Formate dehydrogenase mutant and use thereof

Abstract

Provided in the present invention are a formate dehydrogenase mutant and use thereof. The formate dehydrogenase mutant comprises: (a) a protein having an amino acid sequence set forth in SEQ ID NO: 1; or (b) a protein that has been subjected to amino acid mutation at at least one of the following sites of the amino acid sequence in (a): A10, C23, K47, E53, I103, K109, V120, H148, V152, A168, L184, N187, E202, F285, Q287, T321, or K328, and that possesses formate dehydrogenase activity; and (c) a protein having no less than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having a formate dehydrogenase function. The formate dehydrogenase mutant of the present application exhibits good tolerance and high activity under extreme conditions and demonstrates good suitability for industrial scale-up with low costs and high yield.

Description

Formate dehydrogenase mutants and their applications

[0001] This application is based on and claims priority to Chinese application CN application number 202411430302.2 filed on October 14, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of enzyme reaction technology, and more specifically, to a formate dehydrogenase mutant and its applications. Background Technology

[0003] Most oxidoreductases rely on coenzymes NADPH or NADH for catalytic reactions and are important biocatalysts for the synthesis of chiral compounds. However, coenzymes are consumed as products are generated, and the high cost of NAD(P)H hinders its large-scale production. Therefore, a second oxidoreductase can be added to the reaction system to recover cofactors by sacrificing inexpensive substrates, thus constructing an efficient and low-cost cofactor regeneration system.

[0004] Formate dehydrogenase (FDH) is an important coenzyme regeneration tool that uses formate as a cosubstrate to reduce NAD. + Obtain NADH, or reduce NADP + When NADPH is obtained, the generated byproduct CO2 overflows from the reaction system, thereby driving the reaction toward the formation of NADH or NADPH.

[0005] In patent (CN 115354034 A), a NADP-dependent formate dehydrogenase mutant (different from the enzyme used in this application), a coenzyme regeneration system, and its application in the preparation of L-glufosinate ammonium salt are disclosed. The substrate conversion rate of 200mM is as high as 100%, the byproduct carbon dioxide is easily discharged from the reaction system, and the product separation and purification are more convenient, showing good application prospects. In patent (CN 112662637 A), a formate dehydrogenase mutant and its application in biocatalytic reaction to prepare (3R,5R)-3,5-dihydroxyhexanoate compounds are disclosed. This can significantly improve the substrate conversion rate, obtain products with high optical purity, and greatly reduce the production cost of pharmaceutical intermediates.

[0006] After formate dehydrogenase dehydrogenates, the byproduct is carbon dioxide, which does not increase the amount of wastewater to be treated. Moreover, the reaction conditions are mild and environmentally friendly, making it a suitable coenzyme for production. However, existing formate dehydrogenases have problems such as low conversion rate and poor tolerance. Summary of the Invention

[0007] The main objective of this invention is to provide a formate dehydrogenase mutant and its application, in order to solve the problem of poor activity of wild-type formate dehydrogenase in the prior art.

[0008] To achieve the above objectives, according to a first aspect of the present invention, a formate dehydrogenase mutant is provided, comprising: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having formate dehydrogenase activity at at least one of the following sites in the amino acid sequence of (a): A10, C23, K47, E53, I103, K109, V120, H148, V152, A168, L184, N187, E202, F285, Q287, T321 or K328; and (c) a protein having more than 80% homology with the amino acid sequence defined in either (a) or (b).

[0009] Furthermore, the amino acid mutations in (b) above are each independently selected from the following: A10C; C23S; K47C or K47T or K47V; E53V or E53T or E53A or E53I; I103G; K109I or K109R or K109P or K109N; V120T or V120A; H148G or H148K or H148Y or H148R; V152I; A168G; L184V or L184K or L184R; N187R; E202A or E202D; F285Q or F285S or F285M or F285W; Q287E; T321K or T321R or T321S or T321A or T321Q; K328S or K328R or K328M; wherein the letter before the number represents the original amino acid and the letter after the number represents the mutant amino acid; preferably, in (c), the protein has more than 85%, more preferably more than 90%, more preferably more than 95%, and even more preferably more than 99% homology with the amino acid sequence defined in (a) or (b) and has formate dehydrogenase function.

[0010] Furthermore, the mutations in the aforementioned formate dehydrogenase mutants include any one of the following amino acid mutations: N187R; N187R+A10C; N187R+C23S; N187R+K47C; N187R+K47T; N187R+K47V; N187R+E53V; N187R+E53T; N187R+E53A; N187R+E53I; N187R+I103G; N187R+K109I; N187R+K109R; N187R+K109P; N187R+K109N; N187R+V120T; N187R+V120A; N187R+H148G; N187R+H148K; N1 87R+H148Y; N187R+H148R; N187R+V152I; N187R+A168G; N187R+L184V; N18 7R+L184K; N187R+L184R; N187R+E202A; N187R+E202D; N187R+F285Q; N187R +F285S; N187R+F285M; N187R+F285W; N187R+Q287E; N187R+T321K; N187R+T 321R; N187R+T321S; N187R+T321A; N187R+T321Q; N187R+K328S; N187R+K32 8R; N187R+K328M; N187R+K328S+A10C; N187R+K328S+K47C; N187R+K328S+ K47T; N187R+K328S+K47V; N187R+K328S+E53V; N187R+K328S+E53A; N187R+ K328S+E53I; N187R+K328S+I103G; N187R+K328S+K109I; N187R+K328S+K10 9R; N187R+K328S+K109P; N187R+K328S+K109N; N187R+K328S+V120L; N187R +K328S+V120G; N187R+K328S+H148G; N187R+K328S+H148K; N187R+K328S+ H148Y; N187R+K328S+H148R; N187R+K328S+V152I; N187R+K328S+A168G; N1 87R+K328S+L184V; N187R+K328S+L184K; N187R+K328S+L184R; N187R+K328 S+F285Q; N187R+K328S+F285S; N187R+K328S+F285M; N187R+K328S+F285W;N187R+K328S+Q287E; N187R+K328S+T321K; N187R+K328S+T321A; N187R+K328S+T321Q; N187R+K328S+K109R+A10C; N187R+K328S+K109R+K47C; N187 R+K328S+K109R+K47T; N187R+K328S+K109R+K47V; N187R+K328S+K109R+E53V; N187R+K328S+K109R+E53T; N187R+K328S+K109R+E53A; N187R+K328S+ K109R+E53I;N187R+K328S+K109R+H148G;N187R+K328S+K109R+H148K;N1 87R+K328S+K109R+H148Y;N187R+K328S+K109R+H148R;N187R+K328S+K10 9R+V152I;N187R+K328S+K109R+A168G;N187R+K328S+K109R+L184V;N187 R+K328S+K109R+L184K;N187R+K328S+K109R+L184R;N187R+K328S+K109R+ F285Q; N187R+K328S+K109R+F285S; N187R+K328S+K109R+F285M; N187R+K328S+K109R+F285W; N187R+K328S+K109R+T321K; N187R+K328S+K109R+T3 21R;N187R+K328S+K109R+T321S;N187R+K328S+K109R+T321A;N187R+K32 8S+K109R+T321Q;N187R+K328S+K109R+L184K+A10C;N187R+K328S+K109R+ L184K+K47C;N187R+K328S+K109R+L184K+K47T;N187R+K328S+K109R+L18 4K+K47V;N187R+K328S+K109R+L184K+E53V;N187R+K328S+K109R+L184K+ E53T; N187R+K328S+K109R+L184K+E53A; N187R+K328S+K109R+L184K+E53I; N187R+K328S+K109R+L184K+I103G; N187R+K328S+K109R+L184K+H148G;N187R+K328S+K109R+L184K+H148K;N187R+K328S+K109R+L184K+H148Y;N187R+K328S+K109R+L184K+H148R;N187R+K328S+K109R+L184K+V152I;N187R+K328S+K109R+L184K+A168G;N187R+K328S+K109R+L184K+F285Q;N187R+K328S+K109R+L184K+F285S;N187R+K328S+K109R+L184K+F285M;N187R+K328S+K109R+L184K+F285W;N187R+K328S+K109R+L184K+Q287E;N187R+K328S+K109R+L184K+T321K;N187R+K328S+K109R+L184K+T321A;N187R+K328S+K109R+L184K+T321Q;N187R+K328S+K109R+L184K+I103G+A10C;N187R+K328S+K109R+L184K+I103G+K47C;N187R+K328S+K109R+L184K+I103G+K47T;N187R+K328S+K109R+L184K+I103G+K47V;N187R+K328S+K109R+L184K+I103G+E53V;N187R+K328S+K109R+L184K+I103G+E53T;N187R+K328S+K109R+L184K+I103G+E53A;N187R+K328S+K109R+L184K+I103G+E53I;N187R+K328S+K109R+L184K+I103G+V120A;N187R+K328S+K109R+L184K+I103G+H148G;N187R+K328S+K109R+L184K+I103G+H148K;N187R+K328S+K109R+L184K+I103G+H148Y;N187R+K328S+K109R+L184K+I103G+H148R;N187R+K328S+K109R+L184K+I103G+E202D;N187R+K328S+K109R+L184K+I103G+F285S;N187R+K328S+K109R+L184K+I103G+F285M;N187R+K328S+K109R+L184K+I103G+F285W;N187R+K328S+K109R+L184K+I103G+T321K;N187R+K328S+K109R+L184K+I103G+T321S;N187R+K328S+K109R+L184K+I103G+T321A;N187R+K328S+K109R+L184K+I103G+T321Q;N187R+K328S+K109R+L184K+I103G+H148G+A10C;N187R+K328S+K109R+L184K+I103G+H148G+K47C;N187R+K328S+K109R+L184K+I103G+H148G+K47T;N187R+K328S+K109R+L184K+I103G+H148G+K47V;N187R+K328S+K109R+L184K+I103G+H148G+E53V;N187R+K328S+K109R+L184K+I103G+H148G+E53T;N187R+K328S+K109R+L184K+I103G+H148G+E53A;N187R+K328S+K109R+L184K+I103G+H148G+E53I;N187R+K328S+K109R+L184K+I103G+H148G+V120G;N187R+K328S+K109R+L184K+I103G+H148G+V152I;N187R+K328S+K109R+L184K+I103G+H148G+A168G;N187R+K328S+K109R+L184K+I103G+H148G+E202D;N187R+K328S+K109R+L184K+I103G+H148G+F285Q;N187R+K328S+K109R+L184K+I103G+H148G+F285M;N187R+K328S+K109R+L184K+I103G+H148G+F285W;N187R+K328S+K109R+L184K+I103G+H148G+T321S;N187R+K328S+K109R+L184K+I103G+H148G+T321A;N187R+K328S+K109R+L184K+I103G+H148G+T321Q;N187R+K328S+K109R+L184K+I103G+H148G+K47T+A10C;N187R+K328S+K109R+L184K+I103G+H148G+K47T+E53V;N187R+K328S+K109R+L184K+I103G+H148G+K47T+E53I; N187R+K328S+K109R+ L184K+I103G+H148G+K47T+V120A; N187R+K328S+K109R+L184K+I103G+H148G+ K47T+A168G; N187R+K328S+K109R+L184K+I103G+H148G+K47T+E202D; N187R+ K328S+K109R+L184K+I103G+H148G+K47T+F285Q; N187R+K328S+K109R+L184K+ I103G+H148G+K47T+F285S; N187R+K328S+K109R+L184K+I103G+H148G+K47T+ F285M; N187R+K328S+K109R+L184K+I103G+H148G+K47T+F285W; N187R+K328S+ K109R+L184K+I103G+H148G+K47T+T321K; N187R+K328S+K109R+L184K+I103G +H148G+K47T+T321A; N187R+K328S+K109R+L184K+I103G+H148G+K47T+T321Q. ;

[0011] To achieve the above objectives, according to a second aspect of the present invention, a DNA molecule is provided that encodes the aforementioned formate dehydrogenase mutant.

[0012] Furthermore, the DNA molecule is selected from: 1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; 2) a polynucleotide having 80% or more, preferably 90% or more, and more preferably 95% or more homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2.

[0013] To achieve the above objectives, according to a third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is linked with the aforementioned DNA molecule.

[0014] To achieve the above objectives, according to a fourth aspect of the present invention, a host cell is provided, wherein the host cell is transformed with the above-described recombinant plasmid.

[0015] To achieve the above objectives, according to a fifth aspect of the present invention, a method for energy cycling using the aforementioned formate dehydrogenase mutant is provided, the method comprising: formate and NAD... + Under the action of the aforementioned formate dehydrogenase mutant, NADH is formed.

[0016] To achieve the above objectives, according to a sixth aspect of the present invention, a method for preparing non-natural amino acids is provided, the method comprising using the aforementioned formate dehydrogenase mutant, in the presence of amino acid dehydrogenase, formate, and NAD+. + Under the influence of [the substance], the α-keto acid substrate shown in Formula I is reacted to obtain non-natural amino acids;

[0017] R is selected from substituted aryl or unsubstituted aryl, C2-C8 substituted heterocyclic or unsubstituted heterocyclic, C3-C8 substituted cycloalkyl or unsubstituted cycloalkyl, C1-C 10 Alkyl, C2-C 10 olefinic group or C2-C 10 The naphthyl group; the substituted heterocyclic group or unsubstituted heteroatom is selected from S atom, O atom or N atom; the substituted aryl group, substituted heterocyclic group, and substituted cycloalkyl group are each independently selected from halogen, O atom or C1-C5 alkyl group.

[0018] Furthermore, the R mentioned above is selected from substituted aryl groups or C4-C8 substituted heterocyclic groups; the heteroatom of the substituted heterocyclic group is an S atom; the substituents of the substituted aryl group and the heterocyclic group are each independently selected from halogens or C1-C5 alkyl groups.

[0019] Furthermore, the above-mentioned α-keto acid substrates are selected from...

[0020] By applying the technical solution of this invention, the formate dehydrogenase mutant of this application has good tolerance and high activity under extreme conditions, can efficiently synthesize non-natural amino acids, can be well used for industrial scale-up, has low cost and high yield, and realizes true green chemistry. Detailed Implementation

[0021] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0022] As mentioned in the background section, wild-type formate dehydrogenase has low efficiency in synthesizing non-natural amino acids and poor tolerance, requiring large quantities in practical applications. This is especially problematic when the reaction substrate requires high temperatures or organic solvents to dissolve, hindering efficient industrial production. Therefore, in this application, the inventors attempted to modify formate dehydrogenase through directed evolution to improve its enzymatic activity and tolerance to extreme conditions, thereby increasing production efficiency in industrial production. This led to the proposed series of protective solutions in this application.

[0023] In a first typical embodiment of this application, a formate dehydrogenase mutant is provided, comprising: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having formate dehydrogenase activity by mutating at least one of the following sites in the amino acid sequence of (a): A10, C23, K47, E53, I103, K109, V120, H148, V152, A168, L184, N187, E202, F285, Q287, T321 or K328; and (c) a protein having more than 80% homology with the amino acid sequence defined in either (a) or (b).

[0024] The amino acid sequence shown in SEQ ID NO:1 is a formate dehydrogenase derived from Candida boidinii. Computer simulation analysis of the enzyme's model structure using homology modeling of this amino acid sequence predicted the active site for non-natural amino acid synthesis reactions, identifying 16 amino acid residues including: A10, C23, K47, E53, I103, K109, V120, H148, V152, A168, L184, E202, F285, Q287, T321, and K328. These amino acid sites may affect the protein's catalytic activity and stability. Mutating these amino acid sites can yield proteins with formate dehydrogenase function, or even enhanced formate dehydrogenase function. For the obtained proteins, changes can be made at non-critical mutation sites and active sites to obtain proteins with over 80% homology to the above amino acid sequence and formate dehydrogenase function.

[0025] The sequence of SEQ ID NO:1 is as follows:

[0026] In a preferred embodiment, the amino acid mutations in (b) are each independently selected from the following: A10C; C23S; K47C or K47T or K47V; E53V or E53T or E53A or E53I; I103G; K109I or K109R or K109P or K109N; V120T or V120A; H148G or H148K or H148Y or H148R; V152I; A168G; L184V or L184K or L184R; N187R; E202A or E202 D; F285Q or F285S or F285M or F285W; Q287E; T321K or T321R or T321S or T321A or T321Q; K328S or K328R or K328M; wherein, the letter before the number represents the original amino acid, and the letter after the number represents the mutant amino acid; preferably, in (c), the protein has more than 85%, more preferably more than 90%, more preferably more than 95%, and even more preferably more than 99% homology with the amino acid sequence defined in (a) or (b) and has formate dehydrogenase function.

[0027] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0028] Substitution and replacement rules generally apply to amino acids with similar properties; the effects of substitution are similar. For example, conserved amino acid substitutions can occur in the aforementioned homologous proteins. "Conserved amino acid substitutions" include, but are not limited to:

[0029] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0030] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0031] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0032] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0033] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0034] In this application, the applicant further investigated the aforementioned active site and discovered that mutations to different amino acids at the active site resulted in varying protein activities, with specific mutations enhancing formate dehydrogenase activity. Experimental investigations revealed that specific mutations at the active site could yield proteins with enhanced activity. For the amino acid mutation sites of formate dehydrogenase proteins, flexible selection and combinations of the aforementioned mutations are possible.

[0035] In a preferred embodiment, the formate dehydrogenase mutant mutation includes any one of the following amino acid mutations: N187R; N187R+A10C; N187R+C23S; N187R+K47C; N187R+K47T; N187R+K47V; N187R+E53V; N187R+E53T; N187R+E53A; N187R+E53I; N187R+I103G; N187R+K109I; N187R+K109R; N187R+K109P; N187R+K109N; N187R+V120T; N187R+V120A; N187R+H148G; N187R+H148K ;N187R+H148Y; N187R+H148R; N187R+V152I; N187R+A168G; N187R+L184V; N 187R+L184K; N187R+L184R; N187R+E202A; N187R+E202D; N187R+F285Q; N18 7R+F285S; N187R+F285M; N187R+F285W; N187R+Q287E; N187R+T321K; N187R +T321R; N187R+T321S; N187R+T321A; N187R+T321Q; N187R+K328S; N187R+K 328R; N187R+K328M; N187R+K328S+A10C; N187R+K328S+K47C; N187R+K328S +K47T; N187R+K328S+K47V; N187R+K328S+E53V; N187R+K328S+E53A; N187R +K328S+E53I; N187R+K328S+I103G; N187R+K328S+K109I; N187R+K328S+K1 09R; N187R+K328S+K109P; N187R+K328S+K109N; N187R+K328S+V120L; N187 R+K328S+V120G; N187R+K328S+H148G; N187R+K328S+H148K; N187R+K328S+ H148Y; N187R+K328S+H148R; N187R+K328S+V152I; N187R+K328S+A168G; N1 87R+K328S+L184V; N187R+K328S+L184K; N187R+K328S+L184R; N187R+K328 S+F285Q; N187R+K328S+F285S; N187R+K328S+F285M; N187R+K328S+F285W;N187R+K328S+Q287E; N187R+K328S+T321K; N187R+K328S+T321A; N187R+K328S+T321Q; N187R+K328S+K109R+A10C; N187R+K328S+K109R+K47C; N187 R+K328S+K109R+K47TN187R+K328S+K109R+K47V; N187R+K328S+K109R+E53V; N187R+K328S+K109R+E53T; N187R+K328S+K109R+E53A; N187R+K328S+ K109R+E53I;N187R+K328S+K109R+H148G;N187R+K328S+K109R+H148K;N1 87R+K328S+K109R+H148Y;N187R+K328S+K109R+H148R;N187R+K328S+K10 9R+V152I;N187R+K328S+K109R+A168G;N187R+K328S+K109R+L184V;N187 R+K328S+K109R+L184K;N187R+K328S+K109R+L184R;N187R+K328S+K109R+ F285Q; N187R+K328S+K109R+F285S; N187R+K328S+K109R+F285M; N187R+K328S+K109R+F285W; N187R+K328S+K109R+T321K; N187R+K328S+K109R+T3 21R;N187R+K328S+K109R+T321S;N187R+K328S+K109R+T321A;N187R+K32 8S+K109R+T321Q;N187R+K328S+K109R+L184K+A10C;N187R+K328S+K109R+ L184K+K47C;N187R+K328S+K109R+L184K+K47T;N187R+K328S+K109R+L18 4K+K47V;N187R+K328S+K109R+L184K+E53V;N187R+K328S+K109R+L184K+ E53T; N187R+K328S+K109R+L184K+E53A; N187R+K328S+K109R+L184K+E53I; N187R+K328S+K109R+L184K+I103G; N187R+K328S+K109R+L184K+H148G;N187R+K328S+K109R+L184K+H148K;N187R+K328S+K109R+L184K+H148Y;N187R+K328S+K109R+L184K+H148R;N187R+K328S+K109R+L184K+V152I;N187R+K328S+K109R+L184K+A168G;N187R+K328S+K109R+L184K+F285Q;N187R+K328S+K109R+L184K+F285S;N187R+K328S+K109R+L184K+F285M;N187R+K328S+K109R+L184K+F285W;N187R+K328S+K109R+L184K+Q287E;N187R+K328S+K109R+L184K+T321K;N187R+K328S+K109R+L184K+T321A;N187R+K328S+K109R+L184K+T321Q;N187R+K328S+K109R+L184K+I103G+A10C;N187R+K328S+K109R+L184K+I103G+K47C;N187R+K328S+K109R+L184K+I103G+K47T;N187R+K328S+K109R+L184K+I103G+K47V;N187R+K328S+K109R+L184K+I103G+E53V;N187R+K328S+K109R+L184K+I103G+E53T;N187R+K328S+K109R+L184K+I103G+E53A;N187R+K328S+K109R+L184K+I103G+E53I;N187R+K328S+K109R+L184K+I103G+V120A;N187R+K328S+K109R+L184K+I103G+H148G;N187R+K328S+K109R+L184K+I103G+H148K;N187R+K328S+K109R+L184K+I103G+H148Y;N187R+K328S+K109R+L184K+I103G+H148R;N187R+K328S+K109R+L184K+I103G+E202D;N187R+K328S+K109R+L184K+I103G+F285S;N187R+K328S+K109R+L184K+I103G+F285M;N187R+K328S+K109R+L184K+I103G+F285W;N187R+K328S+K109R+L184K+I103G+T321K;N187R+K328S+K109R+L184K+I103G+T321S;N187R+K328S+K109R+L184K+I103G+T321A;N187R+K328S+K109R+L184K+I103G+T321Q;N187R+K328S+K109R+L184K+I103G+H148G+A10C;N187R+K328S+K109R+L184K+I103G+H148G+K47C;N187R+K328S+K109R+L184K+I103G+H148G+K47T;N187R+K328S+K109R+L184K+I103G+H148G+K47V;N187R+K328S+K109R+L184K+I103G+H148G+E53V;N187R+K328S+K109R+L184K+I103G+H148G+E53T;N187R+K328S+K109R+L184K+I103G+H148G+E53A;N187R+K328S+K109R+L184K+I103G+H148G+E53I;N187R+K328S+K109R+L184K+I103G+H148G+V120G;N187R+K328S+K109R+L184K+I103G+H148G+V152I;N187R+K328S+K109R+L184K+I103G+H148G+A168G;N187R+K328S+K109R+L184K+I103G+H148G+E202D;N187R+K328S+K109R+L184K+I103G+H148G+F285Q;N187R+K328S+K109R+L184K+I103G+H148G+F285M;N187R+K328S+K109R+L184K+I103G+H148G+F285W;N187R+K328S+K109R+L184K+I103G+H148G+T321S;N187R+K328S+K109R+L184K+I103G+H148G+T321A;N187R+K328S+K109R+L184K+I103G+H148G+T321Q;N187R+K328S+K109R+L184K+I103G+H148G+K47T+A10C;N187R+K328S+K109R+L184K+I103G+H148G+K47T+E53V;N187R+K328S+K109R+L184K+I103G+H148G+K47T+E53I; N187R+K328S+K109R+ L184K+I103G+H148G+K47T+V120A; N187R+K328S+K109R+L184K+I103G+H148G+ K47T+A168G; N187R+K328S+K109R+L184K+I103G+H148G+K47T+E202D; N187R+ K328S+K109R+L184K+I103G+H148G+K47T+F285Q; N187R+K328S+K109R+L184K+ I103G+H148G+K47T+F285S; N187R+K328S+K109R+L184K+I103G+H148G+K47T+ F285M; N187R+K328S+K109R+L184K+I103G+H148G+K47T+F285W; N187R+K328S+ K109R+L184K+I103G+H148G+K47T+T321K; N187R+K328S+K109R+L184K+I103G +H148G+K47T+T321A; N187R+K328S+K109R+L184K+I103G+H148G+K47T+T321Q. ;

[0036] All of the above-mentioned amino acid mutations were experimentally investigated in the embodiments of this application, and all of them have formate dehydrogenase activity. Compared with the parent with the amino acid sequence shown in SEQ ID NO: 1, it is possible to obtain formate dehydrogenase mutants with high tolerance to extreme environments (high temperature or organic solvent environment), high enzyme activity, and suitable for industrial scale-up production.

[0037] In a second typical embodiment of this application, a DNA molecule is provided that encodes the aforementioned formate dehydrogenase mutant.

[0038] In a preferred embodiment, the DNA molecule is selected from: 1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; 2) a polynucleotide having 80% or more, preferably 90% or more, and more preferably 95% or more homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2.

[0039] The sequence of SEQ ID NO:2 is as follows:

[0040] In a third typical embodiment of this application, a recombinant plasmid is provided, wherein the recombinant plasmid is linked to the aforementioned DNA molecule.

[0041] The aforementioned DNA can encode the formate dehydrogenase mutant and can be ligated to a recombinant plasmid to form a circular DNA. Both the aforementioned DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosomes, tRNA, etc., to obtain the aforementioned formate dehydrogenase mutant.

[0042] In a fourth typical embodiment of this application, a host cell is provided, wherein the recombinant plasmid described above is transformed within the host cell. This host cell is not of plant origin and can be a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be *Escherichia coli*, and the eukaryotic cell can be yeast.

[0043] Using the aforementioned host cells, recombinant plasmids can be replicated within the host cells, and the DNA molecules carried on the recombinant plasmids can be transcribed and translated to obtain a large number of formate dehydrogenase mutants. Using existing technologies, formate dehydrogenase mutants can be obtained by cleaving and purifying host cells, followed by crude enzyme catalysis or other methods, and then used for subsequent catalysis of substrates to obtain non-natural amino acids.

[0044] In a fifth typical embodiment of this application, a method for energy cycling using the aforementioned formate dehydrogenase mutant is provided, the method comprising: formate and NAD. + Under the action of the aforementioned formate dehydrogenase mutant, NADH is formed. The formate dehydrogenase mutant of this application exhibits good activity and tolerance in any process involving energy cycling (NAD→NADH).

[0045] In a sixth typical embodiment of this application, a method for preparing non-natural amino acids is provided. The method includes using the aforementioned formate dehydrogenase mutant in the reaction of amino acid dehydrogenase, formate, and NAD. + Under the influence of [the substance], the substrate shown in Formula I reacts to obtain non-natural amino acids;

[0046] R is selected from substituted aryl or unsubstituted aryl, C2-C8 substituted heterocyclic or unsubstituted heterocyclic, C3-C8 substituted cycloalkyl or unsubstituted cycloalkyl, C1-C 10 Alkyl, C2-C 10 olefinic group or C2-C 10 The naphthyl group; the substituted heterocyclic group or unsubstituted heteroatom is selected from S atom, O atom or N atom; the substituted aryl group, substituted heterocyclic group, and substituted cycloalkyl group are each independently selected from halogen, O atom or C1-C5 alkyl group.

[0047] Using the above preparation method, the formate dehydrogenase mutant was subjected to formate and NAD+. +Based on its existing structure, it provides NADH for the synthesis of non-natural amino acids from substrates, catalyzing the synthesis of non-natural amino acids. Due to the enhanced activity and tolerance of the formate dehydrogenase mutant, it can catalyze under extreme industrial production conditions, thereby improving production efficiency and reducing industrial production costs.

[0048] In a preferred embodiment, R is selected from substituted aryl groups or C4-C8 substituted heterocyclic groups; the heteroatom of the substituted heterocyclic group is an S atom; the substituents of the substituted aryl group and the heterocyclic group are each independently selected from halogens or C1-C5 alkyl groups.

[0049] In a preferred embodiment, the α-keto acid substrate is selected from... or

[0050] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0051] The formate dehydrogenase mutants obtained in this application were derived through saturation mutagenesis. Saturation mutagenesis is a method that modifies the coding gene of the target protein to obtain mutants in a short period of time in which the target amino acid is replaced by one of 19 other amino acids. This method is not only a powerful tool for targeted protein modification but also an important means of studying protein structure-function relationships. Saturation mutagenesis often yields more ideal evolutionary forms than single-point mutagenesis. Furthermore, saturation mutagenesis excels at addressing problems that site-directed mutagenesis cannot solve. The saturation mutants were constructed using whole-plasmid PCR, followed by digestion of the PCR product with DpnI enzyme to remove the template, and transformation into *E. coli* BL21(DE3).

[0052] The specific process of culturing the formate dehydrogenase mutant enzyme solution in the following embodiments of this application is as follows:

[0053] Take 4 mL of BL21(DE3) mutant strains containing different mutation sites, inoculate them into a 2L Erlenmeyer flask containing 400 mL of LB medium, and incubate at 37℃ with shaking at 200 rpm for 2-3 hours. OD 600 When the pH is 0.6-0.8, add IPTG to a final concentration of 0.06 mM, induce at 25℃ for 18 h, and collect the bacterial cells by centrifugation at 4℃ after induction. Resuspend the bacterial cells in 10 mL of 0.1 M Tris-HCl (pH 8.0) per gram of bacterial sludge, disrupt by sonication, centrifuge, collect the supernatant, and obtain the crude enzyme solution for the catalytic reaction.

[0054] The reaction system used for enzyme activity detection in the following embodiments of this application did not contain the specific substrate for the role of wild-type formate dehydrogenase in the preparation of non-natural amino acids. Since formate dehydrogenase functions as a coenzyme in the preparation of non-natural amino acids, it mainly synthesizes NADH to meet the needs of the main enzyme in the synthesis of non-natural amino acids. After the main enzyme consumes NADH to produce NAD, formate dehydrogenase catalyzes the formation of NADH from NAD, forming a cycle. Therefore, the following embodiments mainly characterize the activity and tolerance of formate dehydrogenase in the NAD→NADH synthesis cycle by detecting its activity and tolerance in the synthesis of non-natural amino acids.

[0055] Example 1

[0056] The enzyme solution was treated at 60℃ and 40% ethanol for 1 h, respectively, and then added to a 0.3 mL reaction system containing 100 mM sodium formate and 10 mM NAD, respectively. The enzyme activity was characterized by the change of OD340 of NADH at 340 nm for 10 min. The results are shown in Table 1.

[0057] Table 1:

[0058] The ratio of decrease to increase in activity compared to the parent material: --- decrease by 10-50 times, -- decrease by 5-10 times, - decrease by 1-5 times, + increase by 1-2 times, ++ increase by 2-10 times, +++ increase by 10-50 times, ++++ increase by 50-100 times, +++++ increase by more than 100 times.

[0059] Continue to mutate to improve activity and tolerance.

[0060] Example 2

[0061] The enzyme solution was treated at 65℃ and 50% ethanol for 1 h, respectively, and then added to a 0.3 mL reaction system containing 100 mM sodium formate and 10 mM NAD, respectively. The enzyme activity was characterized by the change of OD340 of NADH at 340 nm for 10 min. The results are shown in Table 2.

[0062] Table 2:

[0063] The ratio of decrease to increase in activity compared to the parent material: --- decrease by 10-50 times, -- decrease by 5-10 times, - decrease by 1-5 times, + increase by 1-2 times, ++ increase by 2-10 times, +++ increase by 10-50 times, ++++ increase by 50-100 times, +++++ increase by more than 100 times.

[0064] Further combinations of beneficial mutation sites can further enhance activity and tolerance.

[0065] Example 3

[0066] The enzyme solution was treated at 65℃ and 50% ethanol for 3 h, respectively, and then added to a 0.3 mL reaction system containing 100 mM sodium formate and 10 mM NAD, respectively. The enzyme activity was characterized by the change of OD340 of NADH at 340 nm for 10 min. The results are shown in Table 3.

[0067] Table 3:

[0068] The ratio of decrease to increase in activity compared to the parent material: --- decrease by 10-50 times, -- decrease by 5-10 times, - decrease by 1-5 times, + increase by 1-2 times, ++ increase by 2-10 times, +++ increase by 10-50 times, ++++ increase by 50-100 times, +++++ increase by more than 100 times.

[0069] Beneficial mutation sites can be further combined to further enhance activity and tolerance.

[0070] Example 4

[0071] The enzyme solution was treated at 70℃ and 60% ethanol for 3 h, respectively, and then added to a 0.3 mL reaction system containing 100 mM sodium formate and 10 mM NAD, respectively. The enzyme activity was characterized by the change of OD340 of NADH at 340 nm for 10 min. The results are shown in Table 4.

[0072] Table 4:

[0073] The ratio of decrease to increase in activity compared to the parent material: --- decrease by 10-50 times, -- decrease by 5-10 times, - decrease by 1-5 times, + increase by 1-2 times, ++ increase by 2-10 times, +++ increase by 10-50 times, ++++ increase by 50-100 times, +++++ increase by more than 100 times.

[0074] Enzyme activity was measured under other conditions:

[0075] Example 5

[0076] The enzyme solution was treated under other conditions and then added to a 0.3 mL reaction system containing 100 mM sodium formate and 10 mM NAD, respectively. The enzyme activity was characterized by the change in OD340 of NADH at 340 nm for 10 min. The results are shown in Table 5.

[0077] Table 5:

[0078] The ratio of decrease to increase in activity compared to the parent material: --- decrease by 10-50 times, -- decrease by 5-10 times, - decrease by 1-5 times, + increase by 1-2 times, ++ increase by 2-10 times, +++ increase by 10-50 times, ++++ increase by 50-100 times, +++++ increase by more than 100 times.

[0079] A scale-up reaction was carried out with 1g of substrate.

[0080] Example 6

[0081] Based on the best enzyme obtained through evolution, the reaction was scaled up, and 1g was added. or A 100 mL reaction system was prepared using 100 mg amino acid dehydrogenase, 3 eq sodium formate, 5 eq ammonium chloride, 5 mg formate dehydrogenase, 100 mg NAD, and 0.1 M Tris-HCl at pH 9.0. The reaction was carried out at 37 °C, and samples were taken to monitor the reaction time. The specific conversion rates and ee values ​​are shown in Table 6 below.

[0082] Table 6:

[0083] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: by evolving formate dehydrogenase, mutants with improved activity and tolerance are obtained, which can be used to synthesize non-natural amino acids efficiently under high temperature and high organic solvent conditions, with high product yield, greatly reducing waste, saving production costs, and realizing green chemistry.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A mutant formate dehydrogenase enzyme, characterized in that, include: (a) A protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) A protein having undergone an amino acid mutation and possessing formate dehydrogenase activity at at least one of the following sites in the amino acid sequence described in (a): A10, C23, K47, E53, I103, K109, V120, H148, V152, A168, L184, N187, E202, F285, Q287, T321 or K328; (c) A protein having more than 80% homology with the amino acid sequence defined in either (a) or (b) and having formate dehydrogenase function.

2. The formate dehydrogenase mutant according to claim 1, characterized in that, The amino acid mutations in (b) are each independently selected from the following: A10C; C23S; K47C, K47T, or K47V; E53V or E53T or E53A or E53I; I103G; K109I or K109R or K109P or K109N; V120T or V120A; H148G or H148K or H148Y or H148R; V152I; A168G; L184V, L184K, or L184R; N187R; E202A or E202D; F285Q or F285S or F285M or F285W; Q287E; T321K or T321R or T321S or T321A or T321Q; K328S or K328R or K328M; In this context, the letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid. Preferably, in (c), the protein has 85% or more, more preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more homology with the amino acid sequence defined in (a) or (b) and has formate dehydrogenase function.

3. The formate dehydrogenase mutant according to claim 2, wherein The mutations in the formate dehydrogenase mutant include any one of the following amino acid mutations: N187R; N187R+A10C; N187R+C23S; N187R+K47C; N187R+K47T; N187R+K47V; N187R+E53V; N187R+E53T; N187R+E53A; N187R+E53I; N187R+I103G; N187R+K109I; N187R+K109R; N187R+K109P; N187R+K109N; N187R+V120T; N187R+V120A; N187R+H148G; N187R+H148K; N187R+H148Y; N187R+H148R; N187R+V152I; N187R+A168G; N187R+L184V; N187R+L184K; N187R+L184R; N187R+E202A; N187R+E202D; N187R+F285Q; N187R+F285S; N187R+F285M; N187R+F285W; N187R+Q287E; N187R+T321K; N187R+T321R; N187R+T321S; N187R+T321A; N187R+T321Q; N187R+K328S; N187R+K328R; N187R+K328M; N187R+K328S+A10C; N187R+K328S+K47C; N187R+K328S+K47T; N187R+K328S+K47V; N187R+K328S+E53V; N187R+K328S+E53A; N187R+K328S+E53I; N187R+K328S+I103G; N187R+K328S+K109I; N187R+K328S+K109R; N187R+K328S+K109P; N187R+K328S+K109N; N187R+K328S+V120L; N187R+K328S+V120G; N187R+K328S+H148G; N187R+K328S+H148K; N187R+K328S+H148Y; N187R+K328S+H148R; N187R+K328S+V152I; N187R+K328S+A168G; N187R+K328S+L184V; N187R+K328S+L184K; N187R+K328S+L184R; N187R+K328S+F285Q; N187R+K328S+F285S; N187R+K328S+F285M; N187R+K328S+F285W; N187R+K328S+Q287E; N187R+K328S+T321K; N187R+K328S+T321A; N187R+K328S+T321Q; N187R+K328S+K109R+A10C; N187R+K328S+K109R+K47C; N187R+K328S+K109R+K47T; N187R+K328S+K109R+K47V; N187R+K328S+K109R+E53V; N187R+K328S+K109R+E53T; N187R+K328S+K109R+E53A; N187R+K328S+K109R+E53I; N187R+K328S+K109R+H148G; N187R+K328S+K109R+H148K; N187R+K328S+K109R+H148Y; N187R+K328S+K109R+H148R; N187R+K328S+K109R+V152I; N187R+K328S+K109R+A168G; N187R+K328S+K109R+L184V; N187R+K328S+K109R+L184K; N187R+K328S+K109R+L184R; N187R+K328S+K109R+F285Q; N187R+K328S+K109R+F285S; N187R+K328S+K109R+F285M; N187R+K328S+K109R+F285W; N187R+K328S+K109R+T321K; N187R+K328S+K109R+T321R; N187R+K328S+K109R+T321S; N187R+K328S+K109R+T321A; N187R+K328S+K109R+T321Q; N187R+K328S+K109R+L184K+A10C; N187R+K328S+K109R+L184K+K47C; N187R+K328S+K109R+L184K+K47T; N187R+K328S+K109R+L184K+K47V; N187R+K328S+K109R+L184K+E53V; N187R+K328S+K109R+L184K+E53T; N187R+K328S+K109R+L184K+E53A; N187R+K328S+K109R+L184K+E53I; N187R+K328S+K109R+L184K+I103G; N187R+K328S+K109R+L184K+H148G; N187R+K328S+K109R+L184K+H148K; N187R+K328S+K109R+L184K+H148Y; N187R+K328S+K109R+L184K+H148R; N187R+K328S+K109R+L184K+V152I; N187R+K328S+K109R+L184K+A168G; N187R+K328S+K109R+L184K+F285Q; N187R+K328S+K109R+L184K+F285S; N187R+K328S+K109R+L184K+F285M; N187R+K328S+K109R+L184K+F285W; N187R+K328S+K109R+L184K+Q287E; N187R+K328S+K109R+L184K+T321K; N187R+K328S+K109R+L184K+T321A; N187R+K328S+K109R+L184K+T321Q; N187R+K328S+K109R+L184K+I103G+A10C; N187R+K328S+K109R+L184K+I103G+K47C; N187R+K328S+K109R+L184K+I103G+K47T; N187R+K328S+K109R+L184K+I103G+K47V; N187R+K328S+K109R+L184K+I103G+E53V; N187R+K328S+K109R+L184K+I103G+E53T; N187R+K328S+K109R+L184K+I103G+E53A; N187R+K328S+K109R+L184K+I103G+E53I; N187R+K328S+K109R+L184K+I103G+V120A; N187R+K328S+K109R+L184K+I103G+H148G; N187R+K328S+K109R+L184K+I103G+H148K; N187R+K328S+K109R+L184K+I103G+H148Y; N187R+K328S+K109R+L184K+I103G+H148R; N187R+K328S+K109R+L184K+I103G+E202D; N187R+K328S+K109R+L184K+I103G+F285S; N187R+K328S+K109R+L184K+I103G+F285M; N187R+K328S+K109R+L184K+I103G+F285W; N187R+K328S+K109R+L184K+I103G+T321K; N187R+K328S+K109R+L184K+I103G+T321S; N187R+K328S+K109R+L184K+I103G+T321A; N187R+K328S+K109R+L184K+I103G+T321Q; N187R+K328S+K109R+L184K+I103G+H148G+A10C; N187R+K328S+K109R+L184K+I103G+H148G+K47C; N187R+K328S+K109R+L184K+I103G+H148G+K47T; N187R+K328S+K109R+L184K+I103G+H148G+K47V; N187R+K328S+K109R+L184K+I103G+H148G+E53V; N187R+K328S+K109R+L184K+I103G+H148G+E53T; N187R+K328S+K109R+L184K+I103G+H148G+E53A; N187R+K328S+K109R+L184K+I103G+H148G+E53I; N187R+K328S+K109R+L184K+I103G+H148G+V120G; N187R+K328S+K109R+L184K+I103G+H148G+V152I; N187R+K328S+K109R+L184K+I103G+H148G+A168G; N187R+K328S+K109R+L184K+I103G+H148G+E202D; N187R+K328S+K109R+L184K+I103G+H148G+F285Q; N187R+K328S+K109R+L184K+I103G+H148G+F285M; N187R+K328S+K109R+L184K+I103G+H148G+F285W; N187R+K328S+K109R+L184K+I103G+H148G+T321S; N187R+K328S+K109R+L184K+I103G+H148G+T321A; N187R+K328S+K109R+L184K+I103G+H148G+T321Q; N187R+K328S+K109R+L184K+I103G+H148G+K47T+A10C; N187R+K328S+K109R+L184K+I103G+H148G+K47T+E53V; N187R+K328S+K109R+L184K+I103G+H148G+K47T+E53I; N187R+K328S+K109R+L184K+I103G+H148G+K47T+V120A; N187R+K328S+K109R+L184K+I103G+H148G+K47T+A168G; N187R+K328S+K109R+L184K+I103G+H148G+K47T+E202D; N187R+K328S+K109R+L184K+I103G+H148G+K47T+F285Q; N187R+K328S+K109R+L184K+I103G+H148G+K47T+F285S; N187R+K328S+K109R+L184K+I103G+H148G+K47T+F285M; N187R+K328S+K109R+L184K+I103G+H148G+K47T+F285W; N187R+K328S+K109R+L184K+I103G+H148G+K47T+T321K; N187R+K328S+K109R+L184K+I103G+H148G+K47T+T321A; N187R+K328S+K109R+L184K+I103G+H148G+K47T+T321Q.

4. A DNA molecule, characterized in that, The DNA molecule encodes the formate dehydrogenase mutant according to any one of claims 1 to 3.

5. The DNA molecule of claim 4, wherein, The DNA molecules are selected from: 1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; 2) A polynucleotide that is 80% or more, preferably 90% or more, more preferably 95% or more homologous to a polynucleotide sequence as shown in SEQ ID NO:

2.

6. A recombinant plasmid, characterized in that, The recombinant plasmid is ligated with the DNA molecule as described in claim 4 or 5.

7. A host cell, characterized in that, The host cell is transformed with the recombinant plasmid as described in claim 6.

8. A method for energy recycling using the formate dehydrogenase mutant according to any one of claims 1 to 3, characterized by, The method includes: Formate and NAD + NADH is formed under the action of the formate dehydrogenase mutant of any one of claims 1 to 3.

9. A method for producing a non-natural amino acid, characterized by, The production method includes reacting an α-keto acid substrate represented by Formula I under the action of an amino acid dehydrogenase, a formate salt, and NAD + to obtain the unnatural amino acid. R is selected from substituted aryl or unsubstituted aryl, C2-C8substituted heterocyclyl or unsubstituted heterocyclyl, C3-C8substituted cycloalkyl or unsubstituted cycloalkyl, C1-C 10 alkyl, C2-C 10 alkenyl or C2-C 10 naphthyl; The substituted heterocyclic group or unsubstituted heteroatom is selected from S atom, O atom or N atom; The substituted aryl group, the substituted heterocyclic group, and the substituted cycloalkyl group are each independently selected from halogens, O atoms, or C1-C5 alkyl groups.

10. The method of claim 9, wherein, The R is selected from substituted aryl groups or C4-C8 substituted heterocyclic groups; The heteroatom of the substituted heterocyclic group is an S atom; The substituted aryl group and the substituent of the heterocyclic group are each independently selected from halogens or C1-C5 alkyl groups.

11. The preparation method according to claim 9, characterized in that, The alpha-keto acid substrate is selected from

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