃ and 37 ℃ as a result of SDS-PAGE analysis.

Figure 4 shows the process of culturing MBTL-HJ-001 in hydrogen production titer medium.

Figure 5 shows the hydrogen production of Rhodobacter spheroids and MBTL-HJ-001 over time.

Figure 6 shows a recombinant expression vector comprising the nitrogenase gene of Rhodobacter spheroids.

7 is a view showing a method of co-culturing MBTL-HJ-001 and MBTL-HJ-002 according to the present invention.

8 is a diagram showing the hydrogen yield obtained over time co-culturing MBTL-HJ-001 and MBTL-HJ-002 according to the present invention.

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Host cells transformed with hydrogenase or nitrogenase of          Rhodobacter sphaeroide and method for preparing hydrogen using          the same <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 1107 <212> DNA <213> Rhodobacter sphaeroides <400> 1 atgccccaga tcgaaacctt ctacgatgtg atgcgccgcc aggggatcac ccggcgcagc 60 ttcatgaaat actgctcgct cacggcggcg gcgctggggc tcggcccctc cttcgtgccg 120 aagatcgcgc acgcgatgga gacgaagccg cgcacaccgg tgatctgggt ccatgggctc 180 gaatgcacct gctgctcgga gagcttcatc cgcgcggccc atccgctggc caaggacgtc 240 gtcctgtcga tgatctcgct cgactatgac gacacgctga tggcggcggc gggccatcag 300 gccgaagccg cgctgatgga cacgatcgag aaatacaagg gcaactacat ccttgccgtc 360 gagggcaacc cgccgctgaa cgaggacggg atgtattgca tcatcggcgg caagcccttc 420 gtcgagcagc tgaagatggc ggccgaacat gccaaggcga tcatcagctg gggggcctgc 480 gcctcctacg gctgcgtgca ggcggccgcc cccaacccca cgcgggccac gcccgtgcac 540 aaggtcatcc tcgacaagcc gatcgtcaag gtgccgggct gcccgcccat cgccgaagtc 600 atgaccggcg tcatcaccta catgctgacc ttcgaccggc tgcccgagct cgaccgtcag 660 ggccgcccgg cgatgttcta cagccagcgc atccacgaca aatgctaccg ccgcccgcat 720 ttcgacgcgg gccagttcgt cgaggcctgg gacgacgact acgccaagaa gggctactgc 780 ctctacaaga tgggctgcaa ggggccgacc acctacaacg cctgctcgac cgtgcgctgg 840 aacgagggcg tgagcttccc gatccagtcc ggccacggct gcatcggctg ctcggaggac 900 ggcttctggg atcagggatc cttctacgac cggctgacca ccatcaagca gttcggcgtc 960 gaggccaatg ccgacacgat cggcctcacg gccgtgggcg cgctcggcgc gggcgtggcg 1020 gcccatatcg cggccaccgc cctcaagagc gcgcagaaga aatcgcaggc ggccaatacc 1080 gcgaagacag acgaaaagac ggaggcc 1107 <210> 2 <211> 1788 <212> DNA <213> Rhodobacter sphaeroides <400> 2 atggtcgcga caccgaacgg tttcaacctg gacaacaccg gccgccgtat cgtggtggac 60 ccggtgaccc gcatcgaggg tcacatgcgc tgcgaagtga acgtggacga tcagggcatc 120 atccgcaatg ccgtctcgac ggggacgatg tggcgcgggc tcgaggtcat cctcaagggc 180 cgcgacccac gcgacgcctg ggccttcacc gagcggatct gcggggtctg caccggcacc 240 catgcgctca cctccgtccg cgccgtcgag gatgcgctgg ggatctcgat ccccgacaat 300 gcgaactcga tccgcaacat gatgcagctg aacctgcaga tccacgacca catcgtccat 360 ttctaccatc tgcacgcgct ggactgggtg aacccggtca atgcgctgcg cgccgatccg 420 aaggccacgt ccgagctgca gcagaaggtc tcgccttcgc acccgctctc gtcgccgggc 480 tatttccgcg acgtgcagaa ccggctgaag aagttcgtgg aatcggggca gctgggcctg 540 ttcaagaacg gctactggga caatccggcc tatctgctgc cgcccgaggc ggacctgatg 600 gccaccaccc actatctcga ggcgctcgac ctgcagaagg agatcgtgaa ggtccacacg 660 atcttcggcg gcaagaaccc gcatccgaac tggctggtgg ggggcgtgcc ctgcccgatc 720 aacatcgacg gcgtgggcgc ggtcggcgcg atcaacatgg agcggctgaa tctcgtctcc 780 tcgatcatcg accagtgcat ccagttcacc aacaacgtct atctgcccga cgtggtggcc 840 atcggcggct tctaccgcaa ctggctctat ggcggcgggc tctcgtcgaa gtcggtgatg 900 gcctatggcg acatccccga gcatccgaac gatttctcgc ccgaacagct ccatctgccg 960 cggggcgcga tcatcaacgg caatctcgag gaagtgcatg acgtcgaccc gcgcgacccc 1020 gagcaggtgc aggaattcgt cgatcactcc tggtatgcct atggcgagcc ggggcgcggg 1080 ctgcacccct gggacggcgt gaccgagccg cgctacgaac tcggccccaa tgccaagggc 1140 acgcggacga acatcctcga gctcgacgag gcggcgaaat attcctggat caaggcgccg 1200 cgctggaagg gtcacgcgat ggaggtgggc ccgctcgccc gctacatcgt gggctatgcc 1260 aagggccacg aggacatcaa gaaccaggtc gagggtctct tgcgcaccat ggacctgccg 1320 gtctcggcac tgttctcgac gctgggccgc acggccgccc gcgcgctcga ggcggaatat 1380 tgctgccgcc tgcagaagca cttcttcgac aagctcatca ccaacgtgaa gaacggcgac 1440 agcagcaccg ccaatgtcga gaagtgggag ccgcgcacct ggccgaagga ggccaagggc 1500 gtcggcatga ccgaggcccc gcgcggcgcg ctcggccact ggatccgcat caaggatggc 1560 cggatcgaga actaccagtg cgtggtgccc accacctgga acggcagccc gcgcgacgcg 1620 gccggcaaca tcggcgcctt cgaggcgagc ctgctcgaca ccaagatgga gcgccccgag 1680 gagccggtcg agatcctgcg caccctgcac tcgttcgacc cctgcctcgc ctgctccacc 1740 catgtcctgt cgccggacgg ccaggaactc accaccgtca aggtccgc 1788 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 acccatatgc cccagatcga aaccttc 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tgtgctagcg gcctccgtct tttcgtc 27 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 acagctagca tggtcgcgac accgaac 27 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tgtaagcttg cggaccttga cggtggtga 29 <210> 7 <211> 1521 <212> DNA <213> Rhodobacter sphaeroides <400> 7 atgccgcagt cggccgaaaa ggttctggat cacaaggatc tgttcaagga acccgaatat 60 caggcgatgc tcgagaagaa gcgcgccacc tacgagaatg cgacgcccgc cgagacggtg 120 gccgaaaccg cggactggac gaagtcctgg gactatcgcg agaagaacct cgcccgctcc 180 tgcgtgacca tcaacccggc caaggcctgc cagccgctcg gcgcggtctt cgccgccgcc 240 ggctatgaca gcaccatgag cttcgtgcac ggctcgcagg gctgcgtggc ctactatcgc 300 tcgcacctcg cccgccactt caaggagccg tcctcggcgg tgtcctcctc gatgaccgag 360 gatgcggcgg tgttcggcgg cctgaacaac atggtggaag gcctcgccaa cacctatgcg 420 ctctattcgc cgaagatgat cgccgtttcc accacctgca tggcggaagt catcggcgac 480 gacctcaact cgttcatcat caagtcgaag gagaaggaaa gcgtcccggc cgattttccg 540 gtgcccttcg cccatacgcc ggccttcgtg ggcagccacg tcgacggcta cgacaacatg 600 cagaagggca tcctgtcgaa cttctggaag gacgcgccgc gcaccgcggg cgaaggcctg 660 aacatcatcc cgggctttga cggctactgc gtgggcaacg tccgcgagat gaagcgcatg 720 ctcggcctga tgggcgtcga ggcgaccgtt ctgggcgatg cctcggacgt ctacgacacc 780 ccctcggacg gcgagtaccg catgtatgcg ggcggcacca cgcaggagga gatcaaggag 840 gccctgaacg cgaaggccac cctctcgctg caggaatatt gcacccgcag gacgctcgcc 900 ttctgcgagg aagtgggcca ggagaccgcg tcgttccact atccgatggg cgtcaaggcc 960 accgacgagt tcctgatgaa ggtctcggac ctgaccggca aggagatccc ggaagcgctc 1020 cgcctcgagc gcggccgcct gatcgacgcc atggccgaca gccaggccta cctgcacggc 1080 aagacctacg ccatcttcgg cgacccggac ttcgtctatg ccatggcccg cttcgtcatg 1140 gagatgggcg gcgagccgaa gcactgcctc gccaccaacg gcggcaagga ctgggaagtg 1200 cagatgaagg agctgctggc ctcctcgccc ttcggcgaag gctgccaggt ctgggcgggc 1260 aaggacctct ggcacctgcg ctcgatcctc gccacggaac cggcggacct gctgatcggc 1320 agcagctatg gcaagtatct cgagcgcgac tgcaacgtgc cgctgatccg cctgaccttc 1380 ccgatcttcg accgccacca ccaccaccgc ttcccgacct tcggctatca gggcgcgatc 1440 caggtgctgg tgaagatcct cgacaagatc ttcgacaagc tcgacgacga gtccgacatc 1500 tcgttcgacc tgacccgctg a 1521 <210> 8 <211> 2543 <212> DNA <213> Rhodobacter sphaeroides <400> 8 atgggaaaac tccggcagat cgctttctac ggcaagggcg ggatcggcaa gtcgacgacc 60 tcgcagaaca ccctcgcggc actggtcgag atgggtcaga agatcctcat cgtcggctgc 120 gatcccaagg ccgactcgac ccgcctgatc ctgaacacca agctgcagga caccgtgctt 180 cacctcgccg ccgaagcggg ctccgtcgag gatctcgaac tcgaggatgt ggtcaagatc 240 ggctacaagg gcatcaaatg caccgaagcc ggcgggccgg agccgggcgt gggctgcgcg 300 ggccgcggcg tcatcaccgc catcaacttc ctggaagaga acggcgccta tgacgacgtc 360 gactacgtct cctacgacgt gctgggcgac gtggtctgcg gcggcttcgc catgccgatc 420 cgcgagaaca aggcgcagga aatctacatc gtcatgtcgg gcgagatgat ggcgctctat 480 gcggccaaca acatcgccaa gggcatcctg aaatacgcga actcgggcgg cgtgcgcctc 540 ggcggcctga tctgcaacga gcgcaagacc gaccgcgagc tggaactggc cgaggccctc 600 gccgcgcgtc tgggctgcaa gatgatccac ttcgttccgc gcgacaatat cgtgcagcac 660 gccgagctcc gccgcgagac ggtcatccag tatgcgcccg agagcaagca ggcgcaggaa 720 tatcgcgaac tggcccgcaa gatccacgag aactcgggca agggcgtgat cccgaccccg 780 atcaccatgg aagagctgga agagatgctg atggatttcg gcatcatgca gtccgaggaa 840 gaccggctcg ccgccatcgc cgccgccgag gcctgatccg agcgggggcc gggcgccgcc 900 cggtcccttt ccctccctgc cgaatggagc ccgccccatg gcgaaagata tcgctgactc 960 tgccgagacc aacatgaagc tgatcgagga ggtgctggcc gcctaccccg acaaggccag 1020 gaagaagcgc gccaagcacc tgaatgtcgc agcgcccgtc gccgaggccg aacccggcct 1080 ccagtcgaaa tgcgacaatg tgaaatcgaa catcaagtcg gtccccggcg tgatgaccat 1140 ccgcggctgc gcctatgccg gctcgaaggg cgtggtctgg ggcccggtca aggacatgct 1200 gcacatcagc cacggcccgg tcggctgcgg ccactacagc tggtcccagc gccgcaacta 1260 ctacaccggc acgacgggcg tggattcgtt cgtgaccatg caggtcacca ccgacttcca 1320 ggaaaacgac atcgtcttcg gcggtgacaa gaagctggaa aagaccatcg acgagctgaa 1380 catgctcttc ccgctgaaca aggggatctc gatccagtcg gaatgcccga tcggcctgat 1440 cggcgacgac atcgaggcgg tgtcgaagaa gaaggccaag gacatcggca agcgcgtcgt 1500 tccggtgcgc tgcgagggct tccgcggcgt gtcgcagtcg ctcggccacc atatcgcgaa 1560 cgacatgatc cgcgactggg tgctggaagc gggcgagggc gcgcgcgcgg gctacgagcc 1620 cggcccctat gacgtgaaca tcatcggcga ctacaacatc ggcggcgacg cctggtcgag 1680 ccggatcctg ctggaagaga tcggcctcaa cgtcatcgcg caatggtcgg gcgacgccac 1740 catcgccgag atggagcgcg ctccggcggc gaagctgaac ctcatccact gctaccgttc 1800 gatgagctac atctgccggc acatggaaga gaaccacggc gtgccgtgga tggagtacaa 1860 cttcttcggc ccctcgcaga tcgcggcctc gctgcgcgcc atcgccgcga agttcgacga 1920 caggatccag gccaatgccg aagcggtcat cgcgaaatac cagccgctcg tcgatgcggt 1980 gaacgcgaaa tacaagccgc gcctcgaagg caagaaggtg atgctctatg tgggcggcct 2040 gcgtccgcgc cacgtcgtcg acgcctacca tgacctgggc atggagatcg tgggcaccgg 2100 ctacgaattc gcccacaacg acgactacaa gcgcaccggc cattacatca aggaaggcac 2160 gctgatcttc gacgacgtct cgggctacga gctggagaaa ttcgtcgagg cgatccgtcc 2220 cgatctcgtg ggctcgggca tcaaggagaa atacaacacg cagaagatgg gcatcccgtt 2280 ccgtcagatg cactcctggg attattccgg cccctaccac ggctacgacg gctacgcgat 2340 cttcgcgcgc gacatggatc tcgcgatcaa caaccccgtc tggggcatgt tcgatgcgcc 2400 ctggaagaag acggcctgag gccagcccca aggggggcct gctcccgccc cccgaccctc 2460 ccctccaccc atgcaaggtg cgtcccggaa tgaggacggc cagcagaagg atcatgctca 2520 tgccgcagtc ggccgaaaag gtt 2543 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 atagctagca tgccgcagtc ggc 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 actaagcttt cagcgggtca ggt 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ttccatatgg gaaaactccg gca 23 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 taagctagca accttttcgg ccg 23  

Claims (12)

(1) Transformation with a hydrogenase gene of Rhodobacter sphaeroide comprising a hupS gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a hupL gene consisting of the nucleotide sequence shown in SEQ ID NO: 2 Escherichia spp. Host cell, and NifK gene consisting of the nucleotide sequence shown in SEQ ID NO: 7, the nifD gene consisting of the first to the 876th bases in the nucleotide sequence shown in SEQ ID NO: 8, and the bases 938 to 1482 in the nucleotide sequences shown in SEQ ID NO: 8 Preparing an Escherichia genus host cell transformed with a nitrogenase gene of Rhodobacter spheroid comprising a nifH gene consisting of the first base; And (2) hydrogen production method comprising the step of co-culturing the host cell to produce hydrogen. delete delete delete delete delete delete The method of claim 1, The Escherichia genus host cell is E. coli hydrogen production method. The method of claim 8, E. coli is hydrogen production method characterized in that E. coli BL21 (DE3). The method of claim 1, wherein the host cell is hydrogen production method, characterized in that cultured in the hydrogen production titer medium shown in the following table.

The method of claim 1, The host cell is hydrogen production method characterized in that the shaking culture at 140 to 150rpm at a temperature of 29 to 31 ℃. The method of claim 1, Hydrogen production method characterized in that the host cell transformed with nitrogenase in step (2) is cultured in an immobilized state.

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