Fe-S fusion protein acting as electron transfer chain, carbon monoxide formate redox enzyme mediated through FES fusion protein, strain BCF12 derived from <i>Thermococcus </i>wherein enzyme is transformed, and use thereof

Abstract

The present invention relates to an FeS fusion protein acting as an electron transport chain, a novel carbon monoxide:formate oxidoreductase (CFOR) including the FeS fusion protein, novel Thermococcus strain BCF12 transformed with CFOR, and the use thereof. According to the present invention, two different enzymes may be physically linked directly to each other through the FeS fusion protein of the present invention, and thus electrons generated from any one enzyme may be transported directly to another enzyme through the FeS cluster of the FeS fusion protein. Accordingly, a reaction that produces a target substance with high efficiency by directly supplying electrons necessary for the production of the target substance is possible without leakage of electrons generated in any one enzyme. In addition, the present invention has an advantage in that the overall enzyme reaction rate and yield can be dramatically improved using a new electron transport reaction. Furthermore, it is possible to ensure the stability of each enzyme by allowing the enzymes to exist in a physically fixed state in cells.

Claims

1. An FeS fusion protein comprising: a flexible linker having the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6; and two or more FeS proteins, each comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 11, the two or more FeS proteins covalently linked together through the flexible linker, the FeS fusion protein capable of acting as an electron transport chain functioning as a channel through which electrons move, wherein the FeS fusion protein comprises the amino acid sequence of SEQ ID NO: 13.

2. The FeS fusion protein of claim 1, wherein the FeS fusion protein is formed by covalently linking two to five FeS proteins together through the flexible linker.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the structure of a carbon monoxide:formate oxidoreductase (CFOR) of the present invention, and shows a series of processes in which electrons generated from the oxidation of CO gas by CO dehydrogenase (CODH) move through an FeS cluster () present in the electron transport chain FeS fusion protein and is transported to the CO.sub.2 reductase formate dehydrogenase (FDH), and CO.sub.2 is converted into formate by an enzymatic reaction.

(2) FIG. 2 is a schematic view illustrating the types of FeS clusters that are generally present in FeS protein (iron-sulfur protein).

(3) FIG. 3 shows ferredoxin ((A) of FIG. 3) containing an FeS cluster and the protein structure of formate dehydrogenase (FDH).

(4) FIG. 4 shows a process of producing a carbon monoxide:formate oxidoreductase (CFOR) gene by recombination of CO dehydrogenase (CODH) and formate dehydrogenase.

(5) FIGS. 5A to 5C show the genetic structure of each transformant produced by inserting the carbon monoxide:formate oxidoreductase (CFOR) into the genome of a host cell [FIG. 5A: a schematic view of fdh3 and codh gene clusters present in the T. onnurineus NA1 genome and target genes used for cloning; FIG. 5B: construction of a fusion protein between TON_0541 and TON_1017. A schematic view showing cloning of strains to which a flexible linker consisting of one GGGGS (SEQ ID NO: 1) amino acid moiety (T. onnurineus BCF01), a flexible linker consisting of two GGGGS (SEQ ID NO: 1) amino acid moieties (T. onnurineus BCFO2), and a flexible linker consisting of three GGGGS (SEQ ID NO: 1) amino acid moieties (T. onnurineus BCF03) were applied; FIG. 5C: construction of a fusion protein between TON_0540 and TON_1017. A schematic view showing cloning of a strain to which a flexible linker consisting of one GGGGS (SEQ ID NO: 1) amino acid moiety (T. onnurineus BCF12) was applied].

(6) FIG. 6 depicts graphs showing the results of measuring changes in cell growth (A) and formate production (B) in transformants (T. onnurineus BCF01, BCF02, BCF03, and BCF12), into which the carbon monoxide:formate oxidoreductase (CFOR) has been introduced, under a CO condition.

(7) FIG. 7 depicts graphs showing the results of analyzing the CO gas-to-formate bioconversion performance of a T. onnurineus BCF12 strain through a resting cell experiment.

(8) FIG. 8 is a photograph showing the results of PAGE of the carbon monoxide:formate oxidoreductase (CFOR) synthesized by the T. onnurineus BCF12 strain.

(9) FIG. 9 is a graph showing the results of analyzing the CO gas-to-formate bioconversion enzyme activity of the carbon monoxide:formate oxidoreductase (CFOR) in different types of buffer.

(10) FIG. 10 is a graph showing the results of operating a bioreactor for the mutant strain T. onnurineus BCF12 strain into which the carbon monoxide:formate oxidoreductase (CFOR) has been introduced.

DETAILED DESCRIPTION

(11) Hereinafter, the present invention will be described in more detail with reference to examples. These examples serve merely to illustrate the present invention, and thus the scope of the present invention is not construed as being limited by these examples.

Example 1. Cloning of Carbon Monoxide:Formate Oxidoreductase Fusion Protein

(12) As an expression vector for a carbon monoxide:formate oxidoreductase (CFOR), pNA1comFosC1096 derived from the fosmid vector pCC1FOS was used.

(13) pNAlcomFosC1096 was constructed from pCC1FOS to have a 1-kb flanking region so that an insert DNA could be inserted between TON_1126 and TON_1127 of Thermococcus onnurineus NA1 used as a host cell.

(14) The insert DNA was inserted together with the strong promoter P.sub.0157 promoter and HMG-CoA reductase so as to have resistance to simvastatin.

(15) The fdh3 region (TON_0539-0541) and codh region (TON_1017-1020) of Thermococcus onnurineus NA1 were amplified by PCR, and pFd3CoL1C1118, pFd3CoL2C1119 and pFd3CoL1C1120 recombinant plasmids were constructed so that the FeS proteins TON_0541 and TON_1017 were fused with each other by each of the linkers (GGGGS).sub.1 (SEQ ID NO: 1), (GGGGS).sub.2 (SEQ ID NO: 2) and (GGGGS).sub.3 (SEQ ID NO: 3). The recombinant plasmid pFd3NHisCoL1C1128 had the same structure as that of pFd3CoL1C1118, and His.sub.6-tag was inserted into the N-terminus of Fdh3 so that isolation was possible by His-tag affinity chromatography.

(16) The recombinant plasmid pFd3NHisCoL1C1132 was constructed to simplify the structure of CFOR and increase the efficiency of conversion of CO into formate. TON_0540 and TON_0541 of the fdh3 region are all FeS proteins that are subunits involved in electron transport. While the above-described plasmid was constructed so that TON_0541 and TON_1017 were fused to each other by the linker, pFd3NHisCoL1C1132 was constructed so that TON 0540 and TON_1017 were linked directly to each other. In addition, His.sub.6-tag was inserted into the N-terminus of Fdh3 so that isolation of the CFOR protein was possible.

(17) For construction of mutant strain D04, the fdh3 gene region was removed from the genome. To this end, the recombinant plasmid pldFdh3clusterA derived from a pUC118 vector was constructed. pldFdh3clusterA had the simvastatin-resistance gene HMG-CoA reductase as a selection marker, and a 1-kb region flanking the gene fdh3 region (TON 0539-0542) to be removed was inserted into each of the right arm (RA) and the left arm (LA). The primer sequences inserted into each recombinant plasmid are shown in Table 3 below.

(18) TABLE-US-00003 TABLE3 Identifier Sequence Remarks priFCfosFdh3F TAAAATGCTT Forwardprimer GGGAGATGAC usedforPCR CTAGGATGGC offdh3of ACAGAATAAT BCF01,BCF02, TCACTCG BCF03orBCFI2 (SEQID NO:18) priFCfosFdh3R1 GGCATGCTGC Linker-containing CTCCGCCGCC reverseprimer CCCCAGGTAA usedforPCR GCCTCATATT offdh3ofBCP01 TG (SEQID NO:19) priFCfosFdh3R2 GCTGCCTCCG Linker-containing CCGCCGCTTC reverseprimer CGCCTCCTCC usedforPCR CCCCAGGTAA offdh3ofBCF02 GCCTCATATT TG (SEQID NO:20) priFCfosFdh3R3 GCTGGGCTGC Linker-containing CTCCGCCGCC reverseprimer CCCGAAGTAG usedforPCR GCGAGCG offdh3ofBCF12 (SEQID NO:21) priFCfosCodhFI TGGGGGGCGG Linker-containing CGGAGGCAGC forwardprimer ATGCCAGCTT usedforPCRof TTTCCGGTTC codhofBCFOI (SEQID NO:22) priFCfosCodhF2 GGCGGCGGAG Linker-containing GCAGCGGAGG forwardprimer AGGCGGAAGC usedforPCRof ATGCCAGCTT codhofBCF02 TTTCCGGTTC (SEQID NO:23) priFCfosCodhF3 TCGGGGGCGG Linker-containing CGGAGGCAGC forwardprimer CCAGCTTTTT usedforPCRof CCGGTTCC codhofBCF12 (SEQID NO:24) priFCfosCodhR TGGCCATCGT Reverseprimer TGACGCCACG usedforPCR CATGCGACGT ofcodhof CTCACCTCCT BCF01,BCF02, GAGTTTAAAC BCF03orBCF12 CTCAT (SEQID NO:25) priFd3NhisCoLaR TCCTCGTGAT Leftarm(LA) GGTGGTGATG reverseprimer GTGCATCCGC usedforinsert ACCACCGCCC DNAPCRofBCF09 T andcontaining (SEQID his-tagat NO:26) N-terminusof Fdh3 priFd3NhisCoRaF GGATGCACCA Rightarm(RA) TCACCACCAT reverseprimer CACGAGGAGT usedforinsert TTAAGATTGG DNAPCRofBCF09 CCTG andcontaining (SEQID his-tagat NO:27) N-terminusof Fdh3 priFd3ChisCoLaR CTTCAGTGAT Leftarm(LA) GGTGGTGATG reverseprimer GTGGCACCCC containinghis- CCAATCTTCT tagatC-terminus C(SEQID ofFdh3 NO:28) priFd3ChisCoRaF GGTGCCACCA Rightarm(RA) TCACCACCAT reverseprimer CACTGAAGAT containing GGAGAAAAAG his-tagat CTGTTC C-terminus (SEQID ofFdli3 NO:29) priFdSCoNhisLaR CCGGCGTGAT Leftarm(LA) GGTGGTGATG reverseprimer GTGCATTTTC containinghis- ACCACCTCAA tagatN-terminus TACCAC ofCodh (SEQID NO:30) priFd3CoNhisRaF AAATGCACCA Rightarm(RA) TCACCACCAT reverseprimer CACGCCGGAA containinghis- AGAAGGTTCC tagat C N-terminusof (SEQID Codh NO:31) priFd3CoChisLaR TATTAGTGAT Leftarm(LA) GGTGGTGATG reverseprimer GTGGATGGGC usedforinsert CATCCAAGTT DNAPCRofBCF07 TTTTC andcontaining (SEQID his-tagat NO:32) C-terminusof Codh priFd3CoChisRaF CCATCCACCA Right(RA) TCACCACCAT reverseprimer CACTAATAGT usedforinsert TTCTATTATT DNAPCRofBCF07 TTAACTTTG andcontaining (SEQID his-tagat NO:33) C-terminus ofCodh

Example 2. Construction of Mutant Strain

(19) Mutant strain D01 of Thermococcus onnurineus is a mutant strain constructed by removing fdh2C (TON_1563-1564) and fdh3 (TON_0539) from the genome. Mutant strain D02 was constructed using D01 as a parent strain by removing fdh1C (TON_0280-0281) from the genome, and mutant strain D04 was constructed using D02 as a parent strain by removing fdh3C (TON_0539-0542)).

(20) For transformation, Thermococcus onnurineus was pre-cultured in modified medium 1 (MM1) containing maltodextrin to obtain a culture of Thermococcus onnurineus. The culture was resuspended in 0.8 Artificial Sea Water (ASW), and then 5 g of the recombinant plasmid of Example 1 was added thereto and introduced into the cells by heat shock at 80 C. Thereafter, a small amount of medium was added to the cells which were then stabilized at 80 C. for 2 hours. The stabilized transformed cells were inoculated and cultured in a medium containing 10 M simvastatin, and passaged twice so as to be sufficiently enriched. Thereafter, a single colony was obtained and the genotype thereof was analyzed through PCR.

(21) The mutant strains BCF01, BCF02 and BCF03 were constructed using D02 as a parent strain so that the fdh3 region (TON_0538-0541) and the codh region (TON_1017-1020) were fused with each other by each of the linkers (GGGGS).sub.1(SEQ ID NO: 1), (GGGGS).sub.2(SEQ ID NO: 2) and (GGGGS).sub.3(SEQ ID NO: 3).

(22) The mutant strains BCF01, BCF02 and BCF03 were transformed with pFd3CoL1C1118, pFd3CoL2C1119 and pFd3CoL3C1120 recombinant plasmids, respectively, and the carbon monoxide:formate oxidoreductase (CFOR) introduced into each of the recombinant plasmids was inserted between TON_1126 and TON_1127 of Thermococcus onnurineus.

(23) In addition, the mutant strain BCF09 was constructed using D02 as a parent strain and transformed with a pFd3NHisCoL1C1128 recombinant plasmid, and the same CFOR protein as in BCF01 was introduced therein. In addition, His.sub.6-tag was added to the N-terminus of Fdh3.

(24) In addition, the mutant strain BCF12 was constructed using D04 as a parent strain and transformed with a pFd3NHisCoL1C1132 recombinant plasmid so that the fdh3 region (TON_0538-0540) and the codh region (TON_1017-1020) were fused with each other by the linker (GGGGS).sub.1(SEQ ID NO: 1). Also, His.sub.6-tag was inserted into the N-terminus of Fdh3 (TON_0539).

(25) The kinds of strains and plasmids used in the experiment are summarized in Table 4 below.

(26) TABLE-US-00004 TABLE 4 Table 1. Strains and fosmids used this study. Strains or Plasmids Description Reference Strains E. coli DH5a Cloning host TaKaRa T. onnurineus NA1 Wild-type strain Previous study D01 NA1 derivative, fdh2C TON_0539 Previous study D02 D01 derivative, fdh1C Previous study D04 D02 derivative, fdh3C This study BCF01 D02 derivative, P.sub.0157hmg.sub.pfu::TON_0538-TON_0541:TON_1017- This study TON_1020; fusion of TON_0541 and TON_1017 with linker (GGGGS).sub.1 (SEQ ID NO: 1) BCF02 D02 derivative, P.sub.0157hmg.sub.pfu::TON_0538-TON_0541:TON_1017- This study TON_1020; fusion of TON_0541 and TON_1017 with linker (GGGGS).sub.2 (SEQ ID NO: 2) BCF03 D02 derivative, P.sub.0157hmg.sub.pfu::TON_0538-TON_0541:TON_1017- This study TON_1020; fusion of TON_0541 and TON_1017 with linker (GGGGS).sub.3 (SEQ ID NO: 3) BCF09 D02 derivative, P.sub.0157hmg.sub.pfu::TON_0538-TON_0541:TON_1017- This study TON_1020; fusion of TON_0541 and TON_1017 with linker (GGGGS).sub.1 (SEQ ID NO: 1); His.sub.6-tag inserted in N-terminus of Fdh3 (TON_0539) BCF12 D04 derivative, P.sub.0157hmgpfu::TON_0538-TON_0540:TON_1017- This study TON_1020; fusion of TON_0540 and TON_1017 with linker (GGGGS).sub.1 (SEQ ID NO: 1); His.sub.6-tag inserted in N-terminus of Fdh3 (TON_0539) Fosmids pCC1FOS Backbone fosmid; Cm.sup.r EPICENTRE pNA1comFosC1096 pCC1FOS carrying P.sub.0157 promotor, HMG cassette, and 1 kbp Left- This study arm (LA) and Right-arm (RA) for homologous recombination of T. onnurineus NA1 genome; backbone fosmid for mutant construction; Sim.sup.r pFd3CoL1C1118 pNA1comFosC1096 carrying fdh3 region (TON_0538-TON_0541) This study and codh region (TON_1017-TON_1020) from T. onnurineus NA1; fusion of TON_0541 and TON_1017 with linker (GGGGS).sub.1 (SEQ ID NO: 1) pFd3CoL2C1119 pFd3CoL1C1118 carrying fusion of TON_0541 and TON_1017 This study with linker (GGGGS).sub.2 (SEQ ID NO: 2) pFd3CoL3C1120 pFd3CoL1C1118 carrying fusion of TON_0541 and TON_1017 This study with linker (GGGGS).sub.3 (SEQ ID NO: 3) pFd3NHisCoL1C1128 pFd3CoL1C1118 carrying fusion of TON_0541 and TON_1017 This study with linker (GGGGS).sub.1 (SEQ ID NO: 1); His.sub.6-tag inserted in N- terminus of Fdh3 (TON_0539) pFd3NHisCoL1C1132 pNA1comFosC1096 carrying fdh3 region (TON_0538-TON_0540) This study and codh region (TON_1017-TON_1020) from T. onnurineus NA1; fusion of TON_0540 and TON_1017 with linker (GGGGS).sub.1 (SEQ ID NO: 1); His.sub.6-tag inserted in N-terminus of Fdh3 (TON_0539) * Cm.sup.r, chloramphenicol resistance; Sim.sup.r, simvastatin resistance by HMG-CoA reductase; fdh1C, fdh1-mfh1-mnh1 gene cluster (TON_0280-0281); fdh2C, fdh2-mfh2-mnh2 gene cluster (TON_1563-1564); fdh3C, fdh3 gene cluster (TON_0539-0542); LA (left-arm) and RA (right-arm), 1 kbp DNA flanking region suitable for homologous recombination

Example 3. Culture of Transformant and Measurement of Formate Production

(27) The transformant produced in Example 2 was cultured in modified medium 1 containing 4 g/L yeast extract, 35 g/L NaCl, 0.7 g/L KCl, 3.9 g/L MgSO.sub.4, 0.4 g/L CaCl.sub.2.Math.H.sub.2O, 0.3 g/L NH.sub.4Cl, 0.15 g/L Na.sub.2HPO.sub.4, 0.03 g/L NaSiO.sub.3, 0.5 g/L NaHCO.sub.3, 0.5 g/L cysteine-HCl, 1 ml/L Holden's trace element, 2 ml/L Fe-EDTA solution, 1 ml/L Balch's vitamin solution, and 0.05 g/L Na.sub.3S.Math.9H.sub.2O. The Fe-EDTA solution contained 1.54 g/L FeSO.sub.4.Math.9H.sub.2O and 2.06 g/L Na.sub.2.Math.EDTA. The prepared medium was sterilized and then stored in an anaerobic chamber under anaerobic conditions. Each of the mutant strains D02, D04, BCF01, BCF02, BCF03 and BCF12 was cultured at 80 C. in a 160-ml serum vial containing 80 ml of medium and a head space filled with CO at 3 bar.

(28) To measure the growth curve of each strain, the optical density was measured using a UV-Vis spectrophotometer (Shimadzu, UV-2600). The concentration of formate was analyzed using high-performance liquid chromatography (YL instrument, YL9100) with an ion exclusion chromatography column (Shodex, RSpak, KC-811) and measured using a UV detector. As a mobile phase, a 0.13 phosphoric acid aqueous solution was used. To analyze the gas composition of the final head space, gas chromatography (YL instrument, YL6100) with a Molsieve 5A column (Supelco, Bellefonte, PA) and a Porapack N column (Supelco) was used, and argon gas was used as a mobile phase.

(29) As a result, referring to FIG. 6, it was observed that the peak growth of the transformant including the recombinant plasmid introduced therein was inhibited at a relatively low level compared to the host strain Thermococcus onnurineus D02 strain ((A) of FIG. 6). In addition, it was confirmed that there was no formate production in the host strain Thermococcus onnurineus D02 strain, whereas formate was produced in all the BCF01, BCF02, BCF03 and BCF12 strains into which the recombinant plasmid was introduced ((B) of FIG. 6). From these results, it could be confirmed that electron transport was possible in the carbon monoxide:formate oxidoreductase (CFOR) of the present invention, synthesized to include the FeS fusion protein obtained by covalently linking two or more FeS proteins together through one to three GGGGS (SEQ ID NO: 1) flexible linkers. In addition, it was confirmed that formate production in the BCF12 strain constructed using the (GGGGS).sub.1(SEQ ID NO: 1) flexible linker was the highest (19 mM concentration).

Example 4. Analysis of CO Gas-to-Formate Bioconversion Performance Through Resting Cell Experiment

(30) A cell suspension to be used in a resting cell experiment was subjected to 5-L cell culture in a bioreactor. CO was continuously supplied, and the cells were harvested at an OD of 0.9 and centrifuged at 6,000 rpm for 30 minutes to isolate and harvest only the cells. A wash step of washing the obtained cells with an MM1 base (excluding yeast extract) free of nutrient components to remove components other than the cells was repeated three times. Finally, a resting cell experiment was performed using the cells with OD.sub.600 of 0.5, suspended in an MM1 base.

(31) In the resting cell experiment, 6 ml of the cell suspension with an OD.sub.600 of 0.5 was placed in a 20-ml serum vial and then sealed, and the headspace was filled with 100% CO gas at a pressure of 2 bar, and then culture at 80 C. was performed. Formate production, CO consumption and hydrogen production were analyzed over time.

(32) As a result, it could be confirmed that formate production continuously increased up to 48 hours, and CO gas consumption and hydrogen production were continuously maintained. As a result of stoichiometry at a time point of 48 hours, it could be confirmed that about 10, of CO consumption was converted to formate and about 90% was converted to bio-hydrogen.

Example 5. Isolation and Purification of Protein

(33) All protein isolation and purification procedures were performed under anaerobic conditions. In order to determine at protein level whether CO was converted to formate, the isolation of the corresponding fusion protein from the nadFd3CoHisL1C1127 strain that is the CODH C-terminal his-tag strain including the carbon monoxide:formate oxidoreductase (CFOR) was performed using an affinity column purification method. The mutant strain nadFd3CoHisL1C1127 strain including the fusion protein was cultured 350 ml of MMC (bis-Tris pH 6.5) medium and inoculated into a bioreactor.

(34) 5-L cell culture was performed with the bioreactor. CO was continuously supplied, and the cells were harvested at an OD of 0.9 and centrifuged at 6,000 rpm for 30 minutes to harvest only the cells separately. The obtained cells were suspended well in talon buffer [50 mM Tris-HCl (pH 8.0), 0.1 M KCl, 10% glycerol], and then uniformly disrupted using a sonicator. Then, the carbon monoxide:formate oxidoreductase (CFOR) was isolated using a Talon affinity column.

(35) Protein was isolated from talon resin using a talon buffer containing 300 mM imidazole, and the protein concentration was quantified by Bradford assay. The isolated and purified protein was analyzed by 121 SDS-PAGE.

(36) As a result, it could be observed that CO dehydrogenase (CODH) and formate dehydrogenase (Fdh) were isolated together with the TON_0540-TON_1017 protein which is the carbon monoxide:formate oxidoreductase (CFOR). From this result, it could be confirmed that CO dehydrogenase (CODH) and formate dehydrogenase (FDH3) were linked together by the FeS fusion protein to form a single new fusion protein (see FIG. 8).

Example 6. Measurement of Enzymatic Activities

(37) The CODH enzyme activity, Fdh enzyme activity and CO gas-to-formate conversion ability of the carbon monoxide:formate oxidoreductase (CFOR) isolated in Example 5 were measured, and all experiments for measurement of the protein activities were measured under anaerobic conditions.

(38) The CODH enzyme activity was measured using the methyl viologen method that quantifies the concentration of methyl viologen (MV) reduced when CO is given as an electron donor.

(39) For activity measurement, 2 mM DTT, 10 mM MV and 0.5 g CFOR protein were added to 1 ml of 50 mM Tris-HCl (pH 8.0) buffer in a cuvette sealed with a screw-cap, and then the cuvette was sealed by closing the lid. Then, the headspace was purged with CO gas and finally filled with CO gas at 1 bar, thus preparing a reaction.

(40) The cuvette containing the mixture solution was placed on a heat block at 80 C., and the reaction was performed for 1 minute. Then, the reaction was terminated by placing the cuvette on ice, and the absorbance at a wavelength of 578 nm was measured using a spectrophotometer.

(41) Measurement of the activity of formate dehydrogenase (FDH) was performed in the same manner as measurement of the CODH enzyme activity, except that 50 mM potassium phosphate (pH 7.6) buffer was used, formate was used instead of CO as an electron donor, and the enzymatic reaction was performed on a heat block at 80 C. for 5 minutes.

(42) The activity of each of the CODH enzyme and the Fdh enzyme was calculated as the amount of enzyme catalyzing the reduction of 2 mmol methyl viologen, which is equivalent to the amount that catalyzes the oxidation of 1 mmol CO or formate. At this time, the extinction coefficient value is .sub.578=9.7 mM.sup.1.Math.cm.sup.1.

(43) For measurement of the CO conversion/formate production activity of the isolated/purified fusion protein, 2 ml of a mixture solution obtained by adding the isolated fusion protein to each of five types of buffer (50 mM Bis-Tris pH 6.5, 150 mM HEPES pH 7.5, 50 mM potassium phosphate pH 7.6, 100 mM Tris pH 8.0, and 200 mM Bicine-KOH pH 8.5) at a final concentration of 100 g/ml was tested in a 25-ml serum vial. A COCO.sub.2 mixture gas (CO:CO.sub.2=53.5:46.5, vol./vol.) was injected into a 23-ml head space, and then reacted by incubation at 80 C. After 5 hours of the reaction, the concentration of formate produced was measured by LC.

(44) As a result of measuring formate, formate production was found in all the buffer conditions, and the highest formate concentration (about 5 mmol/L) was measured in, 150 mM HEPES (pH 7.5). It was finally confirmed that formate was produced only from CO and CO gases under in vitro conditions (see FIG. 9).

(45) TABLE-US-00005 TABLE 5 Specific activities Species (mmol min.sup.1 mg.sup.1) CO dehydrogenase 1.8 Formate dehydrogenase 120 Formate production 1.67 10.sup.4

(46) From the above-described results, it could be finally confirmed that a new type of fusion protein consisting of a complex of CO dehydrogenase (CODH) and formate dehydrogenase (FDH) linked to each other through the FeS fusion protein acting as an electron transport chain was constructed, and the function of converting CO.sub.2 to formate by the enzymatic reaction induced by electron transfer through the FeS proteins was actually achieved.

Example 7. Measurement of Formate Production Activity

(47) Cells were inoculated into 1.5 L of MMC medium and then purged with 100; CO gas, and a batch culture bioreactor was operated at 80 C., and cell culture was performed under anaerobic conditions. A check valve was provided in a gas outlet to pressurize and regulate the gas pressure in the reactor.

(48) As a result of operating the bioreactor for 9 hours, it was confirmed that formate production was 150 mmol/L and specific formate production rate was 22 mmol/g-DCW/hr (see FIG. 10).

(49) Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.

(50) Thermococcus strain BCF12 strain was deposited in the Korea Research Institute of Bioscience and Biotechnology (having the address of 181, Ipsin-gil, Jeongeup-si, Jeolllabuk-do 56212, Republic of Korea) under the Access number of KCTC 13649BP on Sep. 21, 2018. The deposit has been made under the terms of the Budapest Treaty and all restrictions imposed by the depositor on the availability to the public of the biological material will be irrevocably removed upon the granting of a patent.