KR102129282B1 - Novel Strain from Thermococcus BCF12 and Method for Producing Formic Acid Using the Same - Google Patents

Abstract

The present invention relates to a new strain BCF12 derived from Thermococcus having improved formic acid production ability by introducing a carbon monoxide:formate oxidoreductase (CFOR) gene and a method for producing formic acid using the same.
The thermococcus-derived strain BCF12 of the present invention physically directly binds CO dehydrogenase (CO dehydrogenase, CODH) and formic acid dehydrogenase via a Fe-S fusion protein derived from Fe-S protein to form a CO dehydrogenase A carbon monoxide:formate oxidoreductase (CFOR) transformed by electrons generated directly from the Fe-S fusion protein to the formic acid dehydrogenase through a Fe-S cluster. The new carbon monoxide:formic acid oxidoreductase (CFOR) reaction has the advantage of dramatically improving the reaction rate and yield of formic acid production. In addition, by allowing enzymes to exist in a physically fixed state in a cell, stability of each enzyme can also be secured.

Description

{Novel Strain from Thermococcus BCF12 and Method for Producing Formic Acid Using the Same}

The present invention relates to a production method of thermococcus-derived new strain BCF12 and formic acid using the same, and more specifically, carbon monoxide:formate oxidoreductase (CFOR) gene is introduced to introduce thermococcus with improved formic acid production It relates to a new strain BCF12 and a method for producing formic acid using the same.

With the development of genetic engineering technology, the production of biological materials by biological processes is gradually replacing petrochemical processes. Since the production of biological materials by the method of applying the bioconversion process can use cheap renewable resources as a raw material and minimize the generation of greenhouse gases such as carbon dioxide, it has the advantage of solving environmental problems. For the production of biological materials, research for development of related strains and process improvement is expanding.

Specifically, as a method of manufacturing a metabolic regulatory mutant, a variety of techniques are used to increase the production efficiency of a target substance by creating a new metabolic pathway or manipulating/changing a pathway included in the metabolic process. This technique is presumed to promote or inhibit the expression or activity of one or more target proteins (or enzymes) involved in the synthesis of a specific/purpose product. For example, for recombinant microorganisms with increased butanol production capacity, Korean Patent Publication No. 10-2014-0064469 suppresses the expression or activity of a protein (or enzyme) involved in the pathway for converting acetyl-CoA to acetate, , Microorganisms that promote the expression or activity of proteins (or enzymes) involved in the pathway for converting acetyl-CoA to butyryl-CoA are disclosed. In addition, Japanese Patent Registration No. 4760951 discloses 3-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydrogenase (3), an enzyme involved in the synthesis of butanol (3 Enzymes such as -hydroxybutyryl-CoA dehydratase (CRT), trans-enoyl-CoA reductase (TER), aldehyde/alcohol dehydrogenase (aldehyde and alcohol dehydrogenase, AdhE2) are introduced into the microorganism, butanol There have been attempts to improve the production efficiency of.

However, although the above-described prior arts may accumulate intermediate products necessary for the production path of the substance to be synthesized in the microorganism through reaction, various conversion enzymes in addition to enzymes involved in the synthesis pathway of the substance to be synthesized in the cell There was a disadvantage that the production efficiency was low because they existed, and above all, it was not possible to directly generate a new synthetic route that does not exist in the natural system capable of directly producing the desired substance to be produced, or to generate a reaction to directly synthesize the target substance. .

To solve these problems, Korean Patent Publication No. 10-2017-0024466, etc., narrows the physical gap existing between enzymes related to biosynthesis of the target substance to increase the synthesis efficiency of the target substance, so that the intermediate product immediately participates in the reaction. There has been disclosed a system capable of resolving the problem that accumulation is prevented by other enzymes, but the method of increasing the activity of the enzyme through a simple, indirect method of narrowing the physical gap existing between biosynthesis-related enzymes Since it does not affect the electron transfer or redox reaction of the complex enzyme, there is a fundamental problem that it is not expected to increase the enzyme activity above a certain level.

Accordingly, an object of the present invention is a new carbon monoxide: a new strain BCF12 derived from thermococcus introduced with a formic acid oxidative and reductase, comprising a Fe-S fusion protein linked to two or more Fe-S proteins that function as electron transporters, and It is to provide a method for producing formic acid using this.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a thermococcus-derived strain BCF12 (Accession No.: KCTC 13649BP).

The present inventors can directly and directly transfer electrons between enzymes related to biosynthesis of the target substance to increase the efficiency of synthesis of the target substance and enable a reaction that does not exist in nature that can directly produce the target substance to be produced. The system has been studied, and the Fe-S fusion protein formed by covalently bonding two or more Fe-S proteins through a flexible linker to operate as an electron transport chain that serves as a pathway for electrons to move is intracellular. A new carbon monoxide:formate oxidoreductase (CFOR) was synthesized by direct synthesis of target substances and direct linkage of related enzymes. In addition, the present invention was completed by confirming that the target substance, formic acid, can be produced with high efficiency/high yield by introducing it into a strain of the genus Thermococcus.

Accordingly, the present inventors named the strain Thermococcus Onnurinus BCF12, deposited it on September 21, 2018 at the Korea Research Institute of Bioscience and Biotechnology, and received accession number KCTC 13649BP.

As demonstrated in one embodiment of the present invention, Thermococcus-derived mycobacteria BCF12 (Accession No.: KCTC 13649BP) is a carbon monoxide:formate oxidoreductase (CFOR) gene that is transformed to produce formic acid. It is characterized by an improvement.

In the present invention, the carbon monoxide: formic acid oxidoreductase (carbon monoxide: formate oxidoreductase, CFOR) acts as an electron transport chain that serves as a pathway through which electrons move, and two or more Fe-S proteins have SEQ ID NOs: 1 to Fe-S fusion protein formed by covalent bonding through a flexible linker having any one amino acid sequence selected from 6, and CO dehydrogenase (CODH) operably linked to the C-terminus of the Fe-S fusion protein , And formic dehydrogenase (Fdh) operably linked to the N-terminus of the Fe-S fusion protein, wherein electrons generated in the CO dehydrogenase pass through the Fe-S fusion protein to dehydrogen formate. It is characterized in that it is delivered to an enzyme (fomrate dehydrogenase, Fdh), and carbon monoxide: formic acid oxidation combined with CO dehydrogenase (CODH) and formic dehydrogenase (Fdh) via a Fe-S fusion protein. · It is characterized by a carbon monoxide (formate oxidoreductase, CFOR).

In addition, in the thermococcus-derived new strain BCF12, within the range not affecting the functions of the CO dehydrogenase (CODH), the Fe-S fusion protein, and the formic acid dehydrogenase (Fdh) , Variants, or fragments of amino acids having different sequences by deletion, insertion, substitution or combination of amino acid residues. In addition, amino acid exchanges at the protein and peptide levels that do not entirely alter the activity of the CO dehydrogenase (CODH), the Fe-S fusion protein, and formic acid dehydrogenase (Fdh) are known in the art. It is done. In some cases, phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation may be modified.

The term "peptide" as used herein refers to a polymer in the form of a chain formed by bonding 4 to 1,000 amino acid residues to each other by peptide bonding, and means interchangeable with'polypeptide'.

The term'polynucleotide' referred to in the present invention means a polymer compound in which a nucleotide, a chemical monomer composed of three elements of a base, a sugar, and phosphoric acid, is chained through a plurality of phosphate ester bonds.

In the present invention, the combination of the CO dehydrogenase (CO dehydrogenase, CODH) and the formic acid dehydrogenase (Fmrate dehydrogenase, Fdh) is made through the Fe-S fusion protein, but the Fe-S fusion protein is two different Fe -S protein is characterized by being connected by a flexible linker.

The term "Fe-S protein (Iron-sulfur protein)" as used herein includes sulfides associated with ion centers of di, tri, or tetra in various oxidation states. The term "Fe-S cluster" means a protein characterized by being present.

The "Fe-S cluster" appears in metalloproteins such as ferredoxins, NADH dehydrogenase, hydrogenases, coenzyme Q-cytochrome C reductase (coenzyme Q-cytochrome) c reductase), succinic acid-coenzyme Q reductase and nitrogenase, and nitrogen. The "Fe-S protein" composed of "Fe-S clusters" is best known for its role in the redox reaction of mitochondrial electron transport.

As used herein, the term "electron transport chain" refers to a passage of electron transport in which two or more "Fe-S proteins" are linked in a covalent bond through a flexible linker in the form of a chain in series. The term "Fe-S fusion protein" serving as a delivery chain is composed of two or more "Fe-S proteins" covalently linked through a flexible linker, and the inventors acting as unit elements in forming the electron transport chain Means a newly synthesized fusion protein.

In the thermococcus-derived strain BCF12 of the present invention, the Fe-S fusion protein is a flexible linker having two or more Fe-S proteins having any one amino acid sequence selected from SEQ ID NOs: 1 to 6 shown in Table 1 below. It is characterized in that it is covalently linked to, an example of a flexible linker that plays a role similar to the flexible linker is known by Chen X et al. (Chen X, et al. 2013. Adv Drug Deliv Rev 65:1357-69 .).

[Table 1]

In addition, the content of the peptide linker including the flexible linker is disclosed in detail in the document "Toon H. Evers et al., 2006, J. Christopher Anderson et al., 2010", the content disclosed in this document It is incorporated by reference into the specification.

On the other hand, "When the Fe-S protein accepts electrons and is in a reduced state, an ultra-fine magnetic field of about 180 kG is formed. The Fe-S protein that is located at a short distance by being connected to the flexible linker is formed. Due to the influence of the ultra-fine magnetic field, strong binding is induced with the Fe-S protein that accommodates the electrons, and thereby the physical distance between the electron transport chains of the Fe-S proteins disappears, resulting in "Fe -S fusion protein" can be used as a single electron transport pathway.

As used herein, the term "operably linked" refers to a functional binding between a nucleic acid expression control sequence (eg, a promoter sequence, a signal sequence, or an array of transcription regulator binding sites) and another nucleic acid sequence, thereby The regulatory sequence regulates the transcription and/or translation of the other nucleic acid sequence.

In addition, in the new strain BCF12 derived from Thermococcus of the present invention, the Fe-S protein is characterized by including any one amino acid sequence selected from SEQ ID NOs: 7 to 11 or a combination thereof. The Fe-S protein includes a cystein motif of SEQ ID NOs: 7 to 11 shown in [Table 2] below to form Fe-S clusters, and the present inventors compared 190 well-known Fe-S proteins. Cysteine motifs having the amino acid sequences of SEQ ID NOs: 7 to 11 were selected, and Fe-S fusion proteins generated by covalently bonding Fe-S proteins containing them as flexible linkers act as electron transport chains. I checked it through.

[Table 2]

In addition, in the new strain BCF12 derived from Thermococcus of the present invention, the Fe-S fusion protein is characterized in that 2 to 5 Fe-S proteins are formed by covalently bonding to each other through a flexible linker.

In the new strain BCF12 derived from Thermococcus of the present invention, the CO dehydrogenase (CODH) is Thermococcus onnurineus NA1), Thermococcus CH5 ( Thermococcus sp. CH5), Thermo Rhodococcus and forehead sensor system (Thermococcus guaymasensis), Thermo Caucus Businesses fun Douce (Thermococcus profundus ), Thermo Lactococcus radio tolreo lance (Thermococcus radiotolerans), Thermo Lactococcus gamma tolreo lance (Thermococcus gammatolerans), Thermo caucus immediately fill Ruth (Thermococcus barophilus), Thermo Lactococcus AM4 (Thermococcus AM4), meta furnace Thermo bakteo Thermo auto trophy kusu (Methanothermobacter thermoautotrophicus), peeling booth Ake Ogle Douce (Archaeoglobus fulgidus ), Clostridium autoethanogenum , Clostridium ljungdahlii , Clostridium carboxidivorans , Oxobacter pfennigii , Pepto Streptococcus a production Tooth (Peptostreptococcus productus ), Acetonitrile tumefaciens Woody (Acetobacterium woodii ), Oil cake Te Solarium Limoges breath (Eubacterium limosum ), Butyribacterium methylotrophicum , Rubrivivax ( Rubrivivax gelatinosus ), Pseudomonas may also Palouse tris (Rhodopseudomonas palustris ), Iam also rilrum Lou beureom (Rhodospirillum rubrum ), Citrobacter Y19 ( Citrobacter sp Y19 ), Methanosarcina ( Methanosarcina) barkeri ), Meta nosal Sinai acetic Lance Thibault (Methanosarcina acetivorans), Non Relais Thermo acetic Utica (Moorella thermoacetica ), Non Pasteurella Thermo auto trophy car (Moorella thermoautotrophica), Pasteurella-free AMP (AMP Moorella strain), Carboxy FIG sseomeoseu hydrogenocarbonate's satiety (Carboxydothermus hydrogenoformans), carboxy-di bra Kiwoom wave As kusu (Carboxydibrachium pacificus ), Carboxydocella Sporoproduce Sense ( Carboxydocella sporoproducens ), Carboxydocella thermoautotrophica , Thermincola ( Thermincola) carboxydiphila ), Sseomin Cola Feria three Utica (Thermincola ferriacetica), Emory Shaw wrote bakteo carboxy diborane Lance (Thermolithobacter carboxydivorans), Thermo When Augustine carboxy diborane Lance (Thermosinus carboxydivorans ), Desulfotomaculum kuznetsovii , Disulfonic photos Do Coolum Thermo benjoyi Qom (Desulfotomaculum thermobenzoicum sub sp. thermosyntrophicum ), Disulfonic photos Do Coolum carboxy Lance Thibault (Desulfotomaculum carboxydivorans ) may be a gene derived from any one strain selected from the group consisting of, and the formic dehydrogenase (Fdh) is Thermococcus onnurineus NA1), Thermococcus fumicolans , Thermococcus CH5 ( Thermococcus sp. CH5), Thermo Caucus Selena Lee Crescent sense (Thermococcus celericrescens), Thermococcus litoralis (Thermococcus litoralis), Thermo Caucus flops you see Syracuse (Thermococcus pacificus), Thermo Caucus Businesses fun Douce (Thermococcus profundus), Thermo Caucus tolreo Radio Esperance (Thermococcus radiotolerans), Thermo Caucus Stephen Terry (Thermococcus stetteri ), Thermococcus waiotapuensis , Thermococcus AM4 ( Thermococcus sp . AM4), Thermo Lactococcus fertilization Li kusu (Thermococcus sibiricus), Thermo Lactococcus cotta Karen sheath (Thermococcus kodakarensis), Thermo Gamma Caucus tolreo Lance (Thermococcus gammatolerans), Thermo caucus immediately fill Ruth (Thermococcus barophilus ), Thermococcus 4557 (Thermococcus sp . 4557 ), Pyro's Caucus Purifying wastewater (Pyrococcus furiosus ), Pyrococcus abyssi , Pyro Caucus Yaya shi (Pyrococcus yayanosii ), Pyrococcus NA2 ( Pyrococcus sp . NA2 ), Carboxy also written mousse hydrogenocarbonate's satiety (Carboxydothermus hydrogenofomans ), Rubrivivax gelatinosus , Escherichia coli (Escherichia coli ), Rhodospirillum rubrum , Non Relais Thermo acetic Utica (Moorella thermoacetica ), Clostridium autoethanogenum , Clostridium ljungdahlii , Acetobacterium woodii , Oil cake Te Solarium Limoges breath (Eubacterium limosum ), Clostridium carboxidivorans , Pseudomonas may also Palouse tris (Rhodopseudomonas palustris ) may be a gene derived from any one strain selected from the group consisting of.

In addition, in the thermococcus-derived strain BCF12 of the present invention, since the Fe-S fusion protein has a function of an electron transport chain that serves as a pathway for electron transport, it can directly mediate an oxidation-reduction reaction. Accordingly, electrons generated from the CO dehydrogenase are directly transferred to formic acid dehydrogenase (Fdh), wherein the formic acid dehydrogenase (Fdh) is succinic dehydrogenase, DMSO reductase (dimethyl sulfoxide) (DMSO) Reductase), when replaced with hydrogenase (hydrogenase), etc., has the effect of maintaining the electron transport function. In this case, the substituted succinic dehydrogenase, DMSO reductase (dimethyl sulfoxide (DMSO) reductase) , Depending on the type of enzyme of the hydrogenase (hydrogenase), there is an effect that the functions of succinic acid production, DMSO reduction, and hydrogen production are newly imparted.

In addition, in the thermococcus-derived strain BCF12 of the present invention, the carbon monoxide:formate oxidoreductase (CFOR) may further include an additional "tag" to facilitate separation and purification. have. The fusion proteins of the invention can be tagged with a variety of detectable labeling factors, the tag including His(n), flag, c-Myc, HA, V5, VSV-G and HSV, It is not limited to this. “Tag” means a polypeptide sequence having 3 to 40 amino acid sequences, and is specific for a fusion protein, peptide, protein ligand (eg, fusion protein of the invention) or non-peptide ligand of the invention It imparts binding affinity. In addition, the tags that can be used in the present invention may include a fluorescent tag, a luminescent tag, and a chromogenic tag.

In the new strain BCF12 derived from Thermococcus of the present invention, the carbon monoxide:formate oxidoreductase (CFOR) is CO dehydrogenase (CO dehydrogenase, CODH) of SEQ ID NO: 12, Fe-S of SEQ ID NO: 13 The fusion protein and formic acid dehydrogenase (Fdh) of SEQ ID NO: 14 are characterized by having an amino acid sequence of SEQ ID NO: 15 operably linked thereto.

The present invention includes proteins having an amino acid sequence substantially identical to a protein comprising the amino acid sequence of SEQ ID NO: 15 and variants or active fragments thereof. The substantially identical protein means, but is not limited to, those having 80% or more, preferably 90% or more, and most preferably 95% or more of amino acid sequence homology, and having 80% or more amino acid sequence homology and identical If it has enzyme activity, it is included in the scope of the present invention.

In the new strain BCF12 derived from Thermococcus of the present invention, the carbon monoxide may be exemplified by 16 nucleotide sequences encoding the carbon monoxide:formate oxidoreductase (CFOR), but is not limited thereto. It is apparent to those skilled in the art that any nucleotide sequence encoding formic acid redox protein can be used.

Genes encoding the carbon monoxide:formate oxidoreductase (CFOR) and variants or active fragments thereof of the present invention are encoded within a range that does not change the amino acid sequence of the protein expressed from the coding region Various modifications can be made to the region, and various mutations can be made by substitution, deletion, insertion, or a combination thereof within a range that does not affect the expression of the gene even in areas other than the coding region. It is included in the scope of the invention. Accordingly, the present invention includes a nucleotide sequence substantially identical to a nucleotide sequence encoding the carbon monoxide:formate oxidoreductase (CFOR) and a fragment thereof. The substantially identical nucleotide sequence means those having sequence homology of 80% or more, preferably 90% or more, and most preferably 95% or more, but is not limited thereto, and has 80% or more sequence homology and is encoded. If the protein has the same enzyme activity, it is included in the present invention.

The thermococcus-derived strain BCF12 of the present invention includes (a) a nucleotide sequence encoding the carbon monoxide:formate oxidoreductase (CFOR); And (b) a recombinant vector comprising a promoter operatively linked to the nucleotide sequence is introduced into a cell and transformed. The term "promoter" as used herein refers to a DNA sequence that regulates the expression of a coding sequence or functional RNA. In the recombinant expression vector of the present invention, an expression target material (ie, carbon monoxide: formic acid oxidation/reductase)-encoding nucleotide sequence is operably linked to the promoter.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001), This document is incorporated herein by reference.

When the expression vector of the present invention is a prokaryotic cell as a host, strong promoters (eg, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL promoter, pR promoter, rac5 promoter, amp promoter, which can progress transcription) It is common to include the recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosome binding sites for initiation of translation, and transcription/detox termination sequences. At this time, the bacterial replication starting point may be selected from replication starting points well known in the art useful for stable bacterial replication of long DNA inserts, ColE1, F-factor and P1 replicon ( replicon). The bacterial selection marker of the present invention can use a bacterial selection marker gene known in the art. For example, bacterial selection marker genes include, but are not limited to, genes that confer resistance to antibiotics such as ampicillin, kanamycin, tetracycline, zeocin, neomycin, hygromycin and chloramphenicol. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024, 1984) and the phage λ leftward promoter (pLλ promoter) , Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)) can be used as regulatory sites.

In addition, a promoter that can be used when the recombinant vector of the present invention is applied to eukaryotic cells can regulate the transcription of the expression target material of the present invention, a promoter derived from a mammalian virus and a genome of a mammalian cell ), for example, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothionine promoter , Beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene and promoter of human GM-CSF gene, but is not limited to this no.

Preferably, the recombinant vector used in the present invention includes a poly adenylation sequence (eg, a bovine growth hormone terminator or a polyadenylation sequence derived from SV40).

The vector can include a selectable marker for selecting a host cell containing the vector. The selection marker is for selecting cells transformed with a vector, and markers conferring a selectable phenotype such as drug resistance, nutritional demand, resistance to cytotoxic agents, or expression of surface proteins can be used. In the environment where the selective agent is treated, only the cells expressing the selection marker survive, so that the transformed cells can be selected. In addition, in the case of a replicable expression vector, the vector may include a replication origin, which is a specific nucleic acid sequence in which replication is initiated.

Expression vectors that can be used for bacterial hosts include bacterial plasmids obtained from Escherichia coli, such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9, and derivatives thereof, and broader hosts such as RP4 Plasmids with a range, λgt10 and λgt11, phage DNA that can be exemplified by a wide variety of phage lambda derivatives such as NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phage. In particular, for expression in E. coli, an anthranylate synthase (TrpE) and a carboxy terminus may include a DNA sequence encoding a polylinker, and other expression vector systems include beta-galactosidase (pEX); Lambda PL maltose binding protein (pMAL); And glutathione S-transferase (pGST) (Gene 67:31, 1988; Peptide Research 3:167, 1990).

The method for transporting the vector of the present invention into a host cell may use various methods known in the art, for example, when the host cell is a prokaryotic cell, the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973); and Hanahan, D ., J. Mol. Biol., 166:557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145 (1988)). In the case of eukaryotic cells, in transduction, electroporation, lipofection, microinjection, particle bombardment, yeast spheroid/cell fusion used in YAC, in plant cells Agrobacterium-mediated transformation can be used.

In addition, the production of animal cells transformed with the recombinant expression vector of the present invention can be carried out by gene transfer methods commonly known in the art. For example, electroporation, liposome-mediated transfer (Wong, et al., 1980) and retrovirus-mediated transfer (Chen, HY, et al., (1990), J. Reprod. Fert. 41: 173-182; Kopchick, JJ et al., (1991) Methods for the introduction of recombinant DNA into chicken embryos.In Transgenic Animals, ed.NL First & FP Haseltine, pp.275-293, Boston; Butterworth-Heinemann; Lee , M.-R. and Shuman, R. (1990) Proc. 4th World Congr. Genet. Appl. Livestock Prod. 16, 107-110).

In addition, according to another aspect of the present invention, the present invention is to cultivate a new strain BCF12 derived from Thermococcus of the present invention (Accession No.: KCTC 13649BP) to carbon monoxide: formic acid oxidoreductase (CFOR). It provides a method for producing carbon monoxide:formate oxidoreductase (CFOR), which includes the step of expressing.

In addition, according to another aspect of the present invention, the present invention is (a) the strain of the strain BCF12 derived from the thermococcus (Accession No.: KCTC 13649BP) or the carbon monoxide: formic acid oxidation and reduction enzyme (carbon monoxide:formate oxidoreductase, CFOR) Supplying CO gas in the presence to synthesize formic acid from the CO gas; And (b) recovering the synthesized formic acid.

As described above, the thermococcus-derived strain BCF12 of the present invention physically directly directs CO dehydrogenase (CODH) and formic acid dehydrogenase via a Fe-S fusion protein derived from Fe-S protein. Carbon monoxide:formate oxidoreductase (CFOR) transformed by electrons generated from CO dehydrogenase and directly transferred to formic acid dehydrogenase through Fe-S cluster of Fe-S fusion protein As a strain, it has the advantage of dramatically improving the reaction rate and yield of formic acid production by using a new carbon monoxide:formate oxidoreductase (CFOR) reaction that was not previously available.

In addition, by allowing enzymes to exist in a physically fixed state in a cell, stability of each enzyme can also be secured.

1 is a diagram illustrating the structure of carbon monoxide:formate oxidoreductase (CFOR) introduced into a strain of the present invention, and CO gas is oxidized by CO dehydrogenase (CODH). the enzymatic reaction whereby electrons are transferred to the electron transport chain of Fe-S cluster (◇) by the movement through CO ₂ reduction enzyme formate dehydrogenase (formate dehydrogenase, FDH) (CO 2 reductase) present in the Fe-S fusion protein It is a picture explaining the process by which carbon dioxide (CO ₂ ) is converted to formic acid.
FIG. 2 is a schematic diagram illustrating the types of Fe-S clusters present in Fe-S protein (Iron-sulfur protein).
Figure 3 is a diagram showing the protein structure of ferredoxin (ferredoxin) (A) and formic acid dehydrogenase (formate dehydrogenase, FDH) containing Fe-S cluster.
FIG. 4 is a diagram showing a process of generating a gene of carbon monoxide:formate oxidoreductase (CFOR) by recombining CO dehydrogenase (CODH) and formic acid dehydrogenase.
5 is a diagram illustrating the gene structure of each transformant prepared by inserting a gene of carbon monoxide:formate oxidoreductase (CFOR) into the genome of a host cell [(A) T. the target gene used for the gene cluster fdh3 and codh schematic view and cloned present in onnurineus NA1 genome; (B) Production of a fusion protein between TON_0541 and TON_1017. Flexible linker'GGGGS' amino acid 1 ( T. onnurineus BCF01), 2 ( T. onnurineus BCF02), and 3 ( T. onnurineus BCF03) cloning schematic diagram of the applied strain; (C) Production of a fusion protein between TON_0540 and TON_1017. Flexible linker'GGGGS' amino acid 1 ( T. onnurineus BCF12) applied cloning schematic].
FIG. 6 shows cell growth (A) and formic acid of transformants ( T. onnurineus BCF01, BCF02, BCF03, and BCF12) introduced with carbon monoxide:formate oxidoreductase (CFOR) under CO conditions. It is a graph measuring changes in production (B).
7 is a graph showing the results of analyzing the bioconversion performance of CO gas to formic acid through the Resting cell experiment of the thermococcus-derived strain BCF12 of the present invention.
8 is a photo of PAGE results of carbon monoxide:formate oxidoreductase (CFOR) synthesized from thermococcus-derived strain BCF12 of the present invention.
9 is a graph showing the results of analyzing the CO gas-formic acid bioconversion enzyme activity of carbon monoxide:formate oxidoreductase (CFOR) with different buffer types.
10 is a graph showing the results of the operation of the bioreactor of the strain BCF12 of the mycobacteria derived from thermococcus of the present invention in which carbon monoxide:formate oxidoreductase (CFOR) is introduced.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are only for illustrating the present invention, it should not be construed that the scope of the present invention is limited by these examples.

Example One. Carbon monoxide: Formic acid Of oxidative and reductase fusion proteins Cloning

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

pNA1comFosC1096 was constructed with a side region of 1 kb so that insert DNA could be inserted between TON_1126 and TON_1127 of Thermococcus onnurineus NA1 used as a host cell from pCC1FOS.

Insert DNA was inserted with HMG-CoA reductase to be resistant to the strong promoter P ₀₁₅₇ promoter and simvastatin.

Thermococcus onnurineus ) The fdh3 region (TON_0539-0541) and codh region (TON_1017-1020) of NA1 were amplified through PCR, and the recombinant plasmids of pFd3CoL1C1118, pFd3CoL2C1119, and pFd3CoL1C1120 were Fe-S proteins, respectively, TON_0541 and TON_1017 linker (GG) _It was constructed to be fused through ₁ , (GGGGS) ₂ , and (GGGGS) ₃ . The recombinant plasmid pFd3NHisCoL1C1128 has the same structure as pFd3CoL1C1118, and His ₆ -tag is inserted into the Fdh3 N-terminus to allow separation through His-tag affinity chromatography.

The recombinant plasmid pFd3NHisCoL1C1132 was constructed to simplify the structure of CFOR and increase the conversion efficiency of CO to formic acid. TON_0540 and TON_0541 of the fdh3 region are both Fe-S proteins and are small units involved in electron transport. Previously, TON_0541 and TON_1017 were manufactured to be fused through a linker, whereas pFd3NHisCoL1C1132 was manufactured to directly connect TON_0540 and TON_1017. In addition, a His _6- tag was inserted into the Fdh3 N-terminus to allow separation of the CFOR protein.

Fdh3 for construction of mutant D04 The gene region was removed from the genome, and for this purpose, a recombinant plasmid pldFdh3clusterA derived from the pUC118 vector was constructed. pldFdh3clusterA is a selection marker, and it has the simvastatin resistance gene HMG-CoA reductase, and the side region 1kb of the gene fdh3 region (TON_0539-0542) to be removed is a right arm (RA). , Left arm (LA) was inserted.

Primer sequences inserted into each recombinant plasmid are shown in Table 3 below.

[Table 3]

Example 2. Construction of mutant strains

The mutant strain D01 of Thermococcus onnurineus is a mutant strain in which fdh2C ( TON_1563-1564) and fdh3 (TON_0539) are removed from the genome, respectively. The mutant D02 was made to remove fdh1C (TON_0280-0281) from the genome with D01 as the parent strain, and the mutant D04 was constructed to remove all fdh3 C (TON_0539-0542) with D02 as the parent strain.

For the transformation of a thermodynamic Lactococcus Onnuri Taunus (Thermococcus onnurineus) and preincubated in a maltodextrin (maltodextrin) is added to the MM1 (modified medium 1) medium was secured cultures of Lactococcus Thermo Onnuri Taunus (Thermococcus onnurineus). After resuspension of it in 0.8X Artificial Sea Water (ASW), 5 μg of the recombinant plasmid of Example 1 was added, and this was introduced into the cell through heat shock at 80°C. Then, a small amount of medium was added to stabilize at 80° C. for 2 hours. The stabilized transformed cells were inoculated into a medium containing 10 μM of simvastatin and cultured, and then passaged twice to ensure sufficient enrichment. After that, a single colony was obtained and genotype was confirmed by PCR.

Mutant strains BCF01, BCF02, and BCF03 are D02 as a parent strain so that the fdh3 region (TON_0538-0541) and the codh region (TON_1017-1020) are fused to linker (GGGGS) ₁ , (GGGGS) ₂ , and (GGGGS) ₃ , respectively. It was produced.

The mutant strains BCF01, BCF02, and BCF03 were transformed with recombinant plasmids of pFd3CoL1C1118, pFd3CoL2C1119, and pFd3CoL3C1120, respectively. ( Thermococcus onnurineus ) was inserted between TON_1126 and TON_1127.

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

In addition, the mutant BCF12 was transformed with the recombinant plasmid of pFd3NHisCoL1C1132 using D04 as a parent strain, so that the fdh3 region (TON_0538-0540) and the codh region (TON_1017-1020) were fused with a linker (GGGGS) ₁ And His ₆ -tag was inserted into the N-terminus of Fdh3 (TON_0539).

The types of strains and plasmids used in the experiment are summarized in [Table 4] below.

[Table 4]

Example 3. Culture of transformants and measurement of formic acid production

The transformant prepared in Example 2, 4g / L yeast extract, 35g / L NaCl, 0.7g / L KCl, 3.9g / L MgSO 4, 0.4g / L CaCl 2 · H 2 O, 0.3g / L NH ₄ Cl, 0.15 g/L Na ₂ HPO ₄ , 0.03 g/L NaSiO ₃ , 0.5 g/L NaHCO ₃ , 0.5 g/L cysteine-HCl, 1 ml/L Holden's trace element, 2 ml/L Fe-EDTA solution , 1ml/L Balch's vitamin solution, and cultured in a modified medium 1 (MM1) medium containing 0.05 g/L Na ₂ S·9H ₂ O. Fe-EDTA solution contains 1.54g/L FeSO ₄ ·9H ₂ O, 2.06g/L Na ₂ ·EDTA. The produced medium was sterilized and stored in an anaerobic chamber to maintain anaerobic conditions. Mutants D02, D04, BCF01, BCF02, BCF03, and BCF12 were cultured at 80° C. by replacing 80 ml of medium and head space with 3 bar of CO in 160 ml serum vial.

To observe the growth curve, the optical density was measured using a UV-Vis spectrophotometer (Shimadzu, UV-2600). The concentration of formic acid was analyzed using Ion exclusion chromatography column (Shodex, RSpak, KC-811) of High performance liquid chromatography (YL instrument, YL9100), and was measured using a UV detector. A 0.1% phosphoric acid aqueous solution was used as the mobile phase. To analyze the gas composition in the final head space, the Molsieve 5A column (Supelco, Bellefonte, PA) and Porapack N column (Supelco) of gas chromatography (YL instrument, YL6100) were used, and Argon gas was used as the mobile phase.

As a result, referring to FIG. 6, the highest growth of the transformant into which the recombinant plasmid has been introduced is Thermococcus , which is a host strain. onnurineus ) It was observed to show inhibition at a relatively low level compared to the D02 strain (FIG. 6A ). In addition, the host strain Thermococcus onnurineus ) There was no formic acid production in the D02 strain, whereas formic acid production was confirmed in all of the BCF01, BCF02, BCF03, and BCF12 strains into which the recombinant plasmid was introduced (FIG. 6B). Through these results, carbon monoxide: formic acid oxidase/reductase of the present invention synthesized by including two or more Fe-S proteins, including Fe-S fusion proteins covalently linked through 1 to 3 flexible linkers of'GGGGS' (carbon monoxide:formate oxidoreductase, CFOR) was confirmed to be capable of electron transfer. In addition, it was confirmed that the production of formic acid of the BCF12 strain produced by the (GGGGS) ₁ flexible linker was highest at a concentration of 19 mM.

Example 4. Analysis of formic acid bioconversion performance of CO gas through Resting cell experiment

The cell suspension used for the Resting cell experiment was cultured with a 5L strain using a bio-cultivator, and the CO was continuously supplied to recover the strain at OD 0.9, and centrifuged at 6000 rpm for 30 minutes to isolate and recover only the strain. The wash step was repeated 3 times to remove the remaining components except the strain by washing the obtained strain in the MM1 base (excluding yeast extract) without nutrients. Finally, resting cell experiments were performed using OD ₆₀₀ 0.5 strain suspended as MM1 base.

Resting cell experiment was carried out while putting 6 ml of the OD ₆₀₀ 0.5 strain suspension in 20 ml serum vial, sealing it, filling the headspace with 100% CO gas at 2 bar pressure, and culturing at 80°C, and formic acid productivity and CO consumption over time. And hydrogen productivity.

As a result, it was confirmed that the production of formic acid continued to increase until 48 hours, and CO gas consumption and hydrogen production were continuously maintained. As a result of stoichiometry analysis at the time of 48 hours, it was confirmed that about 10% of CO consumption was converted to formic acid and about 90% was converted to biohydrogen.

Example 5. Isolation and purification of proteins

All protein separation and purification processes were performed under anaerobic conditions. CODH C-term of carbon monoxide:formate oxidoreductase (CFOR) to determine whether CO is converted to formic acid at the protein level. The fusion protein was separated from the his-tag strain nadFd3CoHisL1C1127 using affinity coloumn purification. The mutant nadFd3CoHisL1C1127 strain containing the fusion protein was cultured in 350 ml of MMC (bis-Tris pH 6.5) medium and inoculated in a biological incubator.

5L strain culture was performed with a bio-incubator, and CO was continuously supplied to recover the strain at OD 0.9, and centrifuged at 6000 rpm for 30 minutes to recover only the strain. After securing the strain well in the talon buffer [50mM Tris-HCl (Ph 8.0), 0.1M KCl, 10% glycerol], use a sonicator to crush the strain uniformly and then Talon affinity column. Carbon monoxide:formate oxidoreductase (CFOR) was isolated by using.

Proteins were separated from talon resin using a talon buffer containing final 300 mM imidazol, protein concentration was quantified by Bradford assay, and separated by 12% SDS-PAGE. · The purified protein was confirmed.

As a result, it was observed that CO dehydrogenase (CODH) and formic acid dehydrogenase (Fdh) were separated together with the TON_0540-TON_1017 protein, carbon monoxide:formate oxidoreductase (CFOR). Could. Through these results, it was confirmed that CO dehydrogenase (CO dehydrogenase, CODH) and formic acid dehydrogenase (FDH3) form one new fusion protein through the Fe-S fusion protein (FIG. 8). Reference).

Example 6. Measurement of enzyme activity

CODH enzyme activity and Fdh enzyme activity of carbon monoxide:formate oxidoreductase (CFOR) isolated in Example 5 were confirmed, and formic acid conversion ability of CO gas was performed. Proceeded under conditions.

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

To measure activity, add 2 mM DTT, 10 mM MV and 0.5 ug CFOR protein to 1 ml of 50 mM Tris-Hcl (pH 8.0) buffer in a cuvette sealed with a screw-cap, close the lid, and then seal the headspace. Purging with CO gas, finally filled with 1 bar CO gas to prepare the reaction.

The reaction was carried out by placing the cuvette containing the mixed solution on an 80° C. heat block, and after reacting for 1 minute, the reaction was terminated by transfer to ice, and absorbance was measured at a wavelength of 578 nm using a spectrophotometer.

Measurement of the activity of formic acid dehydrogenase (FDH) was performed in the same way as the activity measurement of CODH enzyme, but 50mM photassium phosphate (pH 7.6) buffer was used, and formic acid was used as an electron donor instead of CO. The reaction was carried out in an 80° C. heat block for 5 minutes.

The activity of the CODH and Fdh enzymes was calculated as the amount of reduction of 2 mmol of methylviologen, which is the same as the amount of oxidation of 1 mmol of CO or formic acid. At this time, the extinction coefficient value is ε ₅₇₈ = 9.7mM ^-1 ·cm ^-1 .

The CO conversion/formic acid production activity of the separated and purified fusion protein is 5 kinds 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) was used as a final 100 ug / ml concentration of the fusion protein was tested in a mixture of 2ml in a 25ml serum vial (serum vial). After injecting CO-CO ₂ mixed gas (CO:CO ₂ =53.5:46.5, vol./vol.) into the 23ml head space, the reaction was performed by incubating at 80°C. After reacting for 5 hours, the concentration of the resulting formic acid was measured by LC.

As a result of formic acid measurement, formation of formic acid was observed under all buffer conditions, and the highest of about 5 mmol/L of formic acid was measured in 150 mM HEPES (pH 7.5). In vitro conditions, final production of formic acid was confirmed only with CO and CO ₂ gas (see FIG. 9 ).

[Table 5]

Through the above results, a new type of fusion protein is formed by fusion of Fe-S protein, which acts as an electron transporter, to form a complex of CO dehydrogenase (CODH) and formic acid dehydrogenase (FDH). It was finally confirmed that the function of converting CO ₂ into formic acid by an enzymatic reaction by electron transfer through Fe-S protein actually works.

Example 7. Measurement of formic acid production activity

After inoculating the strain in 1.5 L of MMC medium, 100% CO gas was purged, a batch culture bioreactor was operated at 80° C., and strain culture was performed under anaerobic conditions. A check valve was installed in the gas outlet part to pressurize and regulate the gas pressure in the reactor.

As a result of operating for 9 hours, formic acid productivity was confirmed to be 150 mmol/L and specific formate production rate was 22 mmol/g-DCW/hr (see FIG. 10).

Through the above example, a strain of New Coliform Thermococcus Onnurinus BCF12, which was shown to have excellent formic acid production ability, was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on September 21, 2018, and was assigned an accession number KCTC 13649BP.

Since the specific parts of the present invention have been described in detail above, for those skilled in the art, this specific technique is only a preferred embodiment, and it is obvious that the scope of the present invention is not limited thereby. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

[Accession Number]

Depository Name: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center

Accession number: KCTC 13649BP

Date of Deposit: September 21, 2018

<110> KOREA OCEAN RESEARCH AND DEVELOPMENT INSTITUTE <120> Noble Fe-S Fusion Protein Acting as an Electron Transfer Chain, Carbon Monoxide:Formate Oxidoreductase Comprising the Fe-S Fusion Protein, Strain from Thermococcus BCF12 Transformed by the Carbon Monoxide:Formate Oxidoreductase, and Use thereof <130> PP180018AN <150> KR 10-2018-0138475 <151> 2018-11-12 <150> KR 10-2018-0138467 <151> 2018-11-12 <150> KR 10-2018-0138462 <151> 2018-11-12 <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> flexible linker of Fe-S Fusion Protein <400> 1 Gly Gly Gly Gly Ser 1 5 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> flexible linker of Fe-S Fusion Protein <400> 2 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> flexible linker of Fe-S Fusion Protein <400> 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 4 <211> 20 <212> PRT <213> Artificial 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motif of Fe-S cluster <400> 10 Cys Xaa Xaa Cys One <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> cystein motif of Fe-S cluster <400> 11 Cys Xaa Xaa Xaa Cys 1 5 <210> 12 <211> 630 <212> PRT <213> Artificial Sequence <220> <223> CO dehydrogenase part of Noble Carbon Monoxide:Formate Oxidoreductase <400> 12 Met Ala Gly Lys Lys Val Pro Ser Lys Gln Val Ser Ile Thr Pro Gly 1 5 10 15 Val Gly Lys Leu Ile Glu Lys Ala Glu Glu Asp Gly Val Lys Thr Ala 20 25 30 Trp His Arg Phe Leu Glu Gln Gln Pro Gln Cys Gly Phe Gly Leu Leu 35 40 45 Gly Val Cys Cys Lys Asn Cys Thr Met Gly Pro Cys Arg Ile Asp Pro 50 55 60 Phe Gly Val Gly Pro Thr Lys Gly Val Cys Gly Ala Asp Ala Asp Thr 65 70 75 80 Ile Val Ala Arg Asn Ile Val Arg Met Ile Ala Ala Gly Thr Ala Gly 85 90 95 His Ser Asp His Ser Arg Asp Val Val His Val Phe Lys Gly Ile Ala 100 105 110 Glu Gly Lys Phe Lys Asp Tyr Lys Leu Thr Asp Val Glu Lys Leu Lys 115 120 125 Glu Leu Ala Lys Ile Leu Gly Val Glu Thr Glu Gly Lys Ser Glu Asn 130 135 140 Glu Ile Ala Leu Glu Val Ala His Ile Leu Glu Met Glu Phe Gly Lys 145 150 155 160 Gln Asp Glu Glu Pro Val Arg Leu Leu Ala Ala Thr Ala Pro Lys Lys 165 170 175 Arg Ile Lys Val Trp Glu Lys Leu Gly Val Leu Pro Arg Ala Ile Asp 180 185 190 Arg Glu Ile Cys Leu Ser Met His Arg Thr His Ile Gly Cys Asp Ala 195 200 205 Asp Pro Ala Ser Leu Leu Leu His Gly Val Arg Thr Ala Leu Ala Asp 210 215 220 Gly Trp Cys Gly Ser Met Met Ala Thr Tyr Leu Ser Asp Ile Leu Phe 225 230 235 240 Gly Thr Pro Lys Pro Ile Lys Ser Leu Ala Asn Leu Gly Val Leu Lys 245 250 255 Glu Asp Met Val Asn Ile Ile Val His Gly His Asn Pro Ile Leu Ser 260 265 270 Met Lys Ile Ala Glu Ile Ala Gln Ser Glu Glu Met Gln Lys Leu Ala 275 280 285 Glu Gln Tyr Gly Ala Lys Gly Ile Asn Val Ala Gly Met Cys Cys Thr 290 295 300 Gly Asn Glu Val Leu Ser Arg Met Gly Val Gln Val Ala Gly Asn Phe 305 310 315 320 Leu Met Gln Glu Leu Ala Ile Ile Thr Gly Ala Val Glu Ala Val Ile 325 330 335 Val Asp Tyr Gln Cys Leu Met Pro Ser Leu Val Asp Val Ala Ser Cys 340 345 350 Tyr His Thr Lys Ile Ile Thr Thr Glu Pro Lys Ala Arg Ile Pro Gly 355 360 365 Ala Ile His Val Glu Phe Glu Pro Glu Lys Ala Asp Glu Ile Ala Lys 370 375 380 Glu Ile Ile Lys Ile Ala Ile Glu Asn Tyr Lys Asn Arg Val Pro Ala 385 390 395 400 Lys Val Tyr Ile Pro Glu His Lys Met Glu Leu Val Ala Gly Phe Ser 405 410 415 Val Glu Ala Ile Leu Glu Ala Leu Gly Gly Thr Leu Glu Pro Leu Ile 420 425 430 Lys Ala Leu Gln Asp Gly Thr Ile Lys Gly Ile Val Gly Ile Val Gly 435 440 445 Cys Asn Asn Pro Arg Val Lys Gln Asn Tyr Gly His Val Thr Leu Ala 450 455 460 Lys Glu Leu Ile Lys Arg Asp Ile Leu Val Val Gly Thr Gly Cys Trp 465 470 475 480 Gly Ile Ala Ala Ala Met His Gly Leu Leu Thr Pro Glu Ala Ala Glu 485 490 495 Met Ala Gly Pro Gly Leu Lys Ala Val Cys Glu Ala Leu Gly Ile Pro 500 505 510 Pro Cys Leu His Met Gly Ser Cys Val Asp Cys Ser Arg Ile Leu Leu 515 520 525 Val Leu Ser Ala Leu Ala Asn Ala Leu Asn Val Asp Ile Ser Asp Leu 530 535 540 Pro Val Ala Gly Ser Ala Pro Glu Trp Met Ser Glu Lys Ala Val Ala 545 550 555 560 Ile Gly Thr Tyr Phe Val Ala Ser Gly Val Phe Thr His Leu Gly Val 565 570 575 Ile Pro Pro Val Leu Gly Ser Gln Lys Val Thr Lys Leu Leu Thr Asp 580 585 590 Asp Ile Glu Asp Leu Leu Gly Gly Lys Phe Tyr Val Glu Thr Asp Pro 595 600 605 Val Lys Ala Ala Glu Thr Ile Tyr Asn Val Ile Ile Glu Lys Arg Lys 610 615 620 Lys Leu Gly Trp Pro Ile 625 630 <210> 13 <211> 379 <212> PRT <213> Artificial Sequence <220> <223> Fe-S Fusion Protein part of Noble Carbon Monoxide:Formate Oxidoreductase <400> 13 Met Glu Lys Lys Leu Phe Ile Asn Leu Gly Arg Cys Ile Ala Cys Arg 1 5 10 15 Ala Cys Glu Val Ala Cys Glu Lys Glu His Gly Ile Ser Phe Ile Thr 20 25 30 Val Tyr Glu Phe Arg Asp Ile Ala Val Pro Leu Asn Cys Arg His Cys 35 40 45 Glu Lys Ala Pro Cys Ile Glu Val Cys Pro Thr Lys Ala Ile Tyr Arg 50 55 60 Asp Glu Asp Gly Ala Val Val Ile Asp Glu Ser Lys Cys Ile Gly Cys 65 70 75 80 Tyr Met Cys Ser Ala Val Cys Pro Tyr Ala Ile Pro Ile Val Asp Pro 85 90 95 Ile Lys Glu Leu Ala Val Lys Cys Asp Leu Cys Ala Glu Arg Arg Lys 100 105 110 Glu Gly Arg Asp Pro Leu Cys Ala Ala Val Cys Pro Thr Asp Ala Ile 115 120 125 Ile Tyr Ala Asp Leu Asn Glu Leu Met Glu Glu Lys Arg Arg Arg Lys 130 135 140 Ala Glu Arg Ile Val Glu Ala Gln Arg Lys Ala Val Glu Thr Leu Ala 145 150 155 160 Tyr Phe Gly Gly Gly Gly Gly Ser Pro Ala Phe Ser Gly Ser Asn Met 165 170 175 Glu Lys Leu Thr Ile Tyr Ile Asn Pro Glu Arg Cys Thr Gly Cys Arg 180 185 190 Ala Cys Glu Ile Ala Cys Ala Val Glu His Ser Met Ser Lys Asn Leu 195 200 205 Phe Gly Ala Ile Phe Glu Lys Pro Thr Pro Lys Pro Arg Leu Gln Val 210 215 220 Val Val Ala Asp Phe Phe Asn Val Pro Met Arg Cys Gln His Cys Glu 225 230 235 240 Asp Ala Pro Cys Met Glu Ala Cys Pro Thr Gly Ala Ile Ser Arg Thr 245 250 255 Lys Glu Gly Phe Val Val Leu Asn Ala Asn Lys Cys Ile Gly Cys Leu 260 265 270 Met Cys Val Met Ala Cys Pro Phe Gly His Pro Lys Phe Glu Pro Glu 275 280 285 Tyr Lys Ala Val Ile Lys Cys Asp Ser Cys Val Asp Arg Val Arg Glu 290 295 300 Gly Lys Glu Pro Ala Cys Val Glu Ala Cys Pro Thr Arg Ala Leu Lys 305 310 315 320 Phe Gly Thr Leu Gly Glu Ile Leu Glu Glu Val Arg Lys Glu Lys Ala 325 330 335 Glu Ser Leu Ile Ser Gly Leu Lys Ser Gln Gly Met Val Tyr Met Lys 340 345 350 Pro Val Ser Glu Ser Lys Lys Lys Glu Asp Leu Val Arg Pro Met Asp 355 360 365 Leu Tyr Leu Ala Tyr Ser Asn Val Val Trp Tyr 370 375 <210> 14 <211> 680 <212> PRT <213> Artificial Sequence <220> <223> Fomrate dehydrogenase part of Noble Carbon Monoxide:Formate Oxidoreductase <400> 14 Met Glu Glu Phe Lys Ile Gly Leu Cys Pro Tyr Cys Gly Met Gly Cys 1 5 10 15 Arg Phe Tyr Ile Lys Thr Leu Asn Gly Gln Pro Ile Gly Ile Glu Pro 20 25 30 Tyr Pro Gly Gly Val Asn Glu Gly Lys Leu Cys Pro Lys Gly Val Ala 35 40 45 Ala Val Asp Phe Leu Arg His Lys Asp Arg Leu Lys Lys Pro Leu Lys 50 55 60 Arg Thr Glu Asn Gly Phe Val Glu Ile Ser Trp Glu Gln Ala Ile Lys 65 70 75 80 Glu Ile Ala Glu Lys Leu Leu Glu Ile Arg Glu Lys Tyr Gly Pro Asp 85 90 95 Thr Leu Gly Phe Phe Ser Ser Ala Arg Cys Ser Asn Glu Glu Asn Tyr 100 105 110 Leu Leu Gln Lys Ile Ala Arg Leu Leu Gly Thr Asn Asn Val Asp His 115 120 125 Cys Ala Arg Leu Cys His Ala Ser Thr Val Val Gly Leu Ala Gln Thr 130 135 140 Val Gly Ala Ala Ala Gln Ser Gly Ser Tyr Thr Asp Ile Pro Lys Ala 145 150 155 160 Lys Val Leu Leu Ile Trp Gly Tyr Asn Pro Ser Glu Thr His Pro Val 165 170 175 Leu Met Arg Tyr Ile Leu Arg Ala Arg Asp Asn Gly Ala Lys Ile Ile 180 185 190 Val Val Asp Pro Arg Lys Thr Arg Thr Val Trp Phe Ala Asp Met His 195 200 205 Leu Gln Leu Lys Pro Gly Thr Asp Ile Val Leu Ala Asn Ala Met Met 210 215 220 His Val Ile Ile Glu Glu Arg Leu Tyr Asp Arg Glu Phe Ile Met Asn 225 230 235 240 Arg Thr Lys Gly Phe Glu Lys Leu Ile Ala Ala Val Gln Lys Tyr Thr 245 250 255 Pro Glu Tyr Ala Glu Glu Ile Thr Gly Val Pro Ala Lys Leu Ile Arg 260 265 270 Glu Ala Ala Ile Thr Phe Ala Thr Ala Gly Arg Gly Ile Val Met Trp 275 280 285 Ala Met Gly Leu Thr Gln His Val Thr Gly Ala Ala Asn Val Lys Ala 290 295 300 Leu Ala Asp Leu Ala Leu Ile Cys Gly Tyr Val Gly Arg Glu Gly Thr 305 310 315 320 Gly Leu Phe Pro Met Arg Gly Gln Asn Asn Val Gln Gly Ala Cys Asp 325 330 335 Met Ala Ala Leu Pro Asn Val Phe Pro Gly Tyr Gln Lys Val Thr Asp 340 345 350 Asp Glu Lys Arg Lys His Val Ala Glu Ile Trp Gly Val Glu Asp Leu 355 360 365 Pro Ser Lys Pro Gly Leu Thr Ile Pro Glu Met Ile Asp Ala Ala Ala 370 375 380 Lys Gly Glu Leu Lys Ala Leu Tyr Ile Met Gly Glu Asn Pro Val Met 385 390 395 400 Ser Asp Pro Asn Thr Lys His Val Ile Glu Ala Leu Lys Asn Leu Glu 405 410 415 Leu Leu Val Val Gln Asp Ile Phe Leu Thr Glu Thr Ala Glu Leu Ala 420 425 430 His Tyr Val Leu Pro Ala Ala Ala Tyr Ala Glu Lys Glu Gly Ser Phe 435 440 445 Thr Ala Ser Glu Arg Arg Val Gln Trp Asn Phe Lys Ala Ile Glu Pro 450 455 460 Pro Gly Glu Ala Lys Pro Asp Trp Glu Ile Leu Thr Met Leu Gly Lys 465 470 475 480 Ala Leu Gly Leu Pro Lys Phe Asp Tyr Ser Asp Val Glu Asp Ile Thr 485 490 495 Arg Glu Ile Thr Leu Val Ala Pro Gln Tyr Arg Gly Ile Thr Pro Glu 500 505 510 Arg Leu Lys Arg Glu Val Met Gly Val Gln Trp Pro Cys Pro Ser Glu 515 520 525 Asp His Pro Gly Thr Pro Arg Leu His Val Glu Arg Phe Ala Thr Pro 530 535 540 Asp Gly Lys Ala Asn Ile Ile Pro Val Glu Phe Lys Pro Pro Ala Glu 545 550 555 560 Glu Pro Asp Glu Glu Tyr Pro Phe Ile Leu Thr Thr Phe Arg Ile Val 565 570 575 Gly Gln Tyr His Thr Leu Thr Met Ser Asn Arg Ser Glu Ser Leu Lys 580 585 590 Lys Arg Trp Ser Ser Pro Tyr Ala Gln Ile Ser Pro Glu Asp Ala Lys 595 600 605 Lys Leu Gly Ile Gln Asp Gly Glu Met Ile Arg Ile Val Thr Arg Arg 610 615 620 Gly Ser Tyr Thr Cys Arg Ala Val Val Thr Glu Asp Val Ser Glu Gly 625 630 635 640 Val Ile Ala Val Pro Trp His Trp Gly Ala Asn Ile Leu Thr Asn Asp 645 650 655 Val Leu Asp Pro Glu Ala Lys Ile Pro Glu Leu Lys Val Ala Ala Cys 660 665 670 Arg Val Glu Lys Ile Gly Gly Cys 675 680 <210> 15 <211> 1689 <212> PRT <213> Artificial Sequence <220> <223> Noble Carbon Monoxide:Formate Oxidoreductase <400> 15 Met Glu Glu Phe Lys Ile Gly Leu Cys Pro Tyr Cys Gly Met Gly Cys 1 5 10 15 Arg Phe Tyr Ile Lys Thr Leu Asn Gly Gln Pro Ile Gly Ile Glu Pro 20 25 30 Tyr Pro Gly Gly Val Asn Glu Gly Lys Leu Cys Pro Lys Gly Val Ala 35 40 45 Ala Val Asp Phe Leu Arg His Lys Asp Arg Leu Lys Lys Pro Leu Lys 50 55 60 Arg Thr Glu Asn Gly Phe Val Glu Ile Ser Trp Glu Gln Ala Ile Lys 65 70 75 80 Glu Ile Ala Glu Lys Leu Leu Glu Ile Arg Glu Lys Tyr Gly Pro Asp 85 90 95 Thr Leu Gly Phe Phe Ser Ser Ala Arg Cys Ser Asn Glu Glu Asn Tyr 100 105 110 Leu Leu Gln Lys Ile Ala Arg Leu Leu Gly Thr Asn Asn Val Asp His 115 120 125 Cys Ala Arg Leu Cys His Ala Ser Thr Val Val Gly Leu Ala Gln Thr 130 135 140 Val Gly Ala Ala Ala Gln Ser Gly Ser Tyr Thr Asp Ile Pro Lys Ala 145 150 155 160 Lys Val Leu Leu Ile Trp Gly Tyr Asn Pro Ser Glu Thr His Pro Val 165 170 175 Leu Met Arg Tyr Ile Leu Arg Ala Arg Asp Asn Gly Ala Lys Ile Ile 180 185 190 Val Val Asp Pro Arg Lys Thr Arg Thr Val Trp Phe Ala Asp Met His 195 200 205 Leu Gln Leu Lys Pro Gly Thr Asp Ile Val Leu Ala Asn Ala Met Met 210 215 220 His Val Ile Ile Glu Glu Arg Leu Tyr Asp Arg Glu Phe Ile Met Asn 225 230 235 240 Arg Thr Lys Gly Phe Glu Lys Leu Ile Ala Ala Val Gln Lys Tyr Thr 245 250 255 Pro Glu Tyr Ala Glu Glu Ile Thr Gly Val Pro Ala Lys Leu Ile Arg 260 265 270 Glu Ala Ala Ile Thr Phe Ala Thr Ala Gly Arg Gly Ile Val Met Trp 275 280 285 Ala Met Gly Leu Thr Gln His Val Thr Gly Ala Ala Asn Val Lys Ala 290 295 300 Leu Ala Asp Leu Ala Leu Ile Cys Gly Tyr Val Gly Arg Glu Gly Thr 305 310 315 320 Gly Leu Phe Pro Met Arg Gly Gln Asn Asn Val Gln Gly Ala Cys Asp 325 330 335 Met Ala Ala Leu Pro Asn Val Phe Pro Gly Tyr Gln Lys Val Thr Asp 340 345 350 Asp Glu Lys Arg Lys His Val Ala Glu Ile Trp Gly Val Glu Asp Leu 355 360 365 Pro Ser Lys Pro Gly Leu Thr Ile Pro Glu Met Ile Asp Ala Ala Ala 370 375 380 Lys Gly Glu Leu Lys Ala Leu Tyr Ile Met Gly Glu Asn Pro Val Met 385 390 395 400 Ser Asp Pro Asn Thr Lys His Val Ile Glu Ala Leu Lys Asn Leu Glu 405 410 415 Leu Leu Val Val Gln Asp Ile Phe Leu Thr Glu Thr Ala Glu Leu Ala 420 425 430 His Tyr Val Leu Pro Ala Ala Ala Tyr Ala Glu Lys Glu Gly Ser Phe 435 440 445 Thr Ala Ser Glu Arg Arg Val Gln Trp Asn Phe Lys Ala Ile Glu Pro 450 455 460 Pro Gly Glu Ala Lys Pro Asp Trp Glu Ile Leu Thr Met Leu Gly Lys 465 470 475 480 Ala Leu Gly Leu Pro Lys Phe Asp Tyr Ser Asp Val Glu Asp Ile Thr 485 490 495 Arg Glu Ile Thr Leu Val Ala Pro Gln Tyr Arg Gly Ile Thr Pro Glu 500 505 510 Arg Leu Lys Arg Glu Val Met Gly Val Gln Trp Pro Cys Pro Ser Glu 515 520 525 Asp His Pro Gly Thr Pro Arg Leu His Val Glu Arg Phe Ala Thr Pro 530 535 540 Asp Gly Lys Ala Asn Ile Ile Pro Val Glu Phe Lys Pro Pro Ala Glu 545 550 555 560 Glu Pro Asp Glu Glu Tyr Pro Phe Ile Leu Thr Thr Phe Arg Ile Val 565 570 575 Gly Gln Tyr His Thr Leu Thr Met Ser Asn Arg Ser Glu Ser Leu Lys 580 585 590 Lys Arg Trp Ser Ser Pro Tyr Ala Gln Ile Ser Pro Glu Asp Ala Lys 595 600 605 Lys Leu Gly Ile Gln Asp Gly Glu Met Ile Arg Ile Val Thr Arg Arg 610 615 620 Gly Ser Tyr Thr Cys Arg Ala Val Val Thr Glu Asp Val Ser Glu Gly 625 630 635 640 Val Ile Ala Val Pro Trp His Trp Gly Ala Asn Ile Leu Thr Asn Asp 645 650 655 Val Leu Asp Pro Glu Ala Lys Ile Pro Glu Leu Lys Val Ala Ala Cys 660 665 670 Arg Val Glu Lys Ile Gly Gly Cys Met Glu Lys Lys Leu Phe Ile Asn 675 680 685 Leu Gly Arg Cys Ile Ala Cys Arg Ala Cys Glu Val Ala Cys Glu Lys 690 695 700 Glu His Gly Ile Ser Phe Ile Thr Val Tyr Glu Phe Arg Asp Ile Ala 705 710 715 720 Val Pro Leu Asn Cys Arg His Cys Glu Lys Ala Pro Cys Ile Glu Val 725 730 735 Cys Pro Thr Lys Ala Ile Tyr Arg Asp Glu Asp Gly Ala Val Val Ile 740 745 750 Asp Glu Ser Lys Cys Ile Gly Cys Tyr Met Cys Ser Ala Val Cys Pro 755 760 765 Tyr Ala Ile Pro Ile Val Asp Pro Ile Lys Glu Leu Ala Val Lys Cys 770 775 780 Asp Leu Cys Ala Glu Arg Arg Lys Glu Gly Arg Asp Pro Leu Cys Ala 785 790 795 800 Ala Val Cys Pro Thr Asp Ala Ile Ile Tyr Ala Asp Leu Asn Glu Leu 805 810 815 Met Glu Glu Lys Arg Arg Arg Lys Ala Glu Arg Ile Val Glu Ala Gln 820 825 830 Arg Lys Ala Val Glu Thr Leu Ala Tyr Phe Gly Gly Gly Gly Gly Ser 835 840 845 Pro Ala Phe Ser Gly Ser Asn Met Glu Lys Leu Thr Ile Tyr Ile Asn 850 855 860 Pro Glu Arg Cys Thr Gly Cys Arg Ala Cys Glu Ile Ala Cys Ala Val 865 870 875 880 Glu His Ser Met Ser Lys Asn Leu Phe Gly Ala Ile Phe Glu Lys Pro 885 890 895 Thr Pro Lys Pro Arg Leu Gln Val Val Val Ala Asp Phe Phe Asn Val 900 905 910 Pro Met Arg Cys Gln His Cys Glu Asp Ala Pro Cys Met Glu Ala Cys 915 920 925 Pro Thr Gly Ala Ile Ser Arg Thr Lys Glu Gly Phe Val Val Leu Asn 930 935 940 Ala Asn Lys Cys Ile Gly Cys Leu Met Cys Val Met Ala Cys Pro Phe 945 950 955 960 Gly His Pro Lys Phe Glu Pro Glu Tyr Lys Ala Val Ile Lys Cys Asp 965 970 975 Ser Cys Val Asp Arg Val Arg Glu Gly Lys Glu Pro Ala Cys Val Glu 980 985 990 Ala Cys Pro Thr Arg Ala Leu Lys Phe Gly Thr Leu Gly Glu Ile Leu 995 1000 1005 Glu Glu Val Arg Lys Glu Lys Ala Glu Ser Leu Ile Ser Gly Leu Lys 1010 1015 1020 Ser Gln Gly Met Val Tyr Met Lys Pro Val Ser Glu Ser Lys Lys Lys 1025 1030 1035 1040 Glu Asp Leu Val Arg Pro Met Asp Leu Tyr Leu Ala Tyr Ser Asn Val 1045 1050 1055 Val Trp Tyr Met Ala Gly Lys Lys Val Pro Ser Lys Gln Val Ser Ile 1060 1065 1070 Thr Pro Gly Val Gly Lys Leu Ile Glu Lys Ala Glu Glu Asp Gly Val 1075 1080 1085 Lys Thr Ala Trp His Arg Phe Leu Glu Gln Gln Pro Gln Cys Gly Phe 1090 1095 1100 Gly Leu Leu Gly Val Cys Cys Lys Asn Cys Thr Met Gly Pro Cys Arg 1105 1110 1115 1120 Ile Asp Pro Phe Gly Val Gly Pro Thr Lys Gly Val Cys Gly Ala Asp 1125 1130 1135 Ala Asp Thr Ile Val Ala Arg Asn Ile Val Arg Met Ile Ala Ala Gly 1140 1145 1150 Thr Ala Gly His Ser Asp His Ser Arg Asp Val Val His Val Phe Lys 1155 1160 1165 Gly Ile Ala Glu Gly Lys Phe Lys Asp Tyr Lys Leu Thr Asp Val Glu 1170 1175 1180 Lys Leu Lys Glu Leu Ala Lys Ile Leu Gly Val Glu Thr Glu Gly Lys 1185 1190 1195 1200 Ser Glu Asn Glu Ile Ala Leu Glu Val Ala His Ile Leu Glu Met Glu 1205 1210 1215 Phe Gly Lys Gln Asp Glu Glu Pro Val Arg Leu Leu Ala Ala Thr Ala 1220 1225 1230 Pro Lys Lys Arg Ile Lys Val Trp Glu Lys Leu Gly Val Leu Pro Arg 1235 1240 1245 Ala Ile Asp Arg Glu Ile Cys Leu Ser Met His Arg Thr His Ile Gly 1250 1255 1260 Cys Asp Ala Asp Pro Ala Ser Leu Leu Leu His Gly Val Arg Thr Ala 1265 1270 1275 1280 Leu Ala Asp Gly Trp Cys Gly Ser Met Met Ala Thr Tyr Leu Ser Asp 1285 1290 1295 Ile Leu Phe Gly Thr Pro Lys Pro Ile Lys Ser Leu Ala Asn Leu Gly 1300 1305 1310 Val Leu Lys Glu Asp Met Val Asn Ile Ile Val His Gly His Asn Pro 1315 1320 1325 Ile Leu Ser Met Lys Ile Ala Glu Ile Ala Gln Ser Glu Glu Met Gln 1330 1335 1340 Lys Leu Ala Glu Gln Tyr Gly Ala Lys Gly Ile Asn Val Ala Gly Met 1345 1350 1355 1360 Cys Cys Thr Gly Asn Glu Val Leu Ser Arg Met Gly Val Gln Val Ala 1365 1370 1375 Gly Asn Phe Leu Met Gln Glu Leu Ala Ile Ile Thr Gly Ala Val Glu 1380 1385 1390 Ala Val Ile Val Asp Tyr Gln Cys Leu Met Pro Ser Leu Val Asp Val 1395 1400 1405 Ala Ser Cys Tyr His Thr Lys Ile Ile Thr Thr Glu Pro Lys Ala Arg 1410 1415 1420 Ile Pro Gly Ala Ile His Val Glu Phe Glu Pro Glu Lys Ala Asp Glu 1425 1430 1435 1440 Ile Ala Lys Glu Ile Ile Lys Ile Ala Ile Glu Asn Tyr Lys Asn Arg 1445 1450 1455 Val Pro Ala Lys Val Tyr Ile Pro Glu His Lys Met Glu Leu Val Ala 1460 1465 1470 Gly Phe Ser Val Glu Ala Ile Leu Glu Ala Leu Gly Gly Thr Leu Glu 1475 1480 1485 Pro Leu Ile Lys Ala Leu Gln Asp Gly Thr Ile Lys Gly Ile Val Gly 1490 1495 1500 Ile Val Gly Cys Asn Asn Pro Arg Val Lys Gln Asn Tyr Gly His Val 1505 1510 1515 1520 Thr Leu Ala Lys Glu Leu Ile Lys Arg Asp Ile Leu Val Val Gly Thr 1525 1530 1535 Gly Cys Trp Gly Ile Ala Ala Ala Met His Gly Leu Leu Thr Pro Glu 1540 1545 1550 Ala Ala Glu Met Ala Gly Pro Gly Leu Lys Ala Val Cys Glu Ala Leu 1555 1560 1565 Gly Ile Pro Pro Cys Leu His Met Gly Ser Cys Val Asp Cys Ser Arg 1570 1575 1580 Ile Leu Leu Val Leu Ser Ala Leu Ala Asn Ala Leu Asn Val Asp Ile 1585 1590 1595 1600 Ser Asp Leu Pro Val Ala Gly Ser Ala Pro Glu Trp Met Ser Glu Lys 1605 1610 1615 Ala Val Ala Ile Gly Thr Tyr Phe Val Ala Ser Gly Val Phe Thr His 1620 1625 1630 Leu Gly Val Ile Pro Pro Val Leu Gly Ser Gln Lys Val Thr Lys Leu 1635 1640 1645 Leu Thr Asp Asp Ile Glu Asp Leu Leu Gly Gly Lys Phe Tyr Val Glu 1650 1655 1660 Thr Asp Pro Val Lys Ala Ala Glu Thr Ile Tyr Asn Val Ile Ile Glu 1665 1670 1675 1680 Lys Arg Lys Lys Leu Gly Trp Pro Ile 1685 <210> 16 <211> 5076 <212> DNA <213> Artificial Sequence <220> <223> Noble Carbon Monoxide:Formate Oxidoreductase <400> 16 atggaggagt ttaagattgg cctgtgccca tactgtggga tggggtgcag gttttacata 60 aagactctta acgggcagcc cataggaata gagccgtatc ccggtggtgt taatgaagga 120 aagctctgtc caaagggtgt cgccgccgtt gacttcctca gacacaaaga taggctgaaa 180 aagccgctca agagaactga aaacggcttc gtcgagataa gctgggaaca ggcgataaag 240 gagattgctg aaaagcttct ggagatacgc gagaagtacg ggccggatac gttaggcttc 300 ttctcaagtg cccgttgttc caacgaggag aactacctcc tgcagaaaat agcccgcctt 360 ctgggcacca acaacgtcga ccactgcgcg aggctctgtc acgcctcaac ggtcgtcggt 420 cttgctcaga cggttggcgc tgccgctcag agcggctcct acacggacat acccaaggct 480 aaggtactcc tgatatgggg atacaacccg tcagaaaccc acccggttct catgcgctac 540 atcctccgcg cgagggacaa cggggccaag ataatcgtcg tagatccgag gaagacgagg 600 actgtctggt tcgccgatat gcacctccag cttaagcctg gaacggacat agtcctagcc 660 aacgccatga tgcacgtcat cattgaagaa aggctctatg acagggagtt catcatgaac 720 cggacgaagg gctttgagaa gctcatagca gctgtccaga agtacacgcc agaatacgcc 780 gaggaaataa ccggtgttcc cgccaagctc atcagagaag ccgctataac ctttgctact 840 gccggacggg gcatcgtgat gtgggcaatg ggactgacgc agcacgtcac tggggcggcc 900 aacgttaagg ccctcgctga tctggctctg atctgtggct acgtcggaag agaaggaaca 960 ggtctcttcc cgatgcgcgg tcagaacaat gttcagggag catgtgacat ggcagccttg 1020 ccaaacgtct ttccaggcta tcagaaggta actgacgacg agaagaggaa gcacgtggcg 1080 gaaatttggg gcgttgaaga tctgccctcg aagccgggcc ttactattcc agagatgatt 1140 gatgcggctg ctaaaggcga gttgaaggca ctctacataa tgggcgagaa tccggtcatg 1200 agcgatccga acacgaagca cgttatcgag gctctcaaga acctcgaact tctcgttgtt 1260 caggatatat tcctcaccga aacggccgag ctggctcact acgtgctccc agcagccgca 1320 tacgccgaga aggaaggatc attcaccgcg agcgagaggc gcgtccagtg gaacttcaag 1380 gcgattgagc cgccaggaga agccaaaccg gactgggaga tactgacgat gcttggaaag 1440 gctctcggcc tgccaaagtt cgactactca gacgttgaag atattacgag ggagataacc 1500 ctcgttgctc cgcagtaccg tgggataacc cccgagaggc tcaagcgaga ggttatgggt 1560 gtgcagtggc cgtgcccgag cgaggatcat cctggaacgc cgaggctgca cgtcgagcgc 1620 ttcgccaccc ccgacggaaa ggccaacata atccccgtag agttcaagcc acctgcagaa 1680 gagcccgatg aggagtaccc attcatactg acgacattcc gcatcgtcgg ccagtaccac 1740 acactcacga tgagtaacag gagtgaaagc ttgaagaagc gctggtccag cccgtacgcc 1800 cagataagtc cggaagatgc aaagaagctg ggtatacagg atggtgaaat gataaggata 1860 gttacgagac gtggaagcta cacctgcagg gcggtcgtta ctgaagatgt ctcggaaggg 1920 gtgatcgcag ttccgtggca ctggggggcc aatatactca cgaacgatgt cctcgatcca 1980 gaagcaaaga ttcccgagct gaaggtggcc gcatgtaggg tggagaagat tggggggtgc 2040 tgaatggaga aaaagctgtt cataaacctc gggcgctgca ttgcctgccg cgcctgcgag 2100 gtggcctgtg agaaggagca cggaatttca ttcatcacgg tctatgagtt cagggacata 2160 gcggttcccc tcaactgccg ccactgtgag aaggctccgt gtatcgaagt ctgcccgacg 2220 aaggccatct atcgcgacga agatggcgca gttgtgatag acgagtccaa gtgtatcggc 2280 tgctacatgt gttcggccgt ctgcccctac gcgattccga tagttgaccc gataaaggag 2340 ctggctgtga agtgtgacct atgtgccgaa agaaggaagg agggcagaga tccgctctgc 2400 gctgcggtct gtcccaccga tgcgataatc tacgctgacc tcaacgagct gatggaagag 2460 aagaggaggc gcaaggccga gcgcatcgtc gaagcccaga ggaaggcggt cgaaacgctc 2520 gcctacttcg ggggcggcgg aggcagccca gctttttccg gttccaacat ggagaagctt 2580 acaatttaca taaatccaga gagatgcacg gggtgcaggg cctgcgaaat tgcctgtgca 2640 gttgaacatt caatgagcaa aaacctcttt ggcgcaattt ttgaaaaacc aacccccaaa 2700 ccccgactcc aagttgttgt cgccgacttc tttaatgttc caatgagatg ccagcactgt 2760 gaggacgctc cctgtatgga ggcctgccca acaggagcga tctcaaggac caaagaaggc 2820 tttgttgtcc ttaacgccaa caagtgcata ggctgtctca tgtgtgtgat ggcctgtcca 2880 tttggccatc ccaagttcga gcccgaatac aaggctgtga taaaatgcga cagttgtgtt 2940 gatagggtca gagaaggcaa agagccagca tgtgtcgagg cctgtccaac tagagccctg 3000 aagttcggga ctctcggcga aatactggaa gaggttagaa aggagaaggc agagagtctc 3060 atatctgggc tgaaatcgca ggggatggtc tacatgaagc ccgtctccga gtcaaagaag 3120 aaagaggatc ttgttagacc tatggatctg tatcttgcct attcaaatgt agtgtggtat 3180 tgaatggccg gaaagaaggt tccctcaaag caagtctcca taactccagg tgttggaaag 3240 cttattgaga aagccgagga ggatggggtc aagactgcct ggcacagatt tttggagcag 3300 cagcctcagt gtggattcgg tctcttaggt gtctgctgta agaactgtac aatgggacca 3360 tgtagaatcg atccgtttgg ggttggccca actaagggag tttgtggtgc ggatgcagat 3420 acaatagtag caaggaacat tgtaagaatg atagcggctg gtactgccgg tcacagcgat 3480 cactcaagag atgtagtcca tgtattcaag ggcattgctg aaggaaagtt caaggactat 3540 aaactaacag atgttgaaaa gctcaaagag ctggctaaga ttctgggtgt cgaaacagag 3600 ggcaagagcg aaaatgaaat tgcattggaa gtcgcccaca ttcttgagat ggagttcgga 3660 aaacaggatg aggagccagt aagattactt gcagcaacag caccaaagaa gaggattaag 3720 gtctgggaga agctaggagt cttaccaaga gccatcgaca gggagatatg tctcagtatg 3780 cacagaaccc acataggctg tgatgcagac cctgcaagcc ttctactgca tggtgtgagg 3840 actgccctgg ccgacggctg gtgcggctca atgatggcca cttatctgag cgacattctc 3900 tttggaacac caaagccgat aaagtcgctg gcgaacctgg gagtcttgaa ggaagacatg 3960 gtcaacataa tcgttcacgg ccacaacccg attctctcca tgaaaatagc agagattgcc 4020 cagagtgaag agatgcagaa gcttgcagag cagtacggag caaagggaat taacgttgct 4080 ggaatgtgct gtaccggaaa cgaagttctc tcaagaatgg gagttcaggt cgctggaaac 4140 ttcctaatgc aagagctggc gattataact ggtgcagttg aggccgtgat agttgactac 4200 cagtgcctaa tgccctcatt agttgatgtc gcttcatgtt accacactaa gataataact 4260 actgagccaa aggctcgcat tccgggagca atacacgtcg aatttgaacc tgagaaagcg 4320 gacgagatcg ccaaagagat catcaagatt gcaattgaga actataagaa cagagttccg 4380 gcaaaagtct acattccaga gcacaagatg gaattggttg ctggatttag tgtcgaggca 4440 atacttgaag cccttggtgg aacactggag cccctcataa aagccctcca ggacggaaca 4500 ataaagggaa tcgtcggaat cgttggatgt aacaatccaa gggtcaagca gaactacggt 4560 cacgtcacct tggccaagga gctcatcaag agggacatcc tggttgttgg aactggttgc 4620 tggggaattg ctgcagcaat gcatggatta ctaacccccg aagcagctga aatggccggt 4680 ccagggctga aggcagtatg cgaagcgctc ggaattccac catgcctgca catgggaagc 4740 tgtgttgact gttcgagaat cctgctggtc ttgagtgccc ttgccaatgc tctgaatgtt 4800 gacatttcag acttgccagt tgctggctct gctccagaat ggatgagcga gaaggcagtg 4860 gcaataggaa cctacttcgt tgcaagcggc gtcttcacgc acttgggagt tatcccacca 4920 gtccttggaa gccagaaggt taccaaactc cttacggatg acatcgagga tctccttgga 4980 gggaagttct acgttgagac agatccagtg aaagcggcag aaacaatata caacgtgata 5040 attgagaaga ggaaaaaact tggatggccc atctaa 5076 <210> 17 <211> 1140 <212> DNA <213> Artificial Sequence <220> <223> Fe-S Fusion Protein <400> 17 atggagaaaa agctgttcat aaacctcggg cgctgcattg cctgccgcgc ctgcgaggtg 60 gcctgtgaga aggagcacgg aatttcattc atcacggtct atgagttcag ggacatagcg 120 gttcccctca actgccgcca ctgtgagaag gctccgtgta tcgaagtctg cccgacgaag 180 gccatctatc gcgacgaaga tggcgcagtt gtgatagacg agtccaagtg tatcggctgc 240 tacatgtgtt cggccgtctg cccctacgcg attccgatag ttgacccgat aaaggagctg 300 gctgtgaagt gtgacctatg tgccgaaaga aggaaggagg gcagagatcc gctctgcgct 360 gcggtctgtc ccaccgatgc gataatctac gctgacctca acgagctgat ggaagagaag 420 aggaggcgca aggccgagcg catcgtcgaa gcccagagga aggcggtcga aacgctcgcc 480 tacttcgggg gcggcggagg cagcccagct ttttccggtt ccaacatgga gaagcttaca 540 atttacataa atccagagag atgcacgggg tgcagggcct gcgaaattgc ctgtgcagtt 600 gaacattcaa tgagcaaaaa cctctttggc gcaatttttg aaaaaccaac ccccaaaccc 660 cgactccaag ttgttgtcgc cgacttcttt aatgttccaa tgagatgcca gcactgtgag 720 gacgctccct gtatggaggc ctgcccaaca ggagcgatct caaggaccaa agaaggcttt 780 gttgtcctta acgccaacaa gtgcataggc tgtctcatgt gtgtgatggc ctgtccattt 840 ggccatccca agttcgagcc cgaatacaag gctgtgataa aatgcgacag ttgtgttgat 900 agggtcagag aaggcaaaga gccagcatgt gtcgaggcct gtccaactag agccctgaag 960 ttcgggactc tcggcgaaat actggaagag gttagaaagg agaaggcaga gagtctcata 1020 tctgggctga aatcgcaggg gatggtctac atgaagcccg tctccgagtc aaagaagaaa 1080 gaggatcttg ttagacctat ggatctgtat cttgcctatt caaatgtagt gtggtattga 1140 1140

Claims (6)

Thermococcus-derived strain BCF12 (Accession No.: KCTC 13649BP). According to claim 1,
The strain is a thermococcus-derived new strain BCF12, characterized in that the carbon monoxide:formate oxidoreductase (CFOR) gene has been transformed (Accession No.: KCTC 13649BP). According to claim 2,
The carbon monoxide: formic acid oxidoreductase (carbon monoxide: formate oxidoreductase, CFOR) acts as an electron transport chain that serves as a pathway through which electrons move, and two or more Fe-S proteins are selected from SEQ ID NOs: 1 to 6 Fe-S fusion protein formed by covalent bonding through a flexible linker having an amino acid sequence of, and CO dehydrogenase (CODH) operably linked to the C-terminus of the Fe-S fusion protein, and the Fe- It contains a formic acid dehydrogenase (Fdh) operably linked to the N-terminus of the S fusion protein, and electrons generated from the CO dehydrogenase pass through the Fe-S fusion protein to form formate dehydrogenase (fomrate dehydrogenase, Fdh), characterized by being delivered to Thermococcus strain BCF12 (Accession No.: KCTC 13649BP). According to claim 2,
The carbon monoxide: formic acid oxidoreductase (carbon monoxide: formate oxidoreductase, CFOR) is a thermococcus-derived strain BCF12, characterized by having the amino acid sequence of SEQ ID NO: 15 (Accession No.: KCTC 13649BP). Comprising the step of expressing a carbon monoxide: formic acid oxidoreductase (CFOR) by culturing the new strain BCF12 (Accession No.: KCTC 13649BP) from Thermococcus according to any one of claims 1 to 4 Manufacturing method of carbon monoxide:formate oxidoreductase (CFOR). Method of preparing formic acid comprising the following steps:
(a) Carbon monoxide according to the production method of any one of claims 1 to 4, wherein the thermococcus-derived strain BCF12 (Accession No.: KCTC 13649BP) or claim 5: carbon monoxide:formate oxidoreductase, Supplying CO gas in the presence of CFOR) to synthesize formic acid from the CO gas; And
(b) recovering the synthesized formic acid.

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