Formate dehydrogenase mutant and its application

Abstract

The present invention provides a formate dehydrogenase mutant and its application. The formate dehydrogenase mutant includes: (a) a protein having an amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having an amino acid mutation and having formate dehydrogenase activity function at at least one of the following sites: A10, C23, K47, E53, I103, K109, V120, H148, V152, A168, L184, N187, E202, F285, Q287, T321 or K328 in the amino acid sequence in (a); (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having formate dehydrogenase function. The formate dehydrogenase mutant of the application has good tolerance and high activity under extreme conditions, can be well used for industrial amplification, has low cost and high yield.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme reactions, in particular to a formate dehydrogenase mutant and application thereof. Background Art

[0002] Most oxidoreductases rely on coenzymes NADPH or NADH for catalytic reactions and are important biocatalysts for the synthesis of chiral compounds. However, the coenzymes are consumed as the 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 the cofactor at the expense of the cheap substrate, thus constructing an efficient and low-cost cofactor regeneration system.

[0003] Formate dehydrogenase (FDH) is an important coenzyme regeneration enzyme that uses formate as a cosubstrate to reduce NAD + Obtain NADH, or reduce NADP + NADPH is obtained, and the byproduct CO 2 It overflows from the reaction system, thereby pushing the reaction towards the direction of producing NADH or NADPH.

[0004] In the patent (CN 115354034 A), a NADP-dependent formate dehydrogenase mutant (different from the enzyme used in the present application), a coenzyme regeneration system and its application in the preparation of L-glufosinate ammonium salt are disclosed. The conversion rate of 200 mM substrate is as high as 100%, the by-product carbon dioxide is easy to be discharged from the reaction system, and the product separation and purification are more convenient, which has a good application prospect; in the patent (CN 112662637 A), a formate dehydrogenase mutant and its application in the biocatalytic reaction preparation of (3R, 5R)-3,5-dihydroxyhexanoate compounds are disclosed, which can greatly improve the conversion rate of the substrate, obtain products with high optical purity, and greatly reduce the production cost of pharmaceutical intermediates.

[0005] After formate dehydrogenase dehydrogenates, the by-product is carbon dioxide, which will not increase the amount of wastewater treatment. The reaction conditions are mild and environmentally friendly. It is a coenzyme cycle enzyme suitable for production. However, the existing formate dehydrogenase has problems such as low conversion rate and poor tolerance. Summary of the invention

[0006] The main purpose of the present invention is to provide a formate dehydrogenase mutant and its application, so as to solve the problem that the activity of the wild-type formate dehydrogenase in the prior art is poor.

[0007] To achieve the above object, according to a first aspect of the present invention, a formate dehydrogenase mutant is provided, comprising: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein having an amino acid mutation at at least one of the following sites: A10, C23, K47, E53, I103, K109, V120, H148, V152, A168, L184, N187, E202, F285, Q287, T321 or K328 of the amino acid sequence in (a) and having formate dehydrogenase activity; and (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having formate dehydrogenase function.

[0008] Further, 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 letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids; preferably, in (c), the protein has more than 85%, preferably more than 90%, more preferably more than 95%, and further preferably more than 99% homology with the amino acid sequence defined in (a) or (b) and has formate dehydrogenase function.

[0009] Furthermore, the mutation of the formate dehydrogenase mutant includes any 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+H148V; 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;<h2 style=";text-align:left;direction:ltr">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;

[0010] 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; N 187R+K328S+K109R+L184K+I103G+H148G+K47T+A168G; N187R+K328S+K109R+L184K+ I103G+H148G+K47T+E202D; N187R+K328S+K109R+L184K+I103G+H148G+K47T+F285Q; N 187R+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] In order to achieve the above object, according to the second aspect of the present invention, a DNA molecule is provided, which encodes the formate dehydrogenase mutant.

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

[0013] In order to achieve the above object, according to the third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above DNA molecule.

[0014] In order to achieve the above object, according to a fourth aspect of the present invention, a host cell is provided, into which the above recombinant plasmid is transformed.

[0015] In order to achieve the above object, according to a fifth aspect of the present invention, a method for energy cycling using the formate dehydrogenase mutant is provided, the method comprising: + Under the action of the formate dehydrogenase mutant described above, NADH is formed.

[0016] In order to achieve the above object, according to the sixth aspect of the present invention, a method for preparing non-natural amino acids is provided, the preparation method comprising using the formate dehydrogenase mutant to synthesize amino acid dehydrogenase, formate and NAD + Under the action of, reacting the α-keto acid substrate shown in formula I to obtain a non-natural amino acid;

[0017] Formula I;

[0018] R is selected from substituted aryl or unsubstituted aryl, C 2 -C 8 A substituted or unsubstituted heterocyclic group, C 3 -C 8 Substituted cycloalkyl or unsubstituted cycloalkyl, C 1 -C 10 Alkyl, C 2 -C 10 The olefin group or C 2 -C 10 The substituted heterocyclic group or the unsubstituted heteroatom is selected from S atom, O atom or N atom; the substituents of the substituted aryl group, the substituted heterocyclic group and the substituted cycloalkyl group are each independently selected from halogen, O atom or C 1 -C 5 of alkyl.

[0019] Furthermore, the above R is selected from substituted aryl or C 4 -C 8 The heterocyclic group of the substituted heterocyclic group is a S atom; the substituents of the substituted aryl and heterocyclic group are each independently selected from halogen or C 1 -C 5 of alkyl.

[0020] Furthermore, the α-keto acid substrate is selected from , , , , , , , or .

[0021] By applying the technical solution of the present invention, the formate dehydrogenase mutant of the present 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 DESCRIPTION

[0022] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0023] As mentioned in the background technology, the wild-type formate dehydrogenase has low synthesis efficiency for non-natural amino acids and poor tolerance. In practical applications, a large amount of formate dehydrogenase is required, especially when the reaction substrate can only be dissolved under high temperature or organic solvent conditions, and efficient industrial production cannot be carried out. Therefore, in the present application, the inventor attempts to transform formate dehydrogenase by directed evolution, thereby improving the enzyme activity of formate dehydrogenase and its tolerance under extreme conditions, so that it can improve production efficiency in industrial production, and thus proposes a series of protection schemes of the present application.

[0024] In a first typical embodiment of the present application, a formate dehydrogenase mutant is provided, comprising: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein having an 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 having formate dehydrogenase activity; (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having formate dehydrogenase function.

[0025] The amino acid sequence shown in the above SEQ ID NO: 1 is derived from the formate dehydrogenase of Candida boidinii. The model structure of the enzyme was analyzed by computer simulation analysis of homology modeling of the amino acid sequence, and the active site for the non-natural amino acid synthesis reaction was predicted. It was found that 16 amino acid residues included: A10, C23, K47, E53, I103, K109, V120, H148, V152, A168, L184, E202, F285, Q287, T321 and K328. These amino acid sites may affect the catalytic activity and stability of the protein. By mutating the above amino acid sites, a protein with formate dehydrogenase or even enhanced formate dehydrogenase function can be obtained. For the above-obtained protein, changes can be made at non-critical mutation sites and active sites, and a protein with more than 80% homology to the above amino acid sequence and formate dehydrogenase function can be obtained.

[0026] The sequence of SEQ ID NO: 1 is as follows:

[0027] MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGETSELDKHIPDADIIITTPFHPAYITKERLDKAKNLKLVVVAGVGSDHIDLDYINQTGKKISVLEVTGSNVVSVAEHVVMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYDIEGKTIATIGAGRIGYRVLER LLPFNPKELLYYDYQALPKEAEEKVGARRVENIEELVAQADIVTVNAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQTRYAEGTKNILESFFTGKFDYRPQDIILLNGEYVTKAYGKHDKK.

[0028] 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 letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids; preferably, in (c), the protein has more than 85%, preferably more than 90%, more preferably more than 95%, and further preferably more than 99% homology with the amino acid sequence defined in (a) or (b) and has formate dehydrogenase function.

[0029] As used herein, amino acid residues are abbreviated 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).

[0030] Substitution and replacement rules. Generally speaking, amino acids with similar properties will have similar effects after replacement. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" include but are not limited to:

[0031] hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0032] Substitution of hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) by other hydrophobic amino acids with bulky side chains;

[0033] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0034] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0035] A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0036] In the present application, the applicant continues to explore the above active sites and finds that when the active sites are mutated into different amino acids, the corresponding protein activities are also different, and specific mutations can enhance the activity of formate dehydrogenase. Through experimental exploration, it is found that the above specific mutations for the active sites can obtain proteins with enhanced activity. For the amino acid mutation sites of formate dehydrogenase protein, flexible selection and combination can be performed in the above mutations.

[0037] In a preferred embodiment, the mutation of the formate dehydrogenase mutant comprises any 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;<h2 style=";text-align:left;direction:ltr">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. ;

[0038] The above amino acid mutations were all experimentally investigated in the examples of the present application, and all had formate dehydrogenase activity. Compared with the parent having the amino acid sequence shown in SEQ ID NO: 1, formate dehydrogenase mutants with high tolerance to extreme environments (high temperature or organic solvent environment) and high enzyme activity can be obtained, which can be used for industrial scale-up production.

[0039] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the formate dehydrogenase mutant mentioned above.

[0040] In a preferred embodiment, the DNA molecule is selected from: 1) a polynucleotide consisting of the nucleotide sequence as shown in SEQ ID NO: 2; 2) a polynucleotide having more than 80%, more preferably more than 90%, and further preferably more than 95% homology with the polynucleotide consisting of the nucleotide sequence as shown in SEQ ID NO: 2.

[0041] The sequence of SEQ ID NO:2 is as follows:

[0042]

[0043] In a third typical embodiment of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

[0044] The DNA can encode the formate dehydrogenase mutant and can be connected to a recombinant plasmid to form a circular DNA. Both the DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosome, tRNA, etc. to obtain the formate dehydrogenase mutant.

[0045] In a fourth typical embodiment of the present application, a host cell is provided, in which the above-mentioned recombinant plasmid is transformed. The host cell is a host cell of non-plant origin, which can be a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be Escherichia coli, and the eukaryotic cell can be yeast.

[0046] By using the above host cells, the recombinant plasmid can be replicated in the host cells, and the DNA molecules carried on the recombinant plasmid can also be transcribed and translated to obtain a large number of formate dehydrogenase mutants. By using the existing technology, the host cells can be crushed for protein purification, the crude enzyme catalysis after crushing or other methods can be used to obtain formate dehydrogenase mutants, and then the substrate can be catalyzed to obtain non-natural amino acids.

[0047] In a fifth typical embodiment of the present application, a method for energy cycling using the formate dehydrogenase mutant is provided, the method comprising: formate and NAD + Under the action of the formate dehydrogenase mutant, NADH is formed. The formate dehydrogenase mutant of the present application has good activity and tolerance in any process involving energy cycle (NAD→NADH).

[0048] In a sixth typical embodiment of the present application, a method for preparing a non-natural amino acid is provided, the preparation method comprising using the formate dehydrogenase mutant in amino acid dehydrogenase, formate and NAD + Under the action of , the substrate shown in formula Ⅰ is reacted to obtain a non-natural amino acid;

[0049] Formula I;

[0050] R is selected from substituted aryl or unsubstituted aryl, C 2 -C 8 A substituted or unsubstituted heterocyclic group, C 3 -C 8 Substituted cycloalkyl or unsubstituted cycloalkyl, C 1 -C 10 Alkyl, C 2 -C10 The olefin group or C 2 -C 10 The substituted heterocyclic group or the unsubstituted heteroatom is selected from S atom, O atom or N atom; the substituents of the substituted aryl group, the substituted heterocyclic group and the substituted cycloalkyl group are each independently selected from halogen, O atom or C 1 -C 5 of alkyl.

[0051] Using the above preparation method, using the above formate dehydrogenase mutant in formate and NAD + On the basis of its existence, it provides NADH for the synthesis of non-natural amino acids from substrates, catalyzing the synthesis of non-natural amino acids. Due to the improved activity and tolerance of the formate dehydrogenase mutant, it can catalyze under industrial production conditions such as extreme environments, which can improve production efficiency and reduce industrial production costs.

[0052] In a preferred embodiment, R is selected from substituted aryl or C 4 -C 8 The heterocyclic group of the substituted heterocyclic group is a S atom; the substituents of the substituted aryl and heterocyclic group are each independently selected from halogen or C 1 -C 5 of alkyl.

[0053] In a preferred embodiment, the α-keto acid substrate is selected from (Substrate 1), (Substrate 2), (Substrate 3), (Substrate 4), (Substrate 5), (Substrate 6), (Substrate 7), (Substrate 8) or (Substrate 9).

[0054] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0055] The formate dehydrogenase mutants obtained in the present application are obtained by saturation mutation. Saturation mutation is a method of obtaining mutants in which the amino acids at the target site are replaced by other 19 amino acids in a short period of time by modifying the coding gene of the target protein. This method is not only a powerful tool for protein-directed modification, but also an important means for studying the structure-function relationship of proteins. Saturation mutation can often obtain more ideal evolutionary bodies than single-point mutation. These problems that cannot be solved by the site-directed mutagenesis method are precisely the unique features of the saturation mutation method. The construction of saturation mutation adopts the method of whole plasmid PCR, and then the PCR product is digested with DpnI enzyme to remove the template and transformed into Escherichia coli BL21 (DE3).

[0056] The specific process of culturing the formate dehydrogenase mutant enzyme solution in the following examples of the present application is as follows:

[0057] Take 4 mL of BL21(DE3) mutant strains containing different mutation sites and inoculate them into a 2 L Erlenmeyer flask containing 400 mL LB medium. After shaking culture at 37°C and 200 rpm for 2-3 h, the OD 600 When the pH value was 0.6-0.8, add 0.06 mM IPTG at a final concentration, induce at 25°C for 18 h, and collect the cells by centrifugation at 4°C. Resuspend the cells with 10 mL 0.1M Tris-HCl (pH 8.0) per gram of bacterial mud, break them by ultrasonication, centrifuge, collect the supernatant, and obtain the crude enzyme solution for the catalytic reaction.

[0058] The reaction system for enzyme activity detection in the following examples of the present application does not add the specific substrate that the wild-type formate dehydrogenase acts on in the process of preparing non-natural amino acids. Since formate dehydrogenase functions as a coenzyme in the process of preparing non-natural amino acids, it mainly synthesizes NADH to meet the needs of the main enzyme for synthesizing non-natural amino acids. After the main enzyme consumes NADH to produce NAD, formate dehydrogenase catalyzes NAD to form NADH, forming a cycle. Therefore, the following examples mainly detect the activity and tolerance of formate dehydrogenase in the synthesis cycle of NAD→NADH, and then characterize its activity and tolerance in the process of synthesizing non-natural amino acids.

[0059] Example 1

[0060] The enzyme solution was treated at 60°C and 40% ethanol for 1 h, and then added to a 0.3 mL reaction system containing 100 mM sodium formate and 10 mM NAD. The enzyme activity was characterized by monitoring the change in OD340 of NADH at 340 nm for 10 min. The results are shown in Table 1.

[0061] Table 1:

[0062]

[0063]

[0064] The activity is reduced and increased by times compared with the parent, --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-2 times, ++ increased by 2-10 times, +++ increased by 10-50 times, ++++ increased by 50-100 times, +++++ increased by more than 100 times.

[0065] Continue to make mutations to improve activity and tolerance.

[0066] Example 2

[0067] The enzyme solution was treated at 65°C and 50% ethanol for 1 h, and then added to a 0.3 mL reaction system containing 100 mM sodium formate and 10 mM NAD. The enzyme activity was characterized by monitoring the change in OD340 of NADH at 340 nm for 10 min. The results are shown in Table 2.

[0068] Table 2:

[0069]

[0070]

[0071] The activity is reduced and increased by times compared with the parent, --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-2 times, ++ increased by 2-10 times, +++ increased by 10-50 times, ++++ increased by 50-100 times, +++++ increased by more than 100 times.

[0072] Beneficial mutation sites are further combined to further improve activity and tolerance.

[0073] Example 3

[0074] The enzyme solution was treated at 65°C and 50% ethanol for 3 h, and then added to a 0.3 mL reaction system containing 100 mM sodium formate and 10 mM NAD. The enzyme activity was characterized by monitoring the change in OD340 of NADH at 340 nm for 10 min. The results are shown in Table 3.

[0075] Table 3:

[0076]

[0077]

[0078] The activity is reduced and increased by times compared with the parent, --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-2 times, ++ increased by 2-10 times, +++ increased by 10-50 times, ++++ increased by 50-100 times, +++++ increased by more than 100 times.

[0079] Beneficial mutation sites are further combined to further improve activity and tolerance.

[0080] Example 4

[0081] The enzyme solution was treated at 70°C and 60% ethanol for 3 h, and then added to a 0.3 mL reaction system containing 100 mM sodium formate and 10 mM NAD. The enzyme activity was characterized by monitoring the change in OD340 of NADH at 340 nm for 10 min. The results are shown in Table 4.

[0082] Table 4:

[0083]

[0084]

[0085] The activity is reduced and increased by times compared with the parent, --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-2 times, ++ increased by 2-10 times, +++ increased by 10-50 times, ++++ increased by 50-100 times, +++++ increased by more than 100 times.

[0086] Enzyme activity under other conditions:

[0087] Example 5

[0088] The enzyme solution was treated with other conditions and then added to a 0.3 mL reaction system containing 100 mM sodium formate and 10 mM NAD. The enzyme activity was characterized by monitoring the change in OD340 of NADH at 340 nm for 10 min. The results are shown in Table 5.

[0089] Table 5:

[0090]

[0091] The activity is reduced and increased by times compared with the parent, --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-2 times, ++ increased by 2-10 times, +++ increased by 10-50 times, ++++ increased by 50-100 times, +++++ increased by more than 100 times.

[0092] Perform a scale-up reaction using 1 g of substrate.

[0093] Example 6

[0094] Based on the best enzyme obtained by evolution, the reaction was scaled up and 1 g (Substrate 1), (Substrate 2) or (Substrate 3), (Substrate 4), (Substrate 5), (Substrate 6), (Substrate 7), (Substrate 8) or (Substrate 9), 100 mL reaction system, 100 mg amino acid dehydrogenase, 3 eq sodium formate, 5 eq ammonium chloride, 5 mg formate dehydrogenase, 100 mg NAD, 0.1 M Tris-HCl pH 9.0, react at 37°C, follow the reaction time and take samples for detection, the specific conversion rate and ee value are shown in Table 6 below:

[0095] Table 6:

[0096]

[0097] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: by evolving formate dehydrogenase, mutants with improved activity and tolerance are obtained, and efficient non-natural amino acids can be synthesized under high temperature and high organic solvent conditions, with high product yield, greatly reducing the three wastes, saving production costs, and realizing green chemistry.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A formate dehydrogenase mutant, characterized in that: The formate dehydrogenase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO: 1 undergoes amino acid mutations at the following positions: 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; <h2 style=";text-align:left;direction:ltr">N187R+K328S+Q287E; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+T321K; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+T321A; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+T321Q; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+A10C; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+K47C; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+K47T; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+K47V; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+E53V; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+E53T; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+E53A; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+E53I; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+H148G; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+H148K; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+H148Y; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+H148R; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+V152I; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+A168G; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184V; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184K; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184R; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+F285Q; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+F285S; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+F285M; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+F285W; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+T321K; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+T321R; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+T321S; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+T321A; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+T321Q; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184K+A10C; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184K+K47C; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184K+K47T; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184K+K47V; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184K+E53V; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184K+E53T; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184K+E53A; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184K+E53I; <h2 style=";text-align:left;direction:ltr"> N187R+K328S+K109R+L184K+I103G; <h2 style=";text-align:left;direction:ltr"> 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; The letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

2. A DNA molecule, characterized in that The DNA molecule encodes the formate dehydrogenase mutant according to claim 1.

3. A recombinant plasmid, characterized in that: The recombinant plasmid is connected to the DNA molecule according to claim 2.

4. A host cell, characterized in that The host cell is transformed with the recombinant plasmid according to claim 3.

5. A method for energy cycling using the formate dehydrogenase mutant according to claim 1, characterized in that: The method comprises: Formate and NAD + Under the action of the formate dehydrogenase mutant according to claim 1, NADH is formed.

6. A method for preparing a non-natural amino acid, characterized in that: The preparation method comprises using the formate dehydrogenase mutant according to claim 1 to produce amino acid dehydrogenase, formate and NAD + Under the action of , reacting the α-keto acid substrate shown below to obtain the non-natural amino acid; The α-keto acid substrate is selected from , , , , , , , or .

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