Formate dehydrogenase variant and its use

Abstract

By efficiently regenerating NADH, a formate dehydrogenase variant capable of improving the production efficiency of high-value-added chemicals that depend on the NADH regeneration cycle system and reducing the production cost of the high-value-added chemicals, and uses thereof are provided. 【Solution means】The amino acid sequence of this formate dehydrogenase variant is obtained by mutation with at least one of N24D, K185S, M334I, and V377T from the amino acid sequence of the wild-type formate dehydrogenase shown in a specific sequence. The purpose is to improve the catalytic activity of reducing oxidized coenzyme I to reduced coenzyme I.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to formate dehydrogenase variants and their uses. relates to.

Background Art

[0002] In the prior art, the regeneration of NADH is often intervened using formate dehydrogenase (FDH ). FDH belongs to the class of D-2-hydroxyacid dehydrogenases, catalyzes the oxidation of formic acid to produce carbon dioxide, and at the same time can reduce oxidized form of nicotinamide adenine dinucleotide (NAD ) to NADH, and plays an important role in NADH regeneration. Also, in the process of NADH regeneration intervened by FDH, the only by-product generated is carbon dioxide. Since carbon dioxide is likely to escape from the reaction system, it does not affect the enzyme activity in the reaction system or the isolation and purification of the product. + ) to NADH, and plays an important role in NADH regeneration. Also, in the process of NADH regeneration intervened by FDH, the only by-product generated is carbon dioxide. Since carbon dioxide is likely to escape from the reaction system, it does not affect the enzyme activity in the reaction system or the isolation and purification of the product. However, the activity of NADH regenerated through existing FDH is low, the production efficiency of high-value-added chemicals that depend on the NADH regeneration cycle system decreases, and the production cost is high. does not affect the enzyme activity in the reaction system or the isolation and purification of the product. However, the activity of NADH regenerated through existing FDH is low, the production efficiency of high-value-added chemicals that depend on the NADH regeneration cycle system decreases, and the production cost is high.

Summary of the Invention

[0003] The present invention adopts the following technical solutions. In a first aspect, the present invention provides a formate dehydrogenase variant, and the amino acid sequence of the formate dehydrogenase variant is obtained by mutation of at least one of N24D, K185S, M334I, and V377T from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO: 1. The amino acid sequence of the formate dehydrogenase variant is obtained by mutation of at least one of N24D, K185S, M334I, and V377T from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO: 1. obtained. obtained. ​​Here, N24D, K185S, M334I, and V377T are all standard substitutions using the standard one-letter codes for amino acids. The above N24D refers to the amino acid sequence of the formate dehydrogenase variant shown in SEQ ID NO:1, where the asparagine (N) at the 24th position at the N-terminus is substituted with aspartic acid (D). . The above K185S refers to the amino acid sequence of the formate dehydrogenase variant shown in SEQ ID NO:1, where the lysine (K) at the 185th position at the N-terminus is substituted with serine (S). The above M334I refers to the amino acid sequence of the formate dehydrogenase variant shown in SEQ ID NO:1, where the methionine (M) at the 334th position at the N-terminus is substituted with isoleucine (I). . The above V377T refers to the amino acid sequence of the formate dehydrogenase variant shown in SEQ ID NO:1, where the valine (V) at the 377th position at the N-terminus is substituted with threonine (T). In an embodiment of the first aspect, the formate dehydrogenase variant obtained by mutation with at least one of N24D, K185S, M334I, V377T may be as follows: The first formate dehydrogenase variant, the amino acid sequence of the formate dehydrogenase variant, SEQ ID NO :1 is obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 by the N24D mutation, and the first amino acid sequence of the formate dehydrogenase variant is shown in SEQ ID NO:2. The second formate dehydrogenase variant, the amino acid sequence of the formate dehydrogenase variant, SEQ ID NO :1 is obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 by the K185S mutation, and the amino acid sequence of the second formate dehydrogenase variant is shown in SEQ ID NO:3. The third formate dehydrogenase variant, the amino acid sequence of the formate dehydrogenase variant, SEQ ID NO : Obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in Fig. 1 by the M334I mutation, the amino acid sequence of the formate dehydrogenase mutant of the 3rd is shown in SEQ ID NO:4. The 4th formate dehydrogenase mutant, the amino acid sequence of the formate dehydrogenase mutant, SEQ ID NO : Obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in Fig. 1 by the V377T mutation, the amino acid sequence of the 4th formate dehydrogenase mutant is shown in SEQ ID NO:5. The 5th formate dehydrogenase mutant, the amino acid sequence of the formate dehydrogenase mutant, SEQ ID NO : Obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in Fig. 1 by the N24D mutation and the K185S mutation The amino acid sequence of the 5th formate dehydrogenase mutant is shown in SEQ ID NO:6 is shown. The 6th formate dehydrogenase mutant, the amino acid sequence of the formate dehydrogenase mutant, SEQ ID NO : Obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in Fig. 1 by the N24D mutation and the M334I mutation The amino acid sequence of the 6th formate dehydrogenase mutant is shown in SEQ ID NO:7 is shown. The 7th formate dehydrogenase mutant, the amino acid sequence of the formate dehydrogenase mutant, SEQ ID NO : Obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in Fig. 1 by the N24D mutation and the V377T mutation The amino acid sequence of the 7th formate dehydrogenase mutant is shown in SEQ ID NO:8 is shown. The 8th formate dehydrogenase mutant, the amino acid sequence of the formate dehydrogenase mutant, SEQ ID NO : Obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in Fig. 1 by the K185S mutation and the M334I mut ation, the amino acid sequence of the 8th formate dehydrogenase mutant is SEQ ID NO:9 is shown. Ninth formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID NO : obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 by the K185S mutation and the V377T mutation, and the amino acid sequence of the ninth formate dehydrogenase variant is shown in SEQ ID NO:1 0. Tenth formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID N O: obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 by the M334I mutation and the V377T mutation, and the amino acid sequence of the tenth formate dehydrogenase variant is SEQ ID NO :11. Eleventh formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID N O: obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 by the N24D mutation, the K185S mutation and the M334I mutation, and the amino acid sequence of the eleventh formate dehydrogenase variant is SE Q ID NO:12. Twelfth formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID N O: obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 by the N24D mutation, the K185S mutation and the V377T mutation, and the amino acid sequence of the twelfth formate dehydrogenase variant is SE Q ID NO:13. Thirteenth formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID N O: obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 by the K185S mutation, the M334I mutation and the V377T mutation, and the amino acid sequence of the thirteenth formate dehydrogenase variant is S EQ ID NO:14. Fourteenth formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID N The N24D mutation, M334I mutation and V377T mutation are obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in O:1, and the amino acid sequence of the 14th formate dehydrogenase mutant is SE shown in Q ID NO:15. The 15th formate dehydrogenase mutant, the amino acid sequence of the formate dehydrogenase mutant, SEQ ID N The N24D mutation, K185S mutation, M334I mutation and V377T mutation are obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in O:1, and the amino acid sequence of the 15th formate dehydrogenase mutant is shown in SEQ ID NO:16. In a second aspect, the present invention further provides a nucleic acid encoding the above formate dehydrogenase mutant. In a third aspect, the present invention further provides an expression vector containing the above nucleic acid. In a fourth aspect, the present invention further provides a host cell transformed or transfected with the above expression vector. In a fifth aspect, the present invention further provides a method for preparing the above formate dehydrogenase mutant. In a sixth aspect, the present invention further provides the use of the above formate dehydrogenase mutant, nucleic acid, expression vector, host cell or culture prepared by the above preparation method in improving the catalytic activity of reducing oxidized coenzyme I to reduced coenzyme I.

Advantages of the Invention

[0004] The present invention has the following beneficial effects. 1. The present invention provides various formate dehydrogenase mutants that can efficiently catalyze NADH regeneration. These formate dehydrogenase mutants have higher catalytic activity than the original formate dehydrogenase, and as a result, the reduction catalytic effect of formate dehydrogenase mutants from NAD to NADH is further improved. + ​​​​​​The production efficiency of high - value - added chemicals depending on the formate dehydrogenase regeneration cycle system is improved, and the production cost is reduced. 2. The mutant provided by the present invention can effectively improve the regeneration efficiency of reduced coenzyme NADH, and the catalytic efficiency of mutant N24D / K184S / M334I / V377T is 1.5 times that of the wild - type formate dehydrogenase. 3. The formate dehydrogenase mutant provided by the present invention has mild reaction conditions, a wide range of reaction pH values, the reaction temperature is 15 - 40 °C, the reaction pH value is 4 - 10, and its stability is improved compared with that of the wild - type formate dehydrogenase.

Brief Description of the Drawings

[0005]

Figure 1

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Modes for Carrying Out the Invention

[0006] The formate dehydrogenase mutant and its use provided by the examples of the present application relate to the field of biotechnology. By the formate dehydrogenase mutant catalyst, oxidized coenzyme I (Oxdized ​​​form of nicotinamide adenine dinucleoti de, NAD + ) can be reduced to reduced form of nicotin amide adenine dinucleotide, NADH). It can be done. In the existing formate dehydrogenase (FDH) intervention, the NADH regeneration activity is low. As a result, to solve the problem of the background technology that the production efficiency of high-value-added chemicals depending on the NADH regeneration cycle system is reduced and the production cost is high, the examples of the present application provide a formate dehydrogenase variant and its use. This formate dehydrogenase variant is an enzyme with high catalytic activity and can efficiently catalyze a reaction using formate and NAD as substrates to generate carbon dioxide and NADH, improving the reduction of the production efficiency of high-value-added chemicals depending on the NADH regeneration cycle system and reducing its production cost. In the examples of the present application, the term "wild type" refers to a gene + or gene product isolated from a naturally occurring source. A wild-type gene refers to the gene most commonly observed in a population and is thus arbitrarily designed to be the "normal" or "wild-type" form of the gene. Conversely, the terms "modified", "variant" or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitutions, deletions or insertions), post-translational modifications and / or functional properties (e.g., altered properties) compared to the wild-type gene or gene product. It should also be noted that naturally occurring variants can be isolated, and the fact that these variants have properties different from those of the wild-type gene or gene product can improve the reduction of the production efficiency of high-value-added chemicals depending on the NADH regeneration cycle system and reduce its production cost. In the examples of the present application, the term "wild type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene refers to the gene most commonly observed in a population and is thus arbitrarily designed to be the "normal" or "wild-type" form of the gene. Conversely, the terms "modified", "variant" or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitutions, deletions or insertions), post-translational modifications and / or functional properties (e.g., altered properties) compared to the wild-type gene or gene product. It should also be noted that naturally occurring variants can be isolated, and the fact that these variants have properties different from those of the wild-type gene or gene product is noted. Conversely, the terms "modified", "variant" or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitutions, deletions or insertions), post-translational modifications and / or functional properties (e.g., altered properties) compared to the wild-type gene or gene product. Also, it should be noted that naturally occurring variants can be isolated, and the fact that these variants have properties different from those of the wild-type gene or gene product is noted. That is, the terms "modified", "variant" or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitutions, deletions or insertions), post-translational modifications and / or functional properties (e.g., altered properties) compared to the wild-type gene or gene product. Also, it should be noted that naturally occurring variants can be isolated, and the fact that these variants have properties different from those of the wild-type gene or gene product is noted. That is, the fact that these variants have properties different from those of the wild-type gene or gene product It is identified by. Introducing or substituting natural or non-natural amino acids The method is more well-known in this field. In the examples of this application, the relevant information of such common amino acids is shown in Table 1. Note that the examples of this application do not provide the relevant information of all amino acids. When there are other amino acids other than those in Table 1, it should be understood that the relevant information of other amino acids can be obtained by referring to other existing products. Table 1: Abbreviations and corresponding single-letter symbols of common amino acids JPEG0007706046000001.jpg182129 Example 1: The examples of this application provide a formate dehydrogenase mutant, and the amino acid sequence of the formate dehydrogenase mutant is obtained by mutation with at least one of N24D, K185S, M334I, and V377T from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1. SEQ ID NO:1 is shown below. MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH ​​​PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G Here, N24D, K185S, M334I, and V377T all adopt the standard substitution notations of the standard single-letter codes of amino acids. The above N24D refers to the amino acid sequence of the formate dehydrogenase variant shown in SEQ ID NO:1, wherein the asparagine (N) at the 24th position at the N-terminus is substituted with aspartic acid (D). . The above K185S refers to the amino acid sequence of the formate dehydrogenase variant shown in SEQ ID NO:1, wherein the lysine (K) at the 185th position at the N-terminus is substituted with serine (S). The above M334I refers to the amino acid sequence of the formate dehydrogenase variant shown in SEQ ID NO:1, wherein the methionine (M) at the 334th position at the N-terminus is substituted with isoleucine (I). . The above V377T refers to the amino acid sequence of the formate dehydrogenase variant shown in SEQ ID NO:1, wherein the valine (V) at the 377th position at the N-terminus is substituted with threonine (T). In an embodiment of the first aspect, the formate dehydrogenase variant obtained by mutation with at least one of N24D, K185S, M334I, and V377T may be as follows: The first formate dehydrogenase variant, the amino acid sequence of the formate dehydrogenase variant, SEQ ID NO :1 is obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 by the N24D mutation, and the first amino acid sequence of the formate dehydrogenase variant (also called the N24D variant) is shown in SEQ ID NO:2. SEQ ID NO:2 ​MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G The second formate dehydrogenase variant, the amino acid sequence of the formate dehydrogenase variant, SEQ ID NO :1 shows the amino acid sequence of the wild-type formate dehydrogenase obtained by the K185S mutation, and the amino acid sequence of the second formate dehydrogenase variant (also called the K185S variant) is SEQ I D NO:3. SEQ ID NO:3 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G The 3rd formate dehydrogenase variant, the amino acid sequence of the formate dehydrogenase variant, SEQ ID NO : obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO: 1 by the M334I mutation, the amino acid sequence of the 3rd formate dehydrogenase variant (also called the M334I variant) is SEQ I D NO: 4. SEQ ID NO: 4 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G Formate dehydrogenase variant 4, amino acid sequence of formate dehydrogenase variant, SEQ ID NO : obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 by the V377T mutation, the amino acid sequence of formate dehydrogenase variant 4 (also called V377T variant) is SEQ I D NO:5. SEQ ID NO:5 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G Formate dehydrogenase variant 5, amino acid sequence of formate dehydrogenase variant, SEQ ID NO : from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 by the N24D mutation and the K185S mutation Obtained by, and also referred to as the fifth formate dehydrogenase variant (N24D / K185S variant) ) The amino acid sequence is shown in SEQ ID NO:6. SEQ ID NO:6 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G The sixth formate dehydrogenase variant, the amino acid sequence of the formate dehydrogenase variant, SEQ ID NO : The N24D mutation and the M334I mutation from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 Obtained by, and also referred to as the sixth formate dehydrogenase variant (N24D / M334I variant) ) The amino acid sequence is shown in SEQ ID NO:7. SEQ ID NO:7 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G Seventh formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID NO : The N24D mutation and V377T mutation from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 obtained by, and the amino acid sequence of the seventh formate dehydrogenase variant (also referred to as the N24D / V377T variant ) is shown in SEQ ID NO:8. SEQ ID NO:8 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G The 8th formate dehydrogenase variant, the amino acid sequence of the formate dehydrogenase variant, SEQ ID NO : the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO:1 with K185S mutation and M334I mutation, and the amino acid sequence of the 8th formate dehydrogenase variant (also called K185S / M334I variant) is shown in SEQ ID NO:9. SEQ ID NO:9 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G G Ninth formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID NO : obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO: 1 by K185S mutation and V377T mutation, and the amino acid sequence of the ninth formate dehydrogenase variant (also called K185S / V377T variant) is shown in SEQ ID NO: 10. SEQ ID NO: 10 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G Tenth formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID N O: obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO: 1 by M334I mutation and V377T mutation, and the amino acid sequence of the tenth formate dehydrogenase variant (also called M334I / V377T variant) is shown in SEQ ID NO: 11. SEQ ID NO: 11 SEQ ID NO: 11 SEQ ID NO: 11 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G The 11th formate dehydrogenase variant, the amino acid sequence of the formate dehydrogenase variant, SEQ ID N O:1 is obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in O:1 by N24D mutation, K185S mutation and and M334I mutation, and the amino acid sequence of the 11th formate dehydrogenase variant (also called N24D / K185 S / M334I variant) is shown in SEQ ID NO:12. It is shown. SEQ ID NO:12 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G 12th formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID N O: obtained from the amino acid sequence of the wild-type formate dehydrogenase shown in 1 by N24D mutation, K185S mutation and and V377T mutation, the amino acid sequence of the 12th formate dehydrogenase variant (also called N24D / K185 S / V377T variant) is shown in SEQ ID NO:13 as follows. SEQ ID NO:13 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G The 13th formate dehydrogenase variant, the amino acid sequence of the formate dehydrogenase variant, SEQ ID N O: from the amino acid sequence of the wild-type formate dehydrogenase shown in 1, the K185S mutation, the M334I mutation and the V377T mutation, and the amino acid sequence of the 13th formate dehydrogenase variant (also called the K185S / M3 34I / V377T variant) is shown in SEQ ID NO:14 is shown as follows. SEQ ID NO:14 MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G 14th formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID N O:1 shows the N24D mutation, M334I mutation and from the amino acid sequence of the wild-type formate dehydrogenase shown in O:1, and obtained by the V377T mutation. The amino acid sequence of the 14th formate dehydrogenase variant (also called N24D / M334 I / V377T variant) is shown in SEQ ID NO:15 as follows. SEQ ID NO:15 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G 15th formate dehydrogenase variant, amino acid sequence of formate dehydrogenase variant, SEQ ID N O:1 shows the N24D mutation, K185S mutation, from the amino acid sequence of the wild-type formate dehydrogenase shown in O:1, and obtained by the M334I mutation and the V377T mutation. The amino acid sequence of the 15th formate dehydrogenase variant (also called N 24D / K185S / M334I / V377T variant) is S Shown as SEQ ID NO:16. SEQ ID NO:16 MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTP KAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALT AGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYI VNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGR PIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G Example 2: The examples of the present application provide a nucleic acid encoding the formate dehydrogenase variant in Example 1, an expression vector encoding the above nucleic acid, and a host cell transformed or transfected with the above expression vector. Note that the examples of the present application utilize the reported formate dehydrogenase sequence and structure information, perform a non-redundant search from databases such as NCBI, and screen several potential enzyme genes based on principles such as protein structure similarity, conserved site analysis, and host source diversity. These genes are functionally expressed in an E. coli expression system and then purified to obtain a purified formate dehydrogenase variant. Specifically, the above formate dehydrogenase gene is hemizygous and the like. ​​​​​ Transform by directed evolution using a reasonable design, and use formate and NAD + as substrates for catalytic reaction to produce a highly catalytically active formate dehydrogenase mutant that generates carbon dioxide and NADH. The above reaction process is shown in Figure 1. Obtained, and the above reaction process is shown in Figure 1. In the examples of this application, the construction process of the above nucleic acid, the expression vector encoding the above nucleic acid, and the host cell transformed or transfected with the above expression vector is as follows. The construction process of the host cell transformed or transfected with the above expression vector is as follows. as follows. Step 1, construct a nucleic acid encoding the formate dehydrogenase mutant in Example 1 and an expression vector encoding the above nucleic acid. In the examples of this application, by the whole plasmid PCR method, the wild-type gene of formate dehydrogenase of Ancylobacter aquaticus (synthesized by Jinweizhi (Suzhou) Co., Ltd.) was genetically mutated to obtain a target mutant gene (that is, the nucleic acid encoding the formate dehydrogenase mutant in Example 1), and it was constructed on the pET22b plasmid (that is, the expression vector encoding the above nucleic acid). Here, the formate dehydrogenase gene of Ancylobacter aquaticus is shown in SEQ ID NO:17. (that is, the expression vector encoding the above nucleic acid). Here, the formate dehydrogenase gene of Ancylobacter aquaticus is shown in SEQ ID NO:17. gene of Ancylobacter aquaticus is shown in SEQ ID NO:17. SEQ ID NO:17 ATGGCGAAAGTTCTGTGCGTTCTGTACGATGATCCGATCG ATGGTTACCCGACCACCTACGCGCGTGACAACCTGCCGAA AATCGACCACTATCCGGGTGGTCAGACCCTGCCGACCCCG AAAGCGATCGATTTCACTCCGGGCACCATGCTGGGTTCTG TTTCTGGTGAACTGGGCCTGCGTAAATACCTGGAAAGCAA CGGTCACACCCTGGTTGTTACCTCTGATAAAGATGGTCCG GATTCTGTTTTCGAAAAAGAATTGGTTGATGCGGATATTG TTATCAGCCAGCCGTTCTGGCCGGCTTACCTGACCCCGGA ACGCTTCGCTAAAGCTAAAAACCTGAAACTGGCTCTGACC GCGGGCATCGGCTCTGATCACGTTGACCTGCAAAGCGCAA TTGATCGTGGTGTTACCGTTGCGGAAGTGACCTACTGCAA CTCTATCAGCGTGGCGGAACACGTTGTGATGATGATCCTG GGCCTGGTTCGTAACTACCTGCCGGCGCACGACTGGGCGC GTAAAGGTGGCTGGAACATCGCAGATTGCGTGAAACACTC TTACGATCTGGAAGCGATGTCTGTTGGTACTGTGGCGGCG GGCCGCATCGGTCTGGCGGTGCTGCGCCGCCTGGCTCCGT TCGACGTGAAATTACACTATACCGACCGTCACCGCCTGCC GGAAAGCGTTGAAAAAGAACTGAACCTGACCTGGCACGCT TCTCCGACCGATATGTACCCGCACTGCGACGTGGTTACCC TGAACTGCCCGCTGCACCCGGAAACCGAACACATGGTTAA CGAAGAAACCCTGAAACTGTTCAAACGTGGTGCGTACATC GTTAACACCGCGCGTGGTAAACTGTGCGACCGTGATGCGA TCGCGCGCGCGCTGGAAAACGGCACCCTGGCCGGTTATGC GGGCGATGTTTGGTTCCCGCAGCCGGCGCCGGCTGATCAC CCGTGGCGTACTATGGCATGGAACGGCATGACCCCGCACA TGAGCGGCACTAGCCTGACCGCGCAGACCCGTTATGCTGC GGGCACCCGTGAAATCCTGGAATGCTTCTTTGAAGGCCGT CCGATCCGTGATGAATACCTGATCGTTCAGGGCGGTAACC TGGCGGGTGTTGGCGCACACAGCTACTCTAAAGGCAACGC TACCGGTGGTTCTGAAGAAGCGGGTAAATTTAAAAAAGCG GGCTAATCTCTC In the above Ancylobacter aquaticus formate dehydrogenase gene, A represents adenine, T represents thymine, C represents cytosine, G represents guanine, and the primers for the mutation sites N24D, K185S, M334I, and V377T are shown in Table 2. Table 2: Primer table for N24D, K185S, M334I, and V377T JPEG0007706046000002.jpg182129 Hereinafter, the primers for the target mutant gene encoding the formate dehydrogenase mutant in Example 1 will be described. (1) For the primers of the formate dehydrogenase mutants obtained by mutation with any one of N24D, K185S, M334I, and V377T, they are as follows. For the target mutant gene encoding the first formate dehydrogenase mutant (i.e., the nucleic acid encoding the first formate dehydrogenase mutant), its primer is the primer corresponding to N24D (SEQ ​ ID NO: 18 and SEQ ID NO: 19). For the target mutant gene encoding the second formate dehydrogenase variant (i.e., the nucleic acid encoding the second formate dehydrogenase variant), the primers are the primers corresponding to K185S (SEQ ID NO: 20 and SEQ ID NO: 21). For the target mutant gene encoding the third formate dehydrogenase variant (i.e., the nucleic acid encoding the third formate dehydrogenase variant), the primers are the primers corresponding to M334I (SEQ ID NO: 22 and SEQ ID NO: 23). For the target mutant gene encoding the fourth formate dehydrogenase variant (i.e., the nucleic acid encoding the fourth formate dehydrogenase variant), the primers are the primers corresponding to V377T (SEQ ID NO: 24 and SEQ ID NO: 25). For the target mutant gene encoding the fifth formate dehydrogenase variant (i.e., the nucleic acid encoding the fifth formate dehydrogenase variant), the primers are the primers corresponding to N24D and K 185S. For the target mutant gene encoding the sixth formate dehydrogenase variant (i.e., the nucleic acid encoding the sixth formate dehydrogenase variant), the primers are the primers corresponding to N24D and M 334I. For the target mutant gene encoding the seventh formate dehydrogenase variant (i.e., the nucleic acid encoding the seventh formate dehydrogenase variant), the primers are the primers corresponding to N24D and V 377T. (2) The primers for the formate dehydrogenase variants obtained by mutation with any two of N24D, K185S, M334I, and V377T are as follows. For the target mutant gene encoding the fifth formate dehydrogenase variant (i.e., the nucleic acid encoding the fifth formate dehydrogenase variant), the primers are the primers corresponding to N24D and the primers corresponding to K 185S. For the target mutant gene encoding the sixth formate dehydrogenase variant (i.e., the nucleic acid encoding the sixth formate dehydrogenase variant), the primers are the primers corresponding to N24D and the primers corresponding to M 334I. For the target mutant gene encoding the seventh formate dehydrogenase variant (i.e., the nucleic acid encoding the seventh formate dehydrogenase variant), the primers are the primers corresponding to N24D and the primers corresponding to V 377T. For the target mutant gene encoding the seventh formate dehydrogenase variant (i.e., the nucleic acid encoding the seventh formate dehydrogenase variant), the primers are the primers corresponding to N24D and the primers corresponding to V The target mutant gene encoding the 8th formate dehydrogenase variant (i.e., the nucleic acid encoding the 8th formate dehydrogenase variant) has primers corresponding to K185S and primers corresponding to M334I. The target mutant gene encoding the 9th formate dehydrogenase variant (i.e., the nucleic acid encoding the 9th formate dehydrogenase variant) has primers corresponding to K185S and primers corresponding to V377T. The target mutant gene encoding the 10th formate dehydrogenase variant (i.e., the nucleic acid encoding the 10th formate dehydrogenase variant) has primers corresponding to M334I and primers corresponding to V377T. (3) The primers for the formate dehydrogenase variant obtained by the mutation of any three of N24D, K185S, M334I, and V377T are as follows. The target mutant gene encoding the 11th formate dehydrogenase variant (i.e., the nucleic acid encoding the 11th formate dehydrogenase variant) has primers corresponding to N24D, K 185S, and primers corresponding to M334I. The target mutant gene encoding the 12th formate dehydrogenase variant (i.e., the nucleic acid encoding the 12th formate dehydrogenase variant) has primers corresponding to N24D, K 185S, and primers corresponding to V377T. The target mutant gene encoding the 13th formate dehydrogenase variant (i.e., the nucleic acid encoding the 13th formate dehydrogenase variant) has primers corresponding to K185S, primers corresponding to M334I, and primers corresponding to V377T. The target mutant gene encoding the 14th formate dehydrogenase variant (i.e., the nucleic acid encoding the 14th formate dehydrogenase variant) has primers corresponding to N24D, M 334I, and V377T. (4) Primers for the formate dehydrogenase variant obtained by a total of four mutations, N24D, K185S, M334I, and V377T. (4) Primers for the formate dehydrogenase variant obtained by a total of four mutations, N24D, K185S, M334I, and V377T. (4) Primers for the formate dehydrogenase variant obtained by a total of four mutations, N24D, K185S, M334I, and V377T. The target mutant gene encoding the 15th formate dehydrogenase variant (i.e., the nucleic acid encoding the 15th formate dehydrogenase variant) has primers corresponding to N24D, K 185S, M334I, and V377T. (4) Primers for the formate dehydrogenase variant obtained by a total of four mutations, N24D, K185S, M334I, and V377T. (4) Primers for the formate dehydrogenase variant obtained by a total of four mutations, N24D, K185S, M334I, and V377T. The PCR reaction system for the above-mentioned whole plasmid PCR is shown in Table 3. Table 3: PCR reaction system table JPEG0007706046000003.jpg182129 The PCR reaction procedure for the above-mentioned whole plasmid PCR is shown in Table 4. Table 4: PCR reaction procedure table JPEG0007706046000004.jpg182129 After PCR amplification of the target fragment, the amplification products were detected by 0.9% agarose gel electrophoresis. As a result, the amplification products were single bands each about 6000 bp in size. The amplification products were purified and recovered using a DNA gel recovery kit. (4) Primers for the formate dehydrogenase variant obtained by a total of four mutations, N24D, K185S, M334I, and V377T. Finally, the pET22b plasmids of the 1st formate dehydrogenase variant to the 15th formate dehydrogenase variant were obtained, i.e., the expression vectors of the 1st formate dehydrogenase variant, the expression vectors of the 2nd formate dehydrogenase variant, the expression vectors of the 3rd formate dehydrogenase variant, the expression vectors of the 4th formate dehydrogenase variant, and the expression vectors of the 5th formate dehydrogenase variant. (4) Primers for the formate dehydrogenase variant obtained by a total of four mutations, N24D, K185S, M334I, and V377T. (4) Primers for the formate dehydrogenase variant obtained by a total of four mutations, N24D, K185S, M334I, and V377T. (4) Primers for the formate dehydrogenase variant obtained by a total of four mutations, N24D, K185S, M334I, and V377T. Current vector, expression vector of the 6th formate dehydrogenase variant, expression of the 7th formate dehydrogenase variant Current vector, expression vector of the 8th formate dehydrogenase variant, expression of the 9th formate dehydrogenase variant Current vector, expression vector of the 10th formate dehydrogenase variant, 11th formate dehydrogenase variant Expression vector of, expression vector of the 12th formate dehydrogenase variant, 13th formate dehydrogenase variant Expression vector of the variant, expression vector of the 14th formate dehydrogenase variant and expression vector of the 15th formate dehydrogenase Obtain the enzyme variant expression vector. Step 2, construct a host cell that transforms or transfects the above expression vector. Build. In the examples of this application, the Escherichia coli BL21(DE3) strain (hereinafter abbreviated as E. coli BL21 (DE3)) was used as the expression host (i.e., host cell), and the pET22b plasmid, for which successful base sequence analysis was achieved was transfected into E. coli BL21(DE3), and the recombinant mutant expression strain E.coli BL21(DE3) / pET22b-AqFDH was constructed. Specifically, the purified gene fragment was digested with EasyCut endonuclease - DpnI to remove the template, and then recombined with a recombinant enzyme. The recombinant product was transformed into E.coliD H5α recipient cells, spread on the surface of an LB solid medium containing 100 g / mL ampicillin (Ampicillin ), cultured at 37 °C for 14 h, and single colonies were picked out for LB liquid culture, and cultured with shaking at 37 °C and 220 rpm for 16 h. After culturing, sterile glycerol was added to a part of the bacterial solution so that the final glycerol concentration became 25%, then numbered, and stored at 8 0 °C for preparation, and the recombinant mutant plasmid clone strain E.coli DH5α / p was prepared and stored at 80 °C. 0 °C for storage and preparation, and the recombinant mutant plasmid clone strain E.coli DH5α / p Obtain ET22b-AqFDH, centrifuge a portion of the bacterial solution at 8,000 rpm for 3 min to collect cells and extract the plasmid from E. coli DH5α / pET2 2b-AqFDH using a high-purity plasmid mini-extraction kit, and confirm the correctness of the mutation site by sequencing . Finally, the recombinant mutant plasmid clone strains of the first formate dehydrogenase variant to the 15th formate dehydrogenase variant and the corresponding recombinant mutant plasmids were obtained, that is, the host cells of the expression vector transfected with the first formate dehydrogenase variant, the host cells of the expression vector transfected with the second formate dehydrogenase variant, the host cells of the expression vector transfected with the third formate dehydrogenase variant, the host cells of the expression vector transfected with the fourth formate dehydrogenase variant, the host cells of the expression vector transfected with the fifth formate dehydrogenase variant, the host cells of the expression vector transfected with the sixth formate dehydrogenase variant, the host cells of the expression vector transfected with the seventh formate dehydrogenase variant, the host cells of the expression vector transfected with the eighth formate dehydrogenase variant, the host cells of the expression vector transfected with the ninth formate dehydrogenase variant, the host cells of the expression vector transfected with the tenth formate dehydrogenase variant, the host cells of the expression vector transfected with the eleventh formate dehydrogenase variant, the host cells of the expression vector transfected with the twelfth formate dehydrogenase variant, the host cells of the expression vector transfected with the thirteenth formate dehydrogenase variant, the host cells of the expression vector transfected with the fourteenth formate dehydrogenase variant ​​​​​​​​​​​​The host cell of the expression vector to be transfected and the 15th formate dehydrogenase variant are transfected To obtain the host cell of the expression vector to be transfected Examples of this application are the host cells of the expression vector transfected with the above 15th formate dehydrogenase variant (hereinafter referred to as Escherichia coli NJUXR-FX-1) are preserved. The above Escherichia coli NJUXR-FX-1 is a typical Chinese culture preservation center, the preservation location is Wuhan University, Wuhan, China, and the preservation date is January 13, 2025 The preservation number assigned by the preservation unit is CCTCC NO: M 20 25101, and the taxonomic name of the above Escherichia coli NJUXR-FX-1 (also called biological material) is Escherichia coli NJUXR-FX-1, and the Latin name of this taxonomic name is Escherichia coli NJUXR- FX-1 is FX-1 Example 3: The method for preparing the formate dehydrogenase variant described in this example includes constructing a recombinant mutant protein expression strain and obtaining a culture containing the formate dehydrogenase variant In the examples of this application, the successfully analyzed pET22b plasmid of the nucleotide sequence was transfected into E. coli BL21(DE3) as an expression host (i.e., host cell), and the recombinant mutant protein expression strain E. coli BL21(DE3) / pET22b-A qFDH was constructed The above preparation method will be described in detail below The successfully constructed recombinant mutant plasmid was transformed into E. coli BL21(DE3) recipient cells and spread on an ampicillin plate with a final concentration of 100 μg / mL, and single colonies were picked out and transferred to a culture tube of 5 mL of LB liquid medium containing 100 μg / mL ampicillin ​​​​ Inoculate and perform shaking culture at 37 °C and 220 rpm for 16 h to express the recombinant mutant protein The strain E. coli BL21(DE3) / pET22b-AqFDH was obtained. Transfer it to 500 mL of resistant LB medium containing 100 μg / mL ampicillin with an inoculum volume of 3 mL, and when the OD 0 reaches approximately 0.6, add 1 mL of 0.5 M IPTG (i.e., isopropyl-β- 60 D-thiogalactopyranoside), adjust the final concentration of IPTG to 0.5 mM, and induce at 18 °C for about 16 h to obtain a culture containing formate dehydrogenase mutant. Finally, cultures containing the first formate dehydrogenase mutant to the 15th formate dehydrogenase mutant were obtained respectively. After induction, centrifuge to obtain the bacterial cells, resuspend them with buffer, and ultrasonically disrupt the cells under ice bath conditions (operate for 2 s every 5 s, with an operating time of 30 min), and centrifuge at 4 °C and 12,000 rpm / min for 20 min. Collect the supernatant and filter it through a 0.22 μm aqueous filter tip, and use the filtered product as a sample, and subject it to a nickel column to purify the enzyme. From the amino acid sequence of AqFDH, calculate the molar extinction coefficient of the protein, and measure the absorbance of the protein after purification by the A method to calculate the concentration of the protein (a general term for wild-type formate dehydrogenase and formate dehydrogenase mutants). After purification, the absorbance of the protein was measured by the A method to calculate the concentration of the protein (a general term for wild-type formate dehydrogenase and formate dehydrogenase mutants). After purification, the absorbance of the protein was measured by the A method to calculate the concentration of the protein (a general term for wild-type formate dehydrogenase and formate dehydrogenase mutants). Calculate the molar extinction coefficient of the protein, and measure the absorbance of the protein after purification by the A method to calculate the concentration of the protein (a general term for wild-type formate dehydrogenase and formate dehydrogenase mutants). 280 method to calculate the concentration of the protein (a general term for wild-type formate dehydrogenase and formate dehydrogenase mutants). The absorbance of the protein was measured by the A method to calculate the concentration of the protein (a general term for wild-type formate dehydrogenase and formate dehydrogenase mutants). The absorbance of the protein was measured by the A method to calculate the concentration of the protein (a general term for wild-type formate dehydrogenase and formate dehydrogenase mutants). Example 4: The examples of the present application provide the use of the formate dehydrogenase mutants provided by Example 1, the nucleic acids of the formate dehydrogenase mutants provided by Example 2, the expression vectors of the nucleic acids provided by Example 2, the host cells provided by Example 2, or the cultures prepared by the preparation method provided by Example 3 in improving the catalytic activity of reducing nicotinamide adenine dinucleotide phosphate (NADP⁺) to nicotinamide adenine dinucleotide phosphate (NADPH). The absorbance of the protein was measured by the A method to calculate the concentration of the protein (a general term for wild-type formate dehydrogenase and formate dehydrogenase mutants). The absorbance of the protein was measured by the A method to calculate the concentration of the protein (a general term for wild-type formate dehydrogenase and formate dehydrogenase mutants). The absorbance of the protein was measured by the A method to calculate the concentration of the protein (a general term for wild-type formate dehydrogenase and formate dehydrogenase mutants). The absorbance of the protein was measured by the A method to calculate the concentration of the protein (a general term for wild-type formate dehydrogenase and formate dehydrogenase mutants). In one embodiment, a catalyst that reductively catalyzes NAD to NADH is obtained from a culture containing the first formate dehydrogenase variant obtained in Example 3 to the 15th formate dehydrogenase variant. The catalyst may be a whole cell or a pure enzyme solution. to + The catalyst may be a whole cell or a pure enzyme solution. The catalyst may be a whole cell or a pure enzyme solution. In one application scenario, taking the 3rd formate dehydrogenase variant (also called the M334I variant), the 8th formate dehydrogenase variant (also called the K185S / M334I variant), the 6th formate dehydrogenase variant (also called the N24D / M334I variant), the 12th formate dehydrogenase variant (also called the N24D / K185S / V377T variant), and the 15th formate dehydrogenase variant (also called the N24D / K185S / M334I / V377T variant) as examples, the reaction system for reductively catalyzing NAD to NADH is as follows: the final concentration is 0.05 mg / ml of the formate dehydrogenase variant or the whole cell bacterial solution of the recombinant mutant expression strain with OD = 0.1, 1 mM NAD, 5 mM HCOONa, and the reaction buffer is 100 mM potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer with pH = 7. After a 10 - minute reaction, the change in the absorbance value of the product NADH at 340 nm is measured with an enzyme marker, and the relative activities are compared. The results are shown in Figure 2. Compared with the wild - type formate dehydrogenase (corresponding to aqFDH(WT) in Figure 2), in the 3rd formate dehydrogenase variant, the 8th formate dehydrogenase variant, the 6th formate dehydrogenase variant, the 12th formate dehydrogenase variant, and the 15th formate dehydrogenase variant, the efficiency of reductively catalyzing NAD to NADH is significantly improved. to to to to to + to to 600 to to + to to to to to to to + to Here, the results of the catalytic reaction through the whole cell bacterial solution are shown in Table 5. The formate dehydrogenase The results of the catalytic reaction through the pure enzyme solution of the variant are shown in Table 6. Table 5: Catalytic reaction result table of whole cell bacterial solution JPEG0007706046000005.jpg182129 Table 6: Catalytic reaction result table of formate dehydrogenase variant JPEG0007706046000006.jpg182129 As can be seen from Table 5 and Table 6, the catalytic conversion rates of the variants were all higher than that of the wild-type formate dehydrogenase enzyme. In particular, the catalytic efficiency of the 15th formate dehydrogenase variant (also called N24D / K184S / M3 34I / V377T variant) was 1.5 times that of the wild-type formate dehydrogenase catalytic efficiency . From the above, the formate dehydrogenase variants obtained according to the examples of the present application have a higher activity of reducing NAD + to NADH than the wild-type formate dehydrogenase, so the production efficiency of high value-added chemicals dependent on the NADH regeneration cycle system can be improved, and the production cost of the above high value-added chemicals can be reduced. Furthermore, the catalytic conditions of the above catalytic reaction are discussed. (1) Determine the preferred temperature when the formate dehydrogenase variant catalyzes the reduction of NAD + to NADH. Using the whole cell or pure enzyme solution of the formate dehydrogenase variant obtained in Example 2 as a catalyst, the preferred temperature of the catalytic reaction in which the formate dehydrogenase variant catalyzes the reduction of NAD + to NADH was investigated. Taking the 15th formate dehydrogenase variant as an example, the reaction system is as follows: final concentration 0.0 05 mg / ml protein or OD 600 = 0.1 whole cell bacterial solution, 1 mM NAD + , 5 mM HCOONa, the reaction buffer is 100 mM, potassium dihydrogen phosphate / li with pH = 7 It is a potassium hydrogen phosphate buffer. The reaction temperature was controlled at 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C by a water bath, and the absorbance value of the product NADH at 340 nm was detected with an enzyme marker, and the results of comparing the relative activities are shown in Fig. 3. At 50 °C, the optimal catalytic effect was observed, and the enzyme activities were all good within the range of 25 - 55 °C. According to the experiment , the difference in temperature had a great influence on the catalytic action of different mutants, and it was shown that its reaction activity tended to deviate from the normal distribution with the change in temperature. At 50 °C, the optimal catalytic effect was observed, and when deviating from this temperature, the enzyme activity was relatively low. (1) Determine the preferred pH value when the formate dehydrogenase mutant catalytically reduces NAD + to NADH. Using the whole cell or pure enzyme solution of the formate dehydrogenase mutant obtained in Example 2 as a catalyst, the preferred pH value of the catalytic reaction in which the formate dehydrogenase mutant catalytically reduces NAD + to NADH was investigated. Specifically, taking the 15th formate dehydrogenase mutant as an example, the reaction system of the above catalytic reaction is as follows : The whole cell bacterial solution with a final concentration of OD 600 = 0.1, 1 mM NAD + , 5 mM HC OONa, and the reaction buffer is 100 mM potassium dihydrogen phosphate / potassium hydrogen phosphate buffer with pH = 7. The reaction buffer solution is a buffer with different pH values, that is, 100 mM potassium dihydrogen phosphate / potassium hydrogen phosphate buffer (KPi) with pH values of 6, 6.5, 7, 7.5, 8 respectively, 100 mM Tris - HCl buffer with pH values of 8, 8.5, 9, 9.5 respectively, and glycine - sodium hydroxide with pH = 10.25 ( It is a Gly-NaOH buffer solution. The reaction temperature is controlled at 30 °C, and the product N is detected with an enzyme marker The absorbance value of ADH at 340 nm was detected, and the results of comparing the relative activities are shown in Figure 4. The influence of different pH values on the catalytic action of formate dehydrogenase is small. When the pH is 9.5 the optimal catalytic effect was observed. According to the experiment, the influence of different buffer solutions on the enzyme activity is different. When potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer solution is used as the reaction system, the enzyme activity is maintained at a high level. When Tris-HCl buffer solution is used as the reaction system, when pH = 9.5, the activities of wild-type enzyme and mutant reach the maximum value. (2) Determine the preferred kinetic + parameters when the formate dehydrogenase mutant catalytically reduces NAD to NADH. Based on the reaction system of the above catalytic reaction, the reaction rates when the NADH concentrations of wild-type formate dehydrogenase and its mutants are changed are determined. The kinetic parameters are calculated by the double-reciprocal graph method from the reaction rate and the reciprocal of the substrate concentration. The results are shown in Table 7. Compared with wild-type formate dehydrogenase, the Km of the 15th formate dehydrogenase mutant decreases, Kcat increases, and it is shown that the catalytic activity of the mutant is significantly increased. Table 7: Table of kinetic parameters JPEG0007706046000007.jpg182129 Note: Km is the Michaelis constant, r is the reaction rate, and Kcat is the catalytic constant. <Copy of the deposit certificate of the microorganism> JPEG0007706046000008.jpg181129 [Sequence Listing] <st26sequencelisting originalfreetextlanguagecode="ja" dtdversion="V1_3" filenam e="ギ酸脱水素酵素変異体およびその用途.xml" softwarename="WIPO Sequence" software version="2.3.0" productiondate="2025-03-31"> <applicationidentification> <ipofficecode>JP< / ipofficecode> <applicationnumbertext / > <filingdate / > < / applicationidentification> <applicantfilereference> 10284< / applicantfilereference> <earliestpriorityapplicationidentification> <ipofficecode>CN< / ipofficecode> <applicationnumbertext> 202410562403.9< / applicationnumbertext> <filingdate> 2024-05-08< / filingdate> < / earliestpriorityapplicationidentification> <applicantname languagecode="ja">Nanjing University< / applicantname> <applicantnamelatin>Nanjing University< / applicantnamelatin> <inventiontitle languagecode="ja">Formic Acid Dehydrogenase Variant and Its Use< / InventionT itle> <sequencetotalquantity> 25< / sequencetotalquantity> <sequencedata sequenceidnumber="1"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q2"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="2"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q4"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="3"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q6"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="4"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q8"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="5"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q10"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="6"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q12"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="7"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q14"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="8"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q16"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="9"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q18"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="10"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q20"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="11"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q22"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="12"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q24"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="13"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q26"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="14"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q28"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="15"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q30"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="16"> <insdseq> <INSDSeq_length>401< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..401< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q32"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MAKVLCVLYDDPIDGYPTTYARDDLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGP DSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMIL GLVRNYLPAHDWARKGGWNIADCVSHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHA SPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADH PWRTMAWNGMTPHISGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGTGAHSYSKGNATGGSEEAGKFKKA G < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="17"> <insdseq> <INSDSeq_length>1212< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..1212< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q34"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> atggcgaaagttctgtgcgttctgtacgatgatccgatcgatggttacccgaccacctacgcgcgtgacaacctgccgaa aatcgaccactatccgggtggtcagaccctgccgaccccgaaagcgatcgatttcactccgggcaccatgctgggttctg tttctggtgaactgggcctgcgtaaatacctggaaagcaacggtcacaccctggttgttacctctgataaagatggtccg gattctgttttcgaaaaagaattggttgatgcggatattgttatcagccagccgttctggccggcttacctgaccccgga acgcttcgctaaagctaaaaacctgaaactggctctgaccgcgggcatcggctctgatcacgttgacctgcaaagcgcaa ttgatcgtggtgttaccgttgcggaagtgacctactgcaactctatcagcgtggcggaacacgttgtgatgatgatcctg ggcctggttcgtaactacctgccggcgcacgactgggcgcgtaaaggtggctggaacatcgcagattgcgtgaaacactc ttacgatctggaagcgatgtctgttggtactgtggcggcgggccgcatcggtctggcggtgctgcgccgcctggctccgt tcgacgtgaaattacactataccgaccgtcaccgcctgccggaaagcgttgaaaaagaactgaacctgacctggcacgct tctccgaccgatatgtacccgcactgcgacgtggttaccctgaactgcccgctgcacccggaaaccgaacacatggttaa cgaagaaaccctgaaactgttcaaacgtggtgcgtacatcgttaacaccgcgcgtggtaaactgtgcgaccgtgatgcga tcgcgcgcgcgctggaaaacggcaccctggccggttatgcgggcgatgtttggttcccgcagccggcgccggctgatcac ccgtggcgtactatggcatggaacggcatgaccccgcacatgagcggcactagcctgaccgcgcagacccgttatgctgc gggcacccgtgaaatcctggaatgcttctttgaaggccgtccgatccgtgatgaatacctgatcgttcagggcggtaacc tggcgggtgttggcgcacacagctactctaaaggcaacgctaccggtggttctgaagaagcgggtaaatttaaaaaagcg ggctaatctctc < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="18"> <insdseq> <INSDSeq_length>28< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..28< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q36"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gcgcgtgacgacctgccgaaaatcgacc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="19"> <insdseq> <INSDSeq_length>28< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..28< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q38"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>ggtcgattttcggcaggtcgtcacgcgc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="20"> <insdseq> <INSDSeq_length>28< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..28< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q40"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gcagattgcgtgagccactcttacgatc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="21"> <insdseq> <INSDSeq_length>28< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..28< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q42"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gatcgtaagagtggctcacgcaatctgc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="22"> <insdseq> <INSDSeq_length>26< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..26< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q44"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gaccccgcacatcagcggcactagcc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="23"> <insdseq> <INSDSeq_length>26< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..26< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q46"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>ggctagtgccgctgatgtgcggggtc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="24"> <insdseq> <INSDSeq_length>24< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..24< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q48"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>ctggcgggtactggcgcacacagc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="25"> <insdseq> <INSDSeq_length>25< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..25< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q50"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gctgtgtgcgccagtacccgccagg< / INSDSeq_sequence> < / insdseq> < / sequencedata> < / inventiontitle> < / st26sequencelisting>

[0007]

Claims

**Claim 1** A formate dehydrogenase variant, wherein the amino acid sequence of the formate dehydrogenase variant is derived from the amino acid sequence of the wild-type formate dehydrogenase shown in SEQ ID NO: 1 by mutation with at least one of N24D, K185S, M334I, and V377T. A formate dehydrogenase variant characterized by that. **Claim 2** A nucleic acid encoding the formate dehydrogenase variant according to Claim 1. **Claim 3** An expression vector containing the nucleic acid according to Claim 2. ​ ​

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