KR101524340B1 - Recombinant microorganisms, and formate synthesis using recombinant formate dehydrogenase expressed by the same - Google Patents

Abstract

The present invention relates to a recombinant microorganism, a method for producing formic acid using the formate dehydrogenase expressed therefrom, and more particularly to a method for producing formic acid using a nucleotide sequence encoding an acidic formate dehydrogenase derived from a strain of Thiobacillus sp. The present invention relates to a method for producing formic acid by reducing carbon dioxide under an acidic condition of less than pH 7 using a recombinant microorganism and a formate dehydrogenase expressed therefrom to produce formic acid.
According to the present invention, carbon dioxide is reduced in an acidic condition, and the amount of formic acid to produce formic acid is remarkably increased, thereby remarkably reducing carbon dioxide which causes various environmental problems.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a recombinant microorganism and a method for producing formic acid using the recombinant formate dehydrogenase expressed therefrom,

The present invention relates to a recombinant microorganism, a method for producing formic acid using the formate dehydrogenase expressed therefrom, and more particularly, to a recombinant microorganism comprising a nucleotide sequence encoding an acidic formate dehydrogenase, formate dehydrogenase The present invention relates to a process for the production of formic acid, which produces formic acid by reducing carbon dioxide under an acidic condition of less than pH 7 using an enzyme.

Carbon dioxide is mainly produced by the combustion of fossil fuels. The concentration of carbon dioxide in the atmosphere is continuously increasing due to the excessive use of energy due to industrialization. The increase in the concentration of carbon dioxide is absorbed by the carbon dioxide gas in the process of returning to the earth surface through the atmosphere, Causing the greenhouse effect. Such warming is causing global damage such as extreme weather, ecosystem change, and sea level rise. Therefore, global awareness of global environmental preservation has been raised, and countermeasures have been taken to suppress the generation of carbon dioxide or to treat or utilize the generated carbon dioxide. Despite the very small amount of fossil resources, Korea is dependent on fossil fuels mainly for oil, with more than 80% of the energy sources being developed, so it is urgent to develop technologies for the treatment and conversion of carbon dioxide.

At present, as a method for converting carbon dioxide, there have been actively researched methods such as chemical conversion into methanol and fuel and biologically conversion into organic material (see Non-Patent Documents 1 to 7). Methods for reducing carbon dioxide by using isocitrate dehydrogenase, pyruvate dehydrogenase, formate dehydrogenase, and methanol dehydrogenase have been reported. Of these, studies to reduce carbon dioxide to formic acid are known to be quite promising.

On the other hand, the formate dehydrogenase that reduces carbon dioxide to formic acid in the enzyme is not used (NAD-dependent) or not (non-NAD-dependent) by using nicotinamide adenine dinucleotide (NAD) do. Among these, the NDA-independent formate dehydrogenase has excellent activity for removing carbon dioxide, but it contains highly sensitive functional groups such as metal ions such as tungsten and molybdenum, iron-sulfur clusters and selenocysteine 8). It is extremely unstable to oxygen, so it is difficult to use it industrially.

Therefore, enzymes that convert carbon dioxide to formic acid include NAD-dependent formate dehydrogenase, especially Candida Formic acid dehydrogenase (CbFDH) derived from boidinii is most commonly used. The formic acid dehydrogenase derived from Candida albicans also acts as a biocatalyst for reducing carbon dioxide during the reduction reaction through enzymes, and its activity is limited to 0.5 mU / mg, which is extremely low in neutral.

Korea Patent No. 10-068049

 Masuda, S. Energy Conv. Mgmt. 1995, 36, 567-572.  Kakumoto, T. Energy Conv. Mgmt. 1995, 36, 661-664.  Souma, Y .; Ando, H .; Fujiwara, M .; Kieffer, R. Energy Conv. Mgmt. 1995, 36, 593-596.  Michiki, H. Energy Conv. Mgmt. 1995, 36, 701-705.  Murakami, M .; Ikenouchi. M. Energy Conv. Mgmt. 1997, 36, S493-S497.  El-Zahab B, Donnelly D, Wang P. 2008, Biotechnology and Bioengineering 99 (3): 508-514.  Lee HJ, Lee SH, Park CB, Won K. 2011, Chemical Communications 47 (46): 12538-12540.  Reda T, Plugge CM, Abram NJ, Hirst J. 2008. Proceedings of the National Academy of Sciences 105 (31): 10654-10658.  Wood W. L. et al., Proc. Natl. Acad. Sci., Vol. 82, pp. 1585-1588, 1985.

The present invention relates to a method for producing formic acid using formic acid dehydrogenase for reducing formic acid by reducing carbon dioxide and a method for producing formic acid using the formic acid dehydrogenase having high activity even under acidic conditions and its expression And to provide a recombinant microorganism.

In order to achieve the above object, the present invention provides a method for producing formic acid, which comprises a nucleotide sequence encoding an acidic formate dehydrogenase derived from a Thiobacillus sp. Strain and which reduces carbon dioxide in an acidic environment having a pH of less than 7 The nucleotide sequence encoding the acidic formic acid dehydrogenase as a recombinant microorganism comprises (a) a nucleotide sequence encoding a polypeptide of Sequence Listing 4; And (b) a sequence complementary to (a) above.

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According to the present invention, the recombinant microorganism is characterized in that a vector expressing an acidic formate dehydrogenase is introduced into a host cell selected from the group consisting of E. coli, bacteria, yeast and fungi.

According to the present invention, there is provided a method for producing formic acid by reducing carbon dioxide under an acidic condition of less than pH 7 by using a recombinant formate dehydrogenase expressed from any one or more of the above-mentioned recombinant microorganisms.

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According to the present invention, the acidic condition of the above formic acid production method is characterized by a pH of not less than 5.5 and less than pH 7.

According to the present invention,
(i) introducing an expression vector comprising a nucleotide sequence encoding an acidic formate dehydrogenase derived from a strain of the genus Thiobacillus sp. into a host cell,
The nucleotide sequence encoding the acidic formate dehydrogenase comprises (a) a nucleotide sequence encoding a polypeptide of Sequence Listing 4; And (b) a sequence complementary to (a) above,
(ii) culturing the recombinant microorganism obtained above to express the recombinant formate dehydrogenase,
(iii) isolating the recombinant dehydrogenase expressed in the above step, and
(iv) a step of producing formic acid by reducing carbon dioxide under an acidic condition of less than pH 7 using the recombinant formate dehydrogenase obtained above.

According to the present invention, the acidic condition of the above formic acid production method is characterized by a pH of not less than 5.5 and less than pH 7.

According to the present invention, the reducing activity of formic acid dehydrogenase on carbon dioxide is maintained in an acidic environment, so that the amount of formic acid to be synthesized is remarkably increased, and thus carbon dioxide can be remarkably reduced.

1 is a schematic diagram showing the reaction mechanism of formate dehydrogenase.
FIG. 2 is a photograph of an SDS-PAGE gel electrophoresis test showing the expression of formate dehydrogenase in the recombinant microorganism according to the present invention.
3 is a graph showing the temperature stability of the examples and comparative examples according to the present invention.
FIG. 4 is a graph showing enzyme activity for formic acid oxidation in Examples and Comparative Examples according to the present invention. FIG.
FIG. 5 is a graph showing the enzyme activity for the sodium bicarbonate reduction in the examples and the comparative examples according to the present invention.
Figure 6 is a reaction scheme used to determine kinetic parameters of the reaction of formate dehydrogenase. V _max : maximum reaction rate, K _A : Michaelis constant of the cofactor, K _B : Michaelis constant of the substrate, K ' _A : decomposition constant of the enzyme -NADH complex, k _cat : conversion number.
Fig. 7 shows the result of analysis of the amount of formic acid synthesis in Example 4 and Comparative Example 1 according to the present invention by liquid chromatography.

Before describing the specific contents of the present invention, the meaning of the terms used in this specification will be described.

As used herein, the term " polypeptide " is interpreted to include an amino acid sequence that exhibits substantial identity to the amino acid sequence of interest. The above substantial identity is determined by aligning the amino acid sequence of the present invention with any other sequence to the greatest correspondence and analyzing the aligned sequence using algorithms commonly used in the art to obtain a homology of at least 60% , Preferably at least 80% homology, more preferably at least 90% homology.

For example, the polypeptide may comprise at least about 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68% , 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% At least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% 99% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 94% or more, 95% or more, 96% A polypeptide having an amino acid sequence having 99.7% or more, 99.8% or more, or 99.9% or more identity and related to the production of formic acid. Generally, the higher the percent identity, the better.

Also, the polypeptides having the same identity may be used in the polypeptides of the specific amino acid sequence described above in the range of 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 39, 38, 1 to 37, 1 to 36, 1 to 35, 1 to 34, 1 to 33, 1 to 32, 1 to 31, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, And polypeptides which involve deletion, substitution, insertion, and / or addition of an amino acid sequence of residues and which are associated with the production of formic acid. Generally, the smaller the number of elimination, substitution, insertion, and / or addition is, the more preferable.

As used herein, the term "gene" or "polynucleotide" has a meaning inclusive of DNA (gDNA and cDNA) and RNA molecules, and the nucleotide, which is a basic constituent unit in a nucleic acid molecule, includes not only natural nucleotides, Also included are analogs where the site has been modified.

The gene of the present invention is not limited to the nucleic acid molecule encoding the specific amino acid sequence (polypeptide) described above, but may be a polypeptide having an amino acid sequence or a function corresponding to the specific amino acid sequence as described above Encoding nucleic acid molecules. The above substantial identity is determined by aligning the amino acid sequence of the present invention with any other sequence to the greatest correspondence and analyzing the aligned sequence using algorithms commonly used in the art to obtain a homology of at least 60% , Preferably at least 80% homology, more preferably at least 90% homology.

The polypeptide having the corresponding function includes, for example, a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added. Such a polypeptide comprises a polypeptide associated with the formation of formic acid consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted, and / or added, as described above, and is capable of eliminating, substituting, / Or the number of additions is preferably small. In addition, the polypeptide has an amino acid sequence having an identity of about 60% or more with the specific amino acid sequence described above, and includes a polypeptide associated with the production of formic acid. The higher the identity, the more preferable.

As used herein, the term " complementary " means that the purine and pyrimidine nucleotides bind through a hydrogen bond to form double stranded nucleic acid molecules, including partially complementary. Guanine and cytosine, adenine and thymine, and adenine and uracil are involved in the formation of complementary nucleic acid molecules. &Quot; Complementary " is substantially applied to all base pairs in which the above-mentioned relationship includes two single-stranded nucleic acid molecules across the molecule of the total length. By "partially complementary" is meant that a portion of one of the molecules remains as a single strand because the length of one of the two single-stranded nucleic acid molecules is short.

The term " hybridization " as used herein refers to the process by which a single stranded nucleic acid molecule binds to a complementary strand through a nucleotide base pair. Therefore, in the present specification, 'a nucleic acid molecule comprising a nucleotide sequence hybridizing with the first nucleotide sequence of SEQ ID NO: 1' means a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: Refers to a nucleic acid molecule, such as DNA, obtained by, for example, " hybridization under stringent conditions ". A variety of methods known to those skilled in the art can be used for the hybridization method, for example, the method described in Wood W. I. et al. (Non-Patent Document 9).

On the other hand, the 'hybridization' can be interpreted as 'hybridization under strict conditions' within the range necessary for the invention. The term " strict " means a hybridization condition. Highly stringent conditions are undesirable for an asymmetric base pair. Low rigidity has the opposite effect. Strictness can be changed by, for example, temperature, salt concentration. As used herein, the term " stringent conditions " may be any of a low stringency condition, a medium stringency condition, or a high stringency condition. 'Low stringency conditions' are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% Sodium Dodecyl Sulfate (SDS), 50% formamide at 32 ° C. 'Medium stringent conditions' are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 42 ° C. 'High stringency conditions' are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 50 ° C. Under these conditions, it is expected that polynucleotides such as DNA with higher homology will be efficiently obtained at higher temperatures. Nevertheless, many elements are involved in hybridization severity, including temperature, probe concentration, probe length, ionic strength, time, salt concentration, and the like, and those skilled in the art will appreciate that such factors may be selected appropriately to achieve similar stringency . For example, it can be hybridized using a commercial kit, Alkphos Direct Labeling Reagents (Amersham Pharmacia, UK). In this case, according to the attached protocol, incubation with labeled probes overnight, followed by washing of the membrane with primary wash buffer containing 0.1% (w / v) SDS at 55 ° C to detect hybridized nucleic acid molecules such as DNA do.

On the other hand, other nucleic acid molecules that may be hybridized may contain at least about 60%, at least about 61%, at least about 60%, at least about < RTI ID = 0.0 > 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, Or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, or 86% or more , 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% Or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more or 99.9% or more identity.

The identity between amino acid sequences or base sequences can be measured using the algorithm BLAST by Karlin and Altschul, and can be done with the BLASTN and BLASTX programs based on the BLAST algorithm. When the nucleotide sequence is sequenced using BLASTN, the variable is, for example, a score of 100 and a word length = 12. When the amino acid sequence is sequenced using BLASTX, the variable is, for example, a score of 50 and a word length = 3. When BLAST and Gapped BLAST programs are used, default variables are applied for each program.

In order to accomplish the above-mentioned object, the present invention provides an anti- which encodes an acidic formate dehydrogenase derived from one or more strains selected from the group consisting of aquaticus , Moraxella sp., Paracoccus sp., and Thiobacillus sp. A recombinant microorganism producing a formic acid by reducing carbon dioxide in an acidic environment containing a nucleotide sequence of less than pH 7, a recombinant formic acid dehydrogenase derived therefrom, and a method for producing formic acid using the same. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the drawings.

The present invention relates to the use of an Ancylobacter which encodes an acidic formate dehydrogenase derived from one or more strains selected from the group consisting of aquaticus , Moraxella sp., Paracoccus sp., and Thiobacillus sp. A recombinant microorganism comprising at least one nucleotide sequence and reducing carbon dioxide in an acidic environment below pH 7 to produce formic acid.

Formate dehydrogenase in the 'acid formate dehydrogenase' refers to an enzyme that catalyzes the oxidation of formic acid ions to carbon dioxide (CO ₂ ). This enzyme also catalyzes the reduction of carbon dioxide and the isotope exchange reaction between formic acid and carbon dioxide by the reverse reaction (see FIG. 1).

In the 'acidic formic acid dehydrogenase', 'acidic' means that the maximum activity of the enzyme appears under conditions of pH less than 7. The activity of the enzyme may occur not only in acidic conditions but also in neutral conditions. The acidic formic acid dephosphate was prepared from Candida Compared to the activity of the formidase dehydrogenase derived from boidinii , the maximum activity is more acidic and the enzyme activity is better.

According to the present invention, the nucleotide sequence encoding the acidic formate dehydrogenase of the recombinant microorganism comprises (a) a nucleotide sequence encoding any one of the polypeptides selected from the first to fourth sequences of the sequence listing; And (b) a sequence complementary to (a) above.

More specifically, Sequence Listing first sequence is an Ancylobacter < RTI ID = 0.0 > (AaFDH) derived from aquaticus KNK607M and the second sequence is an amino acid sequence of formic acid dehydrogenase (MsFDH) derived from Moraxella sp. strain C-1, The third sequence is the amino acid sequence of formate dehydrogenase (PsFDH) derived from Paracoccus sp. Strain 12-A, and the fourth sequence is the amino acid sequence derived from Thiobacillus sp. Strain KNK65MA It is the amino acid sequence of formate dehydrogenase (TsFDH).

The recombinant microorganism used in the present invention can be prepared by preparing a transformant cassette or a recombinant vector containing a polynucleotide encoding the acidic formate dehydrogenase and introducing it into an appropriate host cell to express the recombinant microorganism. Therefore, the gene group (nucleic acid molecule) encoding the polypeptide of the present invention can be cloned into appropriate vectors such as bacterium, viruses (bacteriophage T17, M-13 derived phage etc.), cosmid, yeast, The vector may be introduced into an appropriate host cell. The vector may further include various nucleic acid molecules such as an initiation regulatory region, a promoter, etc., in order to promote the expression of formate dehydrogenase.

The recombinant microorganism used in the present invention is not limited as long as it is capable of expressing the gene, and may be selected from the group consisting of E. coli, bacteria, yeast and fungi. As for example sheet bakteo (Citrobacter), Enterobacter bakteo (Enterobacter), Clostridium (Clostridium), keulrep when Ella (Klebsiella), Aero bakteo (Aerobacter), Lactobacillus bacteria (Lactobacillus), Aspergillus (Aspergillus), Mai in Saccharomyces a process (Saccharomyces), ski irradiation Caro My process (Schizosaccharomyces), Eisai Kosaka My process (Zygosaccharomyces), Pichia (Pichia), Cluj suberic My process (Kluyveromyces), Candida (Candida), Hanse nyulra (Hansenula), debari Oh, my Seth (Debaryomyces), myuko (Mucor), sat rolrop sheath (Torulopsis), bakteo (Methylobacter), S. cherry Oh (Escherichia), Salmonella (Salmonella), Bacillus (Bacillus), Streptomyces (Streptomyces) methyl, Pseudomonas ( Pseudomonas ) can be used. For example, Escherichia coli was used.

As the introduction method of the recombinant vector, there can be used a known technique such as a transformation method such as a calcium chloride method, a heat shock method, an electric shock method, or a transfection method using recombinant phage virus. It is obvious to those skilled in the art that various vectors and various strains can be easily used depending on the purpose of expression in addition to the above host cells.

The present invention provides a recombinant formate dehydrogenase expressed from any one or more of the above-mentioned recombinant microorganisms. The expression of the recombinant formate dehydrogenase can be obtained by culturing the recombinant microorganism by a method known to the ordinarily skilled artisan, and then isolating, purifying, or filtering the enzyme from known culture mediums known to those skilled in the art.

The present invention provides a method for producing formic acid by reducing carbon dioxide under acidic conditions below pH 7 using a recombinant dephosphate derived from the above recombinant microorganism.

In the present invention, the acidic condition of the method for producing formic acid may be pH 5.5 or higher and lower than pH 7. When carried out at an acidity within the above range, formation of formic acid and carbon dioxide reduction efficiency are excellent.

The present invention
(i) at least one selected from the group consisting of Ancylobacter aquaticus , Moraxella sp., Paracoccus sp., and Thiobacillus sp. Introducing an expression vector containing at least one nucleotide sequence encoding an acidic formate dehydrogenase derived from a strain into a host cell,
Wherein the nucleotide sequence encoding the acidic formate dehydrogenase comprises (a) a nucleotide sequence encoding any one of the polypeptides selected from Sequence Listing Nos. 1 to 4; And (b) a sequence complementary to (a) above,
(ii) culturing the recombinant microorganism obtained above to express the recombinant formate dehydrogenase,
(iii) isolating the recombinant dehydrogenase expressed in the above step, and
(iv) a step of producing formic acid by reducing carbon dioxide under an acidic condition of less than pH 7 using the recombinant formate dehydrogenase obtained in the above step
≪ / RTI > The details of each step are as described above.

According to the present invention, the acidic conditions of the method for producing formic acid may be pH 5.5 or higher and lower than pH 7. When carried out at an acidity within the above range, formation of formic acid and carbon dioxide reduction efficiency are excellent.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

One. Example  And Comparative Example Production

1) Search for genetic information

To identify the formate dehydrogenase peptides with optimal activity in acidic form among the various formate dehydrogenases, we searched using BRENDA (BRaunschweig Enzyme Database, http://www.brenda-enzymes.org). As a result of the search, Ancylobacter aquaticus ) AaFDH (BAC65346.1) of KNK607M, MsFDH (CAA73696.1) of the Moraxella sp. strain C-1, PsFDH (AB071373.1) of the Paracoccus sp. strain 12- And TsFDH (BAC92737.1) of Thiobacillus sp. KNK65MA were to be used. Also, as a comparative example, CbFDH (CAA09466.2) of Candida boidinii and Ceriporiopsis CsFDH ( BAF98206.1 ) of subvermispora was used.

2) Preparation of recombinant microorganisms

Each gene was synthesized and introduced into Escherichia coli DH5α, transformed and amplified. The obtained gene was digested with restriction enzymes (Nde1 and Xho1), and inserted into pET-23b (+) vector (Novagen) to prepare a recombinant vector. The recombinant vector prepared above was introduced into Escherichia coli BL-21 (DE3) and transformed.

division Introduced gene Gene-derived microorganism Used host cell Example 1R AaFDH Ancylobacter aquaticus Escherichia coli BL-21 (DE3) Example 2R MsFDH Moraxella sp. Escherichia coli BL-21 (DE3) Example 3R PsFDH Paracoccus sp. Escherichia coli BL-21 (DE3) Example 4R TsFDH Thiobacillus sp. Escherichia coli BL-21 (DE3) Comparative Example 1R CbFDH Candida boidinii Escherichia coli BL-21 (DE3) Comparative Example 2R CsFDH Ceriporiopsis subvermispora Escherichia coli BL-21 (DE3)

3) Expression of recombinant formate dehydrogenase

Each of the recombinant microorganisms prepared above was inoculated in 15 ml of LB (Luria Bertani) medium and cultured at 200 rpm and 37 ° C in a shaking incubator. A 1 L Erlenmeyer flask was filled with 250 ml of LB medium, and the microorganisms previously cultured in the above were respectively inoculated, followed by culture at 200 rpm and 37 ° C in a shaking incubator.

4) Isolation of recombinant formate dehydrogenase

The cultures of the recombinant microorganisms of the examples and comparative examples prepared above were centrifuged at 4,000 rpm for 20 minutes. The precipitated cells were suspended in BugBuster Master Mix (Novagen) and shaken at room temperature for 30 minutes to disrupt the cell membrane. The cell debris was removed by centrifugation at 12,000 rpm for 20 minutes, and 3 mL of Ni-NTA agarose (QIAGEN) was added to the supernatant, followed by recombinant formate dehydrogenase.

The bound recombinant formate dehydrogenase was washed using a washing buffer (50 mM NaH ₂ PO ₄ , 300 mM sodium chloride, 20 mM imidazole, adjusted to pH 7 by adding sodium hydroxide (NaOH)) and eluted with elution buffer 50mM phosphoric acid and purified by the use of sodium hydrogen (NaH ₂ PO _4), 300mM sodium chloride (NaCl), 20mM imidazole (imidazole), adjusted to pH 7 by addition of sodium hydroxide (NaOH)).

division Introduced gene Gene-derived microorganism Cultured microorganism Example 1D AaFDH Ancylobacter aquaticus Example 1R Example 2D MsFDH Moraxella sp. Example 2R Example 3D PsFDH Paracoccus sp. Example 3R Example 4D TsFDH Thiobacillus sp. Example 4R Comparative Example 1D CbFDH Candida boidinii Comparative Example 1R Comparative Example 2D CsFDH Ceriporiopsis subvermispora Comparative Example 2R

2. Experimental methods and results

1) Formate dehydrogenase expression evaluation

Each of the recombinant microorganisms prepared above was inoculated in 15 ml of LB (Luria Bertani) medium and cultured at 200 rpm and 37 ° C in a shaking incubator. In a 1 L Erlenmeyer flask, 250 ml of LB medium was placed, and the microorganisms previously cultured in the above were respectively inoculated, followed by incubation at 200 rpm and 37 ° C in a shaking incubator. D-1-thiogalactopyranoside (IPTG) at a wavelength of 600 nm was added so that the OD value of the cells became 0.6 to 0.8, and the mixture was added to 1 mM of the isopropyl beta-D-1-thiogalactopyranoside 200 rpm, 23 [deg.] C for 24 hours.

FIG. 2 shows the results of SDS-PAGE gel electrophoresis of the degree of formate dehydrogenase expression of the recombinant microorganism according to the present invention. As a result of the experiment, it was found that the formate dehydrogenase gene was well expressed in all of Examples 1D to 4D as well as Comparative Examples.

2) Structure temperature for formate dehydrogenase

FIG. 3 shows the structural stability of the formate dehydrogenase expressed by the recombinant microorganism according to the present invention.

The experimental results, in Comparative Example 1, the structure melting temperature _{(CbFDH) (T m: Transition} temperature) is, while in each embodiment is the third embodiment structure of the melting temperature of the comparison and 62.0 ℃, Example 2 (CsFDH) is 49.7 ℃ ( PsFDH) was the lowest at 42.0 占 폚, and Example 2 (MsFDH) was found to be 57.3 占 폚. These results indicate that the formate dehydrogenase of the recombinant microorganism according to the present invention is sufficiently stable against temperature.

2) Measurement of formate dehydrogenase activity

The activity of the recombinant formate dehydrogenase according to the present invention was measured using a spectrophotometer.

The oxidation reaction of formic acid is carried out in a reaction buffer (200 mM sodium formate, 2 mM NAD ⁺ and 100 mM sodium phosphate buffer) at different pHs in a 1 cm cell and the final volume is adjusted to 2 ml The reaction was initiated by the addition of 20 ug protein and the increase in NADH was measured at 340 nm.

The reduction reaction of carbon dioxide was carried out by using a reaction buffer (50 mM sodium bicarbonate, NaHCO ₃ , 0.15 mM NADH, 100 mM sodium phosphate buffer) at a different pH and dividing the cells into 1 cm long cells, The reaction was initiated by adding 3 mg protein as much as possible to determine the decrease in NADH at 340 nm.

In experiments on the degree of carbon dioxide reduction, 50 mM sodium bicarbonate (NaHCO ₃ ) was used as a substrate to supply carbon dioxide.

Among the above experimental results, the results for the formic acid oxidation activity are shown in FIG. 4, and the results for the carbon dioxide reduction are shown in FIG.

Formic Acid Oxidation Activity As a result, both Examples 1D to 4D showed sufficient activity for formic acid oxidation. In particular, Example 1D (AaFDH) exhibited the highest activity with a activity of 20.0 units per mg of enzyme (expressed as U / mg) at pH 5.5, which was comparable to that of Comparative Example 1D (CbFDH, 3.8 U / mg, Which is 5.2 times higher. It can be deduced that Example 1D (AaFDH) can be used in various pH ranges as an alternative to NADH regenerating enzyme.

Experiments on carbon dioxide reduction showed that both Examples 1D to 4D exhibit excellent carbon dioxide reducing activity over all pH conditions, respectively, as compared to Comparative Examples 1D and 2D. In particular, Example 4D (TsFDH) showed the highest activity in the order of 12.2, 9.5, 8.7 and 4.0 U / mg at pH 5.5, 6.0, 6.5 and 7.0, 1.6, 1.3, 0.7, 0.5 U / mg at pH 5.5, 6.0, 6.5 and 7.0, respectively).

3) Kinetic parameter

In order to confirm the efficiency of the carbon dioxide reduction and formic acid formation reaction of the recombinant formate dehydrogenase according to the present invention, based on the reaction path as shown in FIG. 6, the forward and reverse reactions were performed under the following conditions, Respectively. On the other hand, kinetic parameters were measured at pH 7 because NADH was extremely unstable to acidity.

(1) regular reaction

- 100 mM phosphate buffer, 25 < 0 > C, pH 7.0

- A: NAD ⁺ (0.1-0.3 mM)

B: Sodium formate (10 to 50 mM)

(2) reverse reaction

- 100 mM phosphate buffer, 25 < 0 > C, pH 7.0

- A: NAD ⁺ (0.1-0.3 mM)

B: Sodium bicarbonate (10 to 50 mM)

The kinetic parameters of Example 4D (TsFDH) are shown in Table 3 below in comparison with Comparative Example 1D.

Example 4D Comparative Example 1D Regular reaction Reverse reaction Regular reaction Reverse reaction K _A (mM) 0.181 0.210 0.086 0.297 K _{A '} (mM) 0.025 0.100 0.100 0.025 K _B (mM) 9.449 7.880 6.034 17.450 k _cat (s ^-1 ) 1.331 0.270 2.600 0.017 K _cat / K _B (mM ^-1 s ^-1 ) 0.140 0.034 0.430 0.974 × 10 ^-3 ( K _cat / K _B ) _forward
( K _cat / K _B ) _reverse 4.111 0.442 x 10 ³

As a result of the experiment, in Comparative Example 1D, the substrate binding force ( K _B ) and the number of conversion (turnover number. K _cat ) were higher and the working efficiency ( K _cat / K _B ) was 3.3 times higher than that in Example 4D.

However, in Example 4D, the substrate binding force ( K _B ) and the number of conversions ( K _cat ) were 2.2 times and 15.9 times higher than those of Comparative Example 1D, respectively, and the operation efficiency ( K _cat / K _B ) Respectively. In particular, in Example 4D, the reduction efficiency ( K _cat / K _B ) was 107.6 times higher than that of Comparative Example 1D in the enzyme catalysis, indicating that the reduction efficiency was remarkably excellent.

4) Liquid chromatography (HPLC) measurement of formic acid

The reaction solution (150 mM sodium bicarbonate, 100 mM sodium phosphate buffer, pH 6.0, 4.4 x 10 ^-8 mol formate dehydrogenase, 7 mM NADH) was used and the final volume of the reaction solution was 5 ml . The reaction was initiated by adding 1 ml of 50 mM NADH to 4 ml of the reaction solution containing all the reagents except NADH, and the reaction was started at 200 rpm. Immediately after the start of the reaction, the reaction vessel was capped to prevent the loss of carbon dioxide.

Using a needle syringe, 190 μl of the reaction solution was mixed with 10 μl of 5 M sulfuric acid to stop the reaction. It was filtered with a 0.20 탆 syringe filter (hydrophilic) and used as a HPLC analysis sample. In the HPLC measurement, 40 μl of the above analytical sample was injected, and 5 mM sulfuric acid was flowed at an elution rate of 0.6 ml / min. The column used the Aminex HPX-87H Ion Exclusion Column (Bio-Rad) at 35 ° C and the detector used a Refractive Index Detector (RID).

The amount of formic acid synthesized by the recombinant formate dehydrogenase (Example 4D) according to the present invention was measured by reaction time. The experimental results are shown in FIG. 7 in comparison with Comparative Example 1D.

As a result of the experiment, it can be seen that the synthesis of formic acid in Comparative Example 1D hardly occurs, whereas the synthesis of formic acid in Example 4D increases with the reaction time. Thus, it can be confirmed that Example 4D is superior in the effect of the formation of formic acid and the reduction of carbon dioxide.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. Do. The actual scope of the invention is thus defined by the appended claims and their equivalents.

<110> Kwangwoon University Industry-Academic Collaboration Foundation <120> Recombinant microorganisms, recombinant formate dehydrogenase          expressed by the same, and formate synthesis using the same <130> DP120355 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 401 <212> PRT <213> Ancylobacter aquaticus <400> 1 Met Ala Lys Val Leu Cys Val Leu Tyr Asp Asp Pro Ile Asp Gly Tyr   1 5 10 15 Pro Thr Thr Tyr Ala Arg Asp Asn Leu Pro Lys Ile Asp His Tyr Pro              20 25 30 Gly Gly Gln Thr Leu Pro Thr Pro Lys Ala Ile Asp Phe Thr Pro Gly          35 40 45 Thr Met Leu Gly Ser Val Ser Gly Glu Leu Gly Leu Arg Lys Tyr Leu      50 55 60 Glu Ser Asn Gly His Thr Leu Val Val Thr Ser Asp Lys Asp Gly Pro  65 70 75 80 Asp Ser Val Phe Glu Lys Glu Leu Val Asp Ala Asp Ile Val Ile Ser                  85 90 95 Gln Pro Phe Trp Pro Ala Tyr Leu Thr Pro Glu Arg Phe Ala Lys Ala             100 105 110 Lys Asn Leu Lys Leu Ala Leu Thr Ala Gly Ile Gly Ser Asp His Val         115 120 125 Asp Leu Gln Ser Ala Ile Asp Arg Gly Val Thr Val Ala Glu Val Thr     130 135 140 Tyr Cys Asn Ser Ile Ser Val Ala Glu His Val Val Met Met Ile Leu 145 150 155 160 Gly Leu Val Arg Asn Tyr Leu Pro Ala His Asp Trp Ala Arg Lys Gly                 165 170 175 Gly Trp Asn Ile Ala Asp Cys Val Lys His Ser Tyr Asp Leu Glu Ala             180 185 190 Met Ser Val Gly Thr Val Ala Ala Gly Arg Ile Gly Leu Ala Val Leu         195 200 205 Arg Arg Leu Ala Pro Phe Asp Val Lys Leu His Tyr Thr Asp Arg His     210 215 220 Arg Leu Pro Glu Ser Val Glu Lys Glu Leu Asn Leu Thr Trp His Ala 225 230 235 240 Ser Pro Thr Asp Met Tyr Pro His Cys Asp Val Val Thr Leu Asn Cys                 245 250 255 Pro Leu His Pro Glu Thr Glu His Met Val Asn Glu Glu Thr Leu Lys             260 265 270 Leu Phe Lys Arg Gly Ala Tyr Ile Val Asn Thr Ala Arg Gly Lys Leu         275 280 285 Cys Asp Arg Asp Ala Ile Ala Arg Ala Leu Glu Asn Gly Thr Leu Ala     290 295 300 Gly Tyr Ala Gly Asp Val Trp Phe Pro Gln Pro Ala Pro Ala Asp His 305 310 315 320 Pro Trp Arg Thr Met Ala Trp Asn Gly Met Thr Pro His Met Ser Gly                 325 330 335 Thr Ser Leu Thr Ala Gln Thr Arg Tyr Ala Ala Gly Thr Arg Glu Ile             340 345 350 Leu Glu Cys Phe Phe Glu Gly Arg Pro Ile Arg Asp Glu Tyr Leu Ile         355 360 365 Val Gln Gly Gly Asn Leu Ala Gly Val Gly Ala His Ser Tyr Ser Lys     370 375 380 Gly Asn Ala Thr Gly Gly Ser Glu Glu Ala Gly Lys Phe Lys Lys Ala 385 390 395 400 Gly     <210> 2 <211> 402 <212> PRT <213> Moraxella sp. <400> 2 Met Ala Lys Val Val Cys Val Leu Tyr Asp Asp Pro Ile Asn Gly Tyr   1 5 10 15 Pro Thr Ser Tyr Ala Arg Asp Asp Leu Pro Arg Ile Asp Lys Tyr Pro              20 25 30 Asp Gly Gln Thr Leu Pro Thr Pro Lys Ala Ile Asp Phe Thr Pro Gly          35 40 45 Ala Leu Leu Gly Ser Val Ser Gly Glu Leu Gly Leu Arg Lys Tyr Leu      50 55 60 Glu Ser Gln Gly His Glu Leu Val Val Thr Ser Ser Lys Asp Gly Pro  65 70 75 80 Asp Ser Glu Leu Glu Lys His Leu His Asp Ala Glu Val Ile Ile Ser                  85 90 95 Gln Pro Phe Trp Pro Ala Tyr Leu Thr Ala Glu Arg Ile Ala Lys Ala             100 105 110 Pro Lys Leu Lys Leu Ala Leu Thr Ala Gly Ile Gly Ser Asp His Val         115 120 125 Asp Leu Gln Ala Ile Asp Asn Asn Ile Thr Val Ala Glu Val Thr     130 135 140 Tyr Cys Asn Ser Asn Ser Val Ala Glu His Val Val Met Met Val Leu 145 150 155 160 Gly Leu Val Arg Asn Tyr Ile Pro Ser His Asp Trp Ala Arg Asn Gly                 165 170 175 Gly Trp Asn Ile Ala Asp Cys Val Ala Arg Ser Tyr Asp Val Glu Gly             180 185 190 Met His Val Gly Thr Val Ala Ala Gly Arg Ile Gly Leu Arg Val Leu         195 200 205 Arg Leu Leu Ala Pro Phe Asp Met His Leu His Tyr Thr Asp Arg His     210 215 220 Arg Leu Pro Glu Ala Val Glu Lys Glu Leu Asn Leu Thr Trp His Ala 225 230 235 240 Thr Arg Glu Asp Met Tyr Gly Ala Cys Asp Val Val Thr Leu Asn Cys                 245 250 255 Pro Leu His Pro Glu Thr Glu His Met Ile Asn Asp Glu Thr Leu Lys             260 265 270 Leu Phe Lys Arg Gly Ala Tyr Leu Val Asn Thr Ala Arg Gly Lys Leu         275 280 285 Cys Asp Arg Asp Ala Ile Val Arg Ala Leu Glu Ser Gly Arg Leu Ala     290 295 300 Gly Tyr Ala Gly Asp Val Trp Phe Pro Gln Pro Ala Pro Asn Asp His 305 310 315 320 Pro Trp Arg Thr Met Pro His Asn Gly Met Thr Pro His Ile Ser Gly                 325 330 335 Thr Ser Leu Ser Ala Gln Thr Arg Tyr Ala Ala Gly Thr Arg Glu Ile             340 345 350 Leu Glu Cys Tyr Phe Glu Gly Arg Pro Ile Arg Asp Glu Tyr Leu Ile         355 360 365 Val Gln Gly Gly Gly Leu Ala Gly Val Gly Ala His Ser Tyr Ser Lys     370 375 380 Gly Asn Ala Thr Gly Gly Ser Glu Glu Ala Ala Lys Tyr Glu Lys Leu 385 390 395 400 Asp Ala         <210> 3 <211> 400 <212> PRT <213> Paracoccus sp. <400> 3 Met Ala Lys Val Val Cys Val Leu Tyr Asp Asp Pro Val Asp Gly Tyr   1 5 10 15 Pro Thr Ser Tyr Ala Arg Asp Ser Leu Pro Val Ile Glu Arg Tyr Pro              20 25 30 Asp Gly Gln Thr Leu Pro Thr Pro Lys Ala Ile Asp Phe Val Pro Gly          35 40 45 Ser Leu Leu Gly Ser Val Ser Gly Glu Leu Gly Leu Arg Asn Tyr Leu      50 55 60 Glu Ala Gln Gly His Glu Leu Val Val Thr Ser Ser Lys Asp Gly Pro  65 70 75 80 Asp Ser Glu Leu Glu Lys His Leu His Asp Ala Glu Val Val Ser Ser                  85 90 95 Gln Pro Phe Trp Pro Ala Tyr Leu Thr Ala Glu Arg Ile Ala Lys Ala             100 105 110 Pro Lys Leu Lys Leu Ala Leu Thr Ala Gly Ile Gly Ser Asp His Val         115 120 125 Asp Leu Gln Ala Ala Ile Asp Arg Gly Ile Thr Val Ala Glu Val Thr     130 135 140 Phe Cys Asn Ser Ile Ser Val Ser Glu His Val Val Met Thr Ala Leu 145 150 155 160 Asn Leu Val Arg Asn Tyr Thr Pro Ser His Asp Trp Ala Val Lys Gly                 165 170 175 Gly Trp Asn Ile Ala Asp Cys Val Thr Arg Ser Tyr Asp Ile Glu Gly             180 185 190 Met His Val Gly Thr Val Ala Ala Gly Arg Ile Gly Leu Ala Val Leu         195 200 205 Arg Arg Phe Lys Pro Phe Gly Met His Leu His Tyr Thr Asp Arg His     210 215 220 Arg Leu Pro Arg Glu Val Glu Leu Glu Leu Asp Leu Thr Trp His Glu 225 230 235 240 Ser Pro Lys Asp Met Phe Pro Ala Cys Asp Val Val Thr Leu Asn Cys                 245 250 255 Pro Leu His Pro Glu Thr Glu His Met Val Asn Asp Glu Thr Leu Lys             260 265 270 Leu Phe Lys Arg Gly Ala Tyr Leu Val Asn Thr Ala Arg Gly Lys Leu         275 280 285 Cys Asp Arg Asp Ala Val Ala Arg Ala Leu Glu Ser Gly Gln Leu Ala     290 295 300 Gly Tyr Gly Gly Asp Val Trp Phe Pro Gln Pro Ala Pro Gln Asp His 305 310 315 320 Pro Trp Arg Thr Met Pro His Asn Ala Met Thr Pro His Ile Ser Gly                 325 330 335 Thr Ser Leu Ser Ala Gln Ala Arg Tyr Ala Ala Gly Thr Arg Glu Ile             340 345 350 Leu Glu Cys His Phe Glu Gly Arg Pro Ile Arg Asp Glu Tyr Leu Ile         355 360 365 Val Gln Gly Gly Ser Leu Ala Gly Val Gly Ala His Ser Tyr Ser Lys     370 375 380 Gly Asn Ala Thr Gly Gly Ser Glu Glu Ala Ala Lys Phe Lys Lys Ala 385 390 395 400 <210> 4 <211> 401 <212> PRT <213> Thiobacillus sp. <400> 4 Met Ala Lys Ile Leu Cys Val Leu Tyr Asp Asp Pro Val Asp Gly Tyr   1 5 10 15 Pro Lys Thr Tyr Ala Arg Asp Asp Leu Pro Lys Ile Asp His Tyr Pro              20 25 30 Gly Gly Gln Thr Leu Pro Thr Pro Lys Ala Ile Asp Phe Thr Pro Gly          35 40 45 Gln Leu Leu Gly Ser Val Ser Gly Glu Leu Gly Leu Arg Lys Tyr Leu      50 55 60 Glu Ala Asn Gly His Thr Phe Val Val Thr Ser Asp Lys Asp Gly Pro  65 70 75 80 Asp Ser Val Phe Glu Lys Glu Leu Val Asp Ala Asp Val Val Ser Ser                  85 90 95 Gln Pro Phe Trp Pro Ala Tyr Leu Thr Pro Glu Arg Ile Ala Lys Ala             100 105 110 Lys Asn Leu Lys Leu Ala Leu Thr Ala Gly Ile Gly Ser Asp His Val         115 120 125 Asp Leu Gln Ser Ala Ile Asp Arg Gly Ile Thr Val Ala Glu Val Thr     130 135 140 Tyr Cys Asn Ser Ile Ser Val Ala Glu His Val Val Met Met Ile Leu 145 150 155 160 Gly Leu Val Arg Asn Tyr Ile Pro Ser His Asp Trp Ala Arg Lys Gly                 165 170 175 Gly Trp Asn Ile Ala Asp Cys Val Glu His Ser Tyr Asp Leu Glu Gly             180 185 190 Met Thr Val Gly Ser Val Ala Ala Gly Arg Ile Gly Leu Ala Val Leu         195 200 205 Arg Arg Leu Ala Pro Phe Asp Val Lys Leu His Tyr Thr Asp Arg His     210 215 220 Arg Leu Pro Glu Ala Val Glu Lys Glu Leu Gly Leu Val Trp His Asp 225 230 235 240 Thr Arg Glu Asp Met Tyr Pro His Cys Asp Val Val Thr Leu Asn Val                 245 250 255 Pro Leu His Pro Glu Thr Glu His Met Ile Asn Asp Glu Thr Leu Lys             260 265 270 Leu Phe Lys Arg Gly Ala Tyr Ile Val Asn Thr Ala Arg Gly Lys Leu         275 280 285 Ala Asp Arg Asp Ala Ile Val Arg Ala Ile Glu Ser Gly Gln Leu Ala     290 295 300 Gly Tyr Ala Gly Asp Val Trp Phe Pro Gln Pro Ala Pro Lys Asp His 305 310 315 320 Pro Trp Arg Thr Met Lys Trp Glu Gly Met Thr Pro His Ile Ser Gly                 325 330 335 Thr Ser Leu Ser Ala Gln Ala Arg Tyr Ala Ala Gly Thr Arg Glu Ile             340 345 350 Leu Glu Cys Phe Phe Glu Gly Arg Pro Ile Arg Asp Glu Tyr Leu Ile         355 360 365 Val Gln Gly Gly Ala Leu Ala Gly Thr Gly Ala His Ser Tyr Ser Lys     370 375 380 Gly Asn Ala Thr Gly Gly Ser Glu Glu Ala Ala Lys Phe Lys Lys Ala 385 390 395 400 Gly     <210> 5 <211> 364 <212> PRT <213> Candida boidinii <400> 5 Met Lys Ile Val Leu Val Leu Tyr Asp Ala Gly Lys His Ala Ala Asp   1 5 10 15 Glu Glu Lys Leu Tyr Gly Cys Thr Glu Asn Lys Leu Gly Ile Ala Asn              20 25 30 Trp Leu Lys Asp Gln Gly His Glu Leu Ile Thr Thr Ser Asp Lys Glu          35 40 45 Gly Gly Asn Ser Val Leu Asp Gln His Ile Pro Asp Ala Asp Ile Ile      50 55 60 Ile Thr Thr Pro Phe His Pro Ala Tyr Ile Thr Lys Glu Arg Ile Asp  65 70 75 80 Lys Ala Lys Lys Leu Lys Leu Val Val Ala Gly Val Gly Ser Asp                  85 90 95 His Ile Asp Leu Asp Tyr Ile Asn Gln Thr Gly Lys Lys Ile Ser Val             100 105 110 Leu Glu Val Thr Gly Ser Asn Val Val Ser Val Ala Glu His Val Val         115 120 125 Met Thr Met Leu Val Leu Val Arg Asn Phe Val Pro Ala His Glu Gln     130 135 140 Ile Ile Asn His Asp Trp Glu Val Ala Ala Ile Ala Lys Asp Ala Tyr 145 150 155 160 Asp Ile Glu Gly Lys Thr Ile Ala Thr Ile Gly Ala Gly Arg Ile Gly                 165 170 175 Tyr Arg Val Leu Glu Arg Leu Val Pro Phe Asn Pro Lys Glu Leu Leu             180 185 190 Tyr Tyr Asp Tyr Gln Ala Leu Pro Lys Asp Ala Glu Glu Lys Val Gly         195 200 205 Ala Arg Arg Val Glu Asn Ile Glu Glu Leu Val Ala Gln Ala Asp Ile     210 215 220 Val Thr Val Asn Ala Pro Leu His Ala Gly Thr Lys Gly Leu Ile Asn 225 230 235 240 Lys Glu Leu Leu Ser Lys Phe Lys Lys Gly Ala Trp Leu Val Asn Thr                 245 250 255 Ala Arg Gly Ala Ile Cys Val Ala Glu Asp Val Ala Ala Ala Leu Glu             260 265 270 Ser Gly Gln Leu Arg Gly Tyr Gly Gly Asp Val Trp Phe Pro Gln Pro         275 280 285 Ala Pro Lys Asp His Pro Trp Arg Asp Met Arg Asn Lys Tyr Gly Ala     290 295 300 Gly Asn Ala Met Thr Pro His Tyr Ser Gly Thr Thr Leu Asp Ala Gln 305 310 315 320 Thr Arg Tyr Ala Gln Gly Thr Lys Asn Ile Leu Glu Ser Phe Phe Thr                 325 330 335 Gly Lys Phe Asp Tyr Arg Pro Gln Asp Ile Ile Leu Leu Asn Gly Glu             340 345 350 Tyr Val Thr Lys Ala Tyr Gly Lys His Asp Lys Lys         355 360 <210> 6 <211> 358 <212> PRT <213> Ceriporiopsis subvermispora <400> 6 Met Lys Val Leu Ala Ile Leu Tyr Lys Gly Gly Asp Ala Ala Thr Gln   1 5 10 15 Glu Pro Arg Leu Leu Gly Thr Ile Glu Asn Gln Leu Gly Ile Arg Gln              20 25 30 Trp Leu Glu Ser Glu Gly His Glu Leu Ile Val Ser Asp Ser Lys Glu          35 40 45 Gly Pro Asp Ser Asp Phe Gln Lys His Ile Val Asp Ala Glu Val Leu      50 55 60 Ile Thr Pro Phe His Pro Gly Tyr Leu Thr Arg Asp Leu Ile Asp  65 70 75 80 Lys Ala Lys Asn Leu Lys Ile Cys Ile Thr Ala Gly Val Gly Ser Asp                  85 90 95 His Ile Asp Leu Asn Ala Ala Val Glu Arg Lys Ile Gln Val Leu Glu             100 105 110 Val Ser Gly Ser Asn Val Val Ser Val Ala Glu His Val Met Met Ser         115 120 125 Ile Leu Leu Leu Val Arg Asn Phe Val Pro Ala His Glu Met Ile Glu     130 135 140 Arg Gly Asp Trp Gln Val Ser Asp Ile Ala Arg Asn Ala Phe Asp Leu 145 150 155 160 Glu Gly Lys Val Val Gly Thr Ile Gly Ala Gly Arg Ile Gly Tyr Arg                 165 170 175 Val Leu Gln Arg Leu Val Pro Phe Asp Cys Lys Glu Leu Leu Tyr Tyr             180 185 190 Asp Tyr Ala Pro Leu Pro Glu His Ala Ala Lys Ala Val Asn Ala Arg         195 200 205 Arg Val Glu Asp Leu Lys Glu Phe Val Ser Gln Cys Asp Val Val Thr     210 215 220 Val Asn Ala Pro Leu His Glu Gly Thr Arg Gly Leu Val Asn Ala Glu 225 230 235 240 Leu Leu Lys His Phe Lys Lys Gly Ala Trp Leu Val Asn Thr Ala Arg                 245 250 255 Gly Ala Ile Cys Asp Lys Asp Ala Val Ala Glu Ala Leu Lys Ser Gly             260 265 270 Gln Leu Ala Gly Tyr Ala Gly Asp Val Trp Asn Val Gln Pro Ala Pro         275 280 285 Lys Asp His Val Trp Arg Thr Met Lys Asn Pro Leu Gly Gly Gly Asn     290 295 300 Gly Met Val Pro His Tyr Ser Gly Thr Thr Leu Asp Ala Gln Ala Arg 305 310 315 320 Tyr Ala Gly Thr Arg Thr Ile Leu Glu Asn Tyr Leu Lys Asn Lys                 325 330 335 Pro Gln Glu Pro Gln Asn Val Val Gly Ile Gly Lys Tyr Glu Thr             340 345 350 Thr Ala Tyr Gly Gln Arg         355

Claims (8)

A recombinant microorganism which contains a nucleotide sequence encoding an acidic formate dehydrogenase derived from the genus Thiobacillus sp. And which produces formic acid by reducing carbon dioxide in an acidic environment below pH 7,
Wherein the nucleotide sequence encoding the acidic formic acid dehydrogenase is at least one selected from the following (a) and (b):
(a) a nucleotide sequence encoding a polypeptide of Sequence Listing 4;
(b) a sequence complementary to (a) above.
delete The method according to claim 1,
Wherein the recombinant microorganism is one wherein a vector expressing an acidic formate dehydrogenase is introduced into a host cell selected from the group consisting of E. coli, bacteria, yeast and fungi.
delete A nucleotide sequence encoding an acidic formate dehydrogenase derived from a Thiobacillus sp. Strain, wherein the nucleotide sequence encoding the acidic formate dehydrogenase is
(a) a nucleotide sequence encoding a polypeptide of Sequence Listing 4;
(b) a sequence complementary to the above (a)
, The method comprising reducing carbon dioxide under acidic conditions below pH 7 to produce formic acid by using the recombinant formate dehydrogenase expressed from the recombinant microorganism.
6. The method of claim 5,
Wherein the acidic condition is pH 5.5 or higher and lower than pH 7.
(i) introducing an expression vector comprising a nucleotide sequence encoding an acidic formate dehydrogenase derived from a strain of the genus Thiobacillus sp. into a host cell,
Wherein the nucleotide sequence encoding the acidic formate dehydrogenase is at least one selected from the following (a) and (b):
(a) a nucleotide sequence encoding a polypeptide of Sequence Listing 4;
(b) a sequence complementary to the above (a)
(ii) culturing the recombinant microorganism obtained above to express the recombinant formate dehydrogenase,
(iii) isolating the recombinant dehydrogenase expressed in the above step, and
(iv) a step of producing formic acid by reducing carbon dioxide under an acidic condition of less than pH 7 using the recombinant formate dehydrogenase obtained in the above step
&Lt; / RTI &gt;
8. The method of claim 7,
Wherein the acidic condition is pH 5.5 or higher and lower than pH 7.

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