Methanol dehydrogenase mutant with improved cofactor affinity

Abstract

The invention discloses a methanol dehydrogenase mutant with improved cofactor affinity, particularly relates to a methanol dehydrogenase mutant with improved cofactor NADP < + > affinity, and belongs to the technical field of enzyme directed evolution and protein engineering. According to the present invention, the 195th site and the 196th site of the methanol dehydrogenase from the bacillus stearothermophilus DSM 2334 are modified so as to improve the affinity of the methanol dehydrogenase to the cofactor NADP < + >, and the 2424th site, the 243 site and the 246th site are modified on the basis so as to construct the methanol dehydrogenase mutant having high catalytic activity on the NADP < + > and low activity on the NAD < + >. V37A mutation is introduced, so that the mutant SM7 which takes NADP < + > as a cofactor and has catalytic activity is further improved. The methanol dehydrogenase mutant provided by the invention can provide a good catalytic element for bioconversion of methanol, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical fields of enzyme directed evolution and protein engineering, and particularly relates to a methanol dehydrogenase mutant with improved cofactor affinity. Background Art

[0002] Methanol, a widely available, inexpensive, and energy-dense organic one-carbon compound, is considered a promising non-sugar carbon source. Currently, methanol has been successfully used as a raw material to produce a range of substances, including amino acids, organic acids, and single-cell proteins.

[0003] Methanol dehydrogenase (MDH) is the key rate-limiting enzyme for the bioutilization of methanol. However, the relatively mediocre catalytic activity and low affinity of MDH for methanol seriously restrict the biotransformation of methanol. + For MDH-dependent MDH, the cofactor NADH is generated during the oxidation of methanol. The extra generation of NADH will undoubtedly lead to an imbalance of cofactors in the cell. On the other hand, NADH mainly enters the respiratory chain. Although it is accompanied by energy generation, it is not conducive to high product yield. In contrast, the cofactor NADPH is mainly involved in intracellular anabolism and plays an important role in the production of many compounds. However, the existing NAD + NAD-dependent MDH + The affinity is high, but the cofactor NADP + The affinity of NADP is very low, resulting in + The catalytic activity of NADP as a cofactor is very low. Therefore, the strategy of directed evolution or semi-rational design is used to improve the cofactor NADP. + The affinity of methanol is of great significance for building an efficient methanol bioconversion platform. Summary of the Invention

[0004] Problems to be solved by the invention

[0005] The current methanol dehydrogenase has low catalytic efficiency and low affinity for methanol, which results in low conversion efficiency. In addition, it has a low affinity for NADP. + Low affinity further restricts the bioconversion of methanol.

[0006] Solutions for solving problems

[0007] In order to make up for the deficiency of poor affinity of methanol dehydrogenase of Bacillus stearothermophilus DSM 2334 for methanol, the present invention provides a method for the synthesis of methanol dehydrogenase. + Methanol dehydrogenase mutants with improved catalytic activity.

[0008] The purpose of the present invention is achieved by the following technical solutions:

[0009] [1]. NADP + A methanol dehydrogenase mutant with improved cofactor catalytic activity, wherein the methanol dehydrogenase mutant is selected from any one of the following groups consisting of (I)-(V):

[0010] (I) the methanol dehydrogenase mutant comprises a mutation at at least one of positions 195 and 196 corresponding to the sequence shown in SEQ ID NO: 2, compared to the sequence shown in SEQ ID NO: 2; preferably, the mutant comprises a mutated amino acid residue at at least one of positions D195S and I196R corresponding to the sequence shown in SEQ ID NO: 2;

[0011] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO: 2;

[0012] (III) A mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions:

[0013] (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (I);

[0014] (b) the full-length complementary polynucleotide of (a);

[0015] (IV) a fragment of the mutant shown by any one of (I), (II) or (III), wherein the fragment still has methanol dehydrogenase activity;

[0016] (V) A polypeptide having an amino acid sequence as shown in (I), (II), (III) or (IV) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus.

[0017] [2] The methanol dehydrogenase mutant according to [1], wherein the methanol dehydrogenase mutant comprises a mutation at at least one of positions 85, 242, 243, 246, and 310 of the sequence shown in SEQ ID NO: 2, and, optionally, comprises a mutation at position 37;

[0018] Preferably, the methanol dehydrogenase mutant has a mutated amino acid residue at at least one of the following positions corresponding to the sequence shown in SEQ ID NO: 2: I85V, K242I, A243N, Q246I, K242Y, K242F, T310A, and, optionally, V37A.

[0019] [3] The methanol dehydrogenase mutant according to [1] or [2], wherein the methanol dehydrogenase mutant corresponds to the sequence shown in SEQ ID NO: 2 and has the following (m1) to (m 10 ) is a mutation shown in any one of:

[0020] (m1)D195S;

[0021] (m2)I196R;

[0022] (m3)D195S, I196R;

[0023] (m4)D195S, I196R, A243N;

[0024] (m5)D195S, I196R, K242I;

[0025] (m6)D195S, I196R, A243N, Q246I;

[0026] (m7)D195S, I196R, K242Y, A243N;

[0027] (m8)D195S, I196R, K242F, A243N;

[0028] (m9)I85V, D195S, I196R, A243N, T310A;

[0029] (m 10 )V37A, D195S, I196R, K242I.

[0030] [4]. An isolated polynucleotide encoding the methanol dehydrogenase mutant according to any one of [1] to [3].

[0031] [5]. A recombinant expression vector, wherein the expression vector comprises the polynucleotide described in [4].

[0032] [6] A recombinant host cell, wherein the recombinant host cell comprises the methanol dehydrogenase mutant according to any one of [1] to [3], the polynucleotide according to [4], or the recombinant host cell comprises the recombinant expression vector according to [5];

[0033] Optionally, the polynucleotide described in [4] is integrated into the genome of the recombinant host cell;

[0034] Optionally, the host cell is a bacterium; preferably, the host cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, or Bacillus methanolicus; more preferably, the host cell is Escherichia coli.

[0035] [7]. A cell culture comprising the recombinant host cell according to [6].

[0036] [8]. A product comprising the methanol dehydrogenase mutant according to any one of [1] to [3], the polynucleotide according to [4], the recombinant expression vector according to [5], the recombinant host cell according to [6], or the cell culture according to [7].

[0037] [9] A method for the bioutilization of methanol, comprising the steps of utilizing the methanol dehydrogenase mutant according to any one of [1] to [3], the isolated polynucleotide according to [4], the recombinant expression vector according to [5], the recombinant host cell according to [6], the cell culture according to [7], or the product according to [8];

[0038] Optionally, products of methanol bioutilization include amino acids, amino acid derivatives, natural secondary metabolites, aldehydes, alcohols, and / or organic acids;

[0039] Optionally, the methanol bioutilization comprises catalytic oxidation of methanol, the product of which is formaldehyde and / or its derivatives;

[0040] Optionally, in a system for methanol bioutilization, NADP is included. + .

[0041]

[10] Use of the methanol dehydrogenase mutant according to any one of [1] to [3], the polynucleotide according to [4], the recombinant expression vector according to [5], the recombinant host cell according to [6], the cell culture according to [7], or the product according to [8] in methanol bioconversion;

[0042] Optionally, the products of the methanol bioconversion include amino acids, amino acid derivatives, natural secondary metabolites, alcohols, aldehydes and / or organic acids;

[0043] Optionally, the products of the methanol bioconversion include formaldehyde and its derivatives.

[0044] Effects of the Invention

[0045] The present invention realizes the conversion of cofactor NADP by modifying the 195th and 196th positions of methanol dehydrogenase derived from Bacillus stearothermophilus DSM 2334. + Based on the mutations at positions 195 and 196, combined mutations were performed at positions 242, 243, and 246, and mutations at positions 85 and 310 were accidentally introduced to obtain a NADP-based + As a cofactor, catalytic activity is enhanced and NAD + The MDH mutants with reduced cofactor catalytic activity were further introduced into the MDHs to improve their catalytic activity. The V37V mutation, which improves the substrate affinity of methanol dehydrogenase, was introduced into the MDHs. The mutants MDH SM2 (D195S / I196R / K242I) and MDH SM7 (V37A / D195S / I196R / K242I) showed a strong affinity for NADP. + showed higher catalytic activity; specifically, mutants MDH SM2 and MDH SM7 used NADP + The catalytic activity of methanol as a cofactor was increased by 13.89 and 30.73 times compared with the wild type, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The enzymatic activities of the MDH mutants towards methanol using two different cofactors are shown in Figure 2 .

[0047] Figure 2 The enzymatic activities of three reconstructed MDH mutants towards methanol with two different cofactors are shown. DETAILED DESCRIPTION

[0048] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0049] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0050] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0051] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0052] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0053] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0054] In this article, the "methanol dehydrogenase", "Methanol dehydrogenase", "MDH" are the meanings commonly understood by those skilled in the art, and are a type of NAD + The nucleotide sequence of the encoding gene is shown in SEQ ID NO: 1. The amino acid sequence is shown in SEQ ID NO: 2.

[0055] Throughout this specification, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0056] As used herein, "wild-type" refers to a naturally occurring amino acid or nucleotide sequence found directly in nature and not artificially modified. A "mutant" refers to an amino acid or nucleotide sequence in which one or more amino acids or nucleotides are substituted, deleted, or inserted compared to the naturally occurring amino acid or nucleotide sequence. As used herein, "naturally occurring" and "wild-type" are synonyms.

[0057] In this specification, the term "mutant" refers to a polynucleotide or polypeptide that contains an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to a "wild type" or "compared" polynucleotide or polypeptide, wherein a substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. A deletion refers to the removal of a nucleotide or amino acid occupying a position. An insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0058] As used herein, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion, or addition of one or more amino acids or nucleotides." In the present invention, the term "mutation" refers to a change in the nucleotide sequence or amino acid sequence. In a specific embodiment, the term "mutation" refers to a "deletion."

[0059] In some embodiments, the "mutation" of the present invention can be selected from "conservative mutations". In the present invention, the term "conservative mutation" refers to a mutation that can maintain the normal function of the protein. A representative example of a conservative mutation is a conservative substitution.

[0060] In this specification, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0061] In light of the teachings of the present invention and the prior art, those skilled in the art will also appreciate that the methanol dehydrogenase of the present invention also encompasses variants thereof. For example, one or more (typically 1-30, preferably 1-10, more preferably 1-6, and most preferably 2, 3, 4, 5, or 6) amino acid substitutions, deletions, or insertions may be included. Another example is the addition of one or more amino acids to the C-terminus and / or N-terminus, such as commonly used peptide isolation tags. Such variants possess the same or similar functions as the methanol dehydrogenase of the present invention.

[0062] As used herein, "methanol bioconversion" or "methanol bioutilization" refers to the conversion of methanol into products such as amino acids, natural secondary metabolites, aldehydes, alcohols, and organic acids via microbial or multi-enzyme cascades. In some exemplary embodiments, "methanol bioconversion" or "methanol bioutilization" includes "catalytic oxidation of methanol," which refers to the conversion of methanol into methanol and its derivatives via microbial or multi-enzyme cascades.

[0063] As used herein, the terms "sequence identity" or "percent identity" in the context of comparing two nucleic acids or polypeptides means that they are identical or have a specified percentage of identical sequences when compared and aligned for maximum correspondence using a nucleotide or amino acid residue sequence comparison algorithm or as measured by visual inspection. In other words, nucleotide or amino acid sequence identity can be defined as the ratio of the number of identical nucleotides or amino acids to the total number of nucleotides or amino acids in the aligned portions when two or more nucleotide or amino acid sequences are aligned to maximize the number of identical nucleotides or amino acids, adding gaps as needed.

[0064] As used herein, the term "recombinant polynucleotide" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be contained in a suitable vector, and the vector can be used to transform into a suitable host cell. A host cell containing the recombinant polynucleotide is referred to as a "recombinant host cell." The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide."

[0065] As used herein, the term "expression" includes any step involved in the production of the polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0066] As used herein, the term "expression vector" refers to a DNA construct containing a DNA sequence operably linked to appropriate control sequences for expressing a gene of interest in a suitable host. A "recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired exogenous polypeptide. A recombinant expression vector may include, for example, i) a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) appropriate transcriptional and translational initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not critical, and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, and DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. For example, the expression vector includes but is not limited to pET series, Duet series, pGEX series, pHY300, pHY300PLK, pTrc or pQlink series, etc., which can be replicated and expressed in prokaryotic cells.

[0067] In this specification, the term "host cell" means any cell type that is easy to transform, transfect, transduce, etc. with a mutant polypeptide, a polynucleotide encoding a mutant polypeptide, or a recombinant expression vector. The term "recombinant host cell" encompasses a host cell that is different from the parent cell after the polynucleotide or recombinant expression vector encoding a mutant polypeptide is introduced, and the recombinant host cell is specifically achieved by transformation. The host cell of the present invention can be a prokaryotic cell or a eukaryotic cell. In one embodiment, the host cell refers to a prokaryotic cell, and specifically, the host cell is derived from a microorganism of the genus Escherichia (Escherichia), the genus Bacillus (Bacillus) or the genus Corynebacterium (Corynebacterium). In some optional embodiments, the host cell is derived from the genus Escherichia (Escherichia) or the genus Bacillus (Bacillus), preferably Escherichia coli (Escherichia coli), for example, Escherichia coli DH5α, Escherichia coli Top10, Escherichia coli Trans T1, Escherichia coli MC1061, Escherichia coli MG1655, etc.

[0068] In this specification, the term "cell culture" refers to a combination of cells and a cell culture medium, wherein the cells are cultured in the cell culture medium outside a living organism.

[0069] As used herein, the terms "transformation," "transfection," and "transduction" have the meanings generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method for introducing nucleic acid into a cell, including, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposomes, and lithium acetate-DMSO.

[0070] As used herein, "conversion" may also refer to the enzymatic conversion (or bioconversion) of one or more substrates into one or more corresponding products. In this context, "methanol bioconversion" or "methanol bioutilization" refers to the conversion of methanol into products such as amino acids, natural secondary metabolites, alcohols, and organic acids by microorganisms or multi-enzyme cascades.

[0071] In this specification, "cultivation" refers to growing a microbial cell population under any suitable conditions (for example, using a liquid, gel or solid culture medium), including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

[0072] In this specification, the terms "isolated" and "purified" are used to refer to molecules (e.g., isolated nucleic acids, polypeptides, etc.) or other components that are removed from at least one other component with which they are naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

[0073] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0074] The technical solution of the present invention is described in detail below. :

[0075] <First Aspect>

[0076] In a first aspect of the present invention, there is provided a method for + A methanol dehydrogenase mutant with improved affinity, wherein the mutant binds to NADP + As a cofactor, the mutant has higher enzyme activity. Furthermore, the mutant + The enzyme activity with methanol as a cofactor is higher than that with NAD + Enzyme activity exhibited towards methanol as a cofactor.

[0077] In some embodiments, the methanol dehydrogenase mutant is selected from any one of the following groups consisting of (I)-(V):

[0078] (I) the methanol dehydrogenase mutant comprises a mutation at at least one of positions 195 and 196 corresponding to the sequence shown in SEQ ID NO: 2, compared to the sequence shown in SEQ ID NO: 2;

[0079] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO: 2;

[0080] (III) A mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions:

[0081] (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (I);

[0082] (b) the full-length complementary polynucleotide of (a);

[0083] (IV) a fragment of the mutant shown by any one of (I), (II) or (III), wherein the fragment still has methanol dehydrogenase activity;

[0084] (V) A polypeptide having an amino acid sequence as shown in (I), (II), (III) or (IV) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus.

[0085] In some specific embodiments, the methanol dehydrogenase mutant has a mutated amino acid residue at at least one of D195S and I196R in the sequence corresponding to SEQ ID NO: 2; preferably, the methanol dehydrogenase mutant has a mutated amino acid residue at D195S in the sequence corresponding to SEQ ID NO: 2; more preferably, the methanol dehydrogenase mutant has a mutated amino acid residue at D195S and I196R in the sequence corresponding to SEQ ID NO: 2.

[0086] In some specific embodiments, the methanol dehydrogenase mutant comprises a mutation at at least one of positions 85, 242, 243, 246, and 310 of the sequence shown in SEQ ID NO: 2, and, optionally, comprises a mutation at position 37.

[0087] In some preferred embodiments, the methanol dehydrogenase mutant has a mutated amino acid residue at at least one of the following positions corresponding to the sequence shown in SEQ ID NO: 2: I85V, K242I, A243N, Q246I, K242Y, K242F, T310A, and, optionally, V37A.

[0088] Furthermore, the methanol dehydrogenase mutant corresponds to the sequence shown in SEQ ID NO: 2 and has the following (m1) to (m 10 ) is a mutation shown in any one of:

[0089] (m1) D195S (SM8 of the present invention, whose amino acid sequence is shown in SEQ ID NO: 10);

[0090] (m2) I196R (SM9 of the present invention, whose amino acid sequence is shown in SEQ ID NO: 11);

[0091] (m3) D195S, I196R (SM10 of the present invention, whose amino acid sequence is shown in SEQ ID NO: 12);

[0092] (m4) D195S, I196R, A243N (SM1 of the present invention, whose amino acid sequence is shown in SEQ ID NO: 3);

[0093] (m5) D195S, I196R, K242I (SM2 of the present invention, whose amino acid sequence is shown in SEQ ID NO: 4);

[0094] (m6) D195S, I196R, A243N, Q246I (SM3 of the present invention, whose amino acid sequence is shown in SEQ ID NO: 5);

[0095] (m7) D195S, I196R, K242Y, A243N (SM4 of the present invention, whose amino acid sequence is shown in SEQ ID NO: 6);

[0096] (m8) D195S, I196R, K242F, A243N (SM5 of the present invention, whose amino acid sequence is shown in SEQ ID NO: 7);

[0097] (m9) I85V, D195S, I196R, A243N, T310A (SM6 of the present invention, whose amino acid sequence is shown in SEQ ID NO: 8);

[0098] (m 10)V37A, D195S, I196R, K242I (SM7 of the present invention, whose amino acid sequence is shown in SEQ ID NO: 9).

[0099] In some specific embodiments, the methanol dehydrogenase mutant is prepared and used in the form of cells expressing the enzyme, as a crude extract, or as an isolated or purified preparation. In some exemplary embodiments, the methanol dehydrogenase mutant is prepared as a lyophilized powder, a powder form (e.g., acetone powder), or as an enzyme solution. In some preferred embodiments, the methanol dehydrogenase mutant is in the form of a substantially pure preparation.

[0100] <Second Aspect>

[0101] In the second aspect of the present invention, an isolated polynucleotide is provided, wherein the polynucleotide encodes the methanol dehydrogenase mutant according to the first aspect of the present invention.

[0102] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0103] The polynucleotide encoding the mutant of the present invention includes: a coding sequence encoding only the mutant; a coding sequence of the mutant and various additional coding sequences; a coding sequence of the mutant (and optional additional coding sequences) and non-coding sequences.

[0104] <Third Aspect>

[0105] In the third aspect of the present invention, a recombinant expression vector is provided, wherein the expression vector comprises the polynucleotide described in the second aspect of the present invention.

[0106] In some embodiments, the polynucleotide of the second aspect is operably linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing a polypeptide.

[0107] <Fourth Aspect>

[0108] In the fourth aspect of the present invention, a recombinant host cell is provided, wherein the recombinant host cell comprises the methanol dehydrogenase mutant described in the first aspect of the present invention, the isolated polynucleotide described in the second aspect of the present invention, or the recombinant expression vector described in the third aspect of the present invention.

[0109] In some optional embodiments, the polynucleotide according to the second aspect of the present invention is integrated into the genome of the recombinant host cell.

[0110] In some alternative embodiments, an expression vector containing a heterologous polynucleotide encoding a methanol dehydrogenase mutant polypeptide is introduced into an appropriate host cell to express the corresponding methanol dehydrogenase mutant polypeptide.

[0111] In some embodiments, the host cell is a bacterium; further, the host cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis or Bacillus methanolicus; preferably, the recombinant host cell is Escherichia coli.

[0112] <Fifth Aspect>

[0113] In a fifth aspect of the present invention, a cell culture comprising the recombinant host cell according to the fourth aspect of the present invention is provided.

[0114] In some specific embodiments of the present invention, the recombinant host cells are cultured in a culture medium at a temperature of 16 to 40° C. and a pH of 6.5 to 7.5 to obtain the cell culture. During the culture process, an inducer for inducing expression of the target protein, such as isopropylthiogalactoside (IPTG), may be added to promote expression of the target protein.

[0115] <Sixth Aspect>

[0116] In the sixth aspect of the present invention, a product is provided, which comprises the methanol dehydrogenase mutant described in the first aspect of the present invention, the polynucleotide described in the second aspect, the expression vector described in the third aspect, the recombinant host cell described in the fourth aspect or the cell culture described in the fifth aspect.

[0117] In some optional embodiments, the product may include enzyme preparations, bacterial preparations, kits, etc.

[0118] <Seventh Aspect>

[0119] In the seventh aspect of the present invention, a method for methanol bioutilization is provided, which includes the steps of utilizing the methanol dehydrogenase mutant described in the first aspect of the present invention, the polynucleotide described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant host cell described in the fourth aspect, the cell culture described in the fifth aspect, or the product described in the sixth aspect.

[0120] In some optional embodiments, products of methanol bioutilization include amino acids, amino acid derivatives, natural secondary metabolites, alcohols, aldehydes and / or organic acids, etc.

[0121] In some exemplary embodiments, the method comprises contacting the methanol dehydrogenase mutant, recombinant host cell and / or cell culture with methanol.

[0122] In some optional embodiments, the methanol bioutilization comprises catalytic oxidation of methanol, and the product thereof is formaldehyde and / or its derivatives.

[0123] In some embodiments, in a system for methanol bioutilization, NADP is included. + .

[0124] <Eighth Aspect>

[0125] In the eighth aspect of the present invention, provided is the use of the methanol dehydrogenase mutant described in the first aspect, the polynucleotide described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant host cell described in the fourth aspect, the cell culture described in the fifth aspect, or the product described in the sixth aspect in methanol bioconversion.

[0126] In some optional embodiments, the products of the methanol bioconversion include amino acids, amino acid derivatives, natural secondary metabolites, alcohols, aldehydes and / or organic acids.

[0127] In some exemplary embodiments, the product of the methanol bioconversion includes formaldehyde and its derivatives.

[0128] Example

[0129] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0130] The culture medium involved in the following examples is as follows:

[0131] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0132] M9 basal medium: 47.8 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 18.7 mM NH4Cl, 2 mM MgSO4 and 100 μM CaCl2, trace elements (134 μM EDTA·2Na, 31 μM FeCl3·6H2O, 6.2 μM ZnCl2, 0.76 μM CuCl2·2H2O, 0.42 μM CoCl2·2H2O, 1.62 μM H3BO3 and 0.081 μM MnCl2·4H2O.

[0133] MDH enzyme activity detection:

[0134] Prepare the enzyme activity detection system according to the system in Table 1. After the reaction system is preheated at 37℃ for 10 minutes, the enzyme and substrate are mixed. Then, the enzyme is quickly placed in the microplate reader Max ABC Plus to detect the OD 412 The absorbance changes and the V max Calculate enzyme activity.

[0135] Table 1. Enzyme activity detection system

[0136]

[0137] Definition and explanation:

[0138] MDH activity is defined as the amount of enzyme required to produce 1 μmol of 3,5-diacetyl-1,4-dihydro-2,6-dimethylpyridine (DDL) per minute at pH 7.4 and 37°C, which is one unit of activity (U).

[0139] In the following examples, the concentrations of antibiotics added were: kanamycin, 50 μg / mL; ampicillin, 100 μg / mL.

[0140] Example 1: NADP + Construction and screening of a methanol dehydrogenase library with improved affinity

[0141] 1. Construction of recombinant vector containing mutants

[0142] The natural cofactor of MDH from Bacillus stearothermophilus is NAD + , in order to find out the influence of NADP + The inventors used NAD as the key amino acid residue for affinity + Docked into MDH. It was subsequently found that residues D195 and K200 interacted with NAD + The 2′-hydroxyl group of MDH forms a hydrogen bond, which promotes the binding of MDH to its natural cofactor, but is not conducive to the cofactor NADP. +I196, N240, K242, A243, and Q246 are located around the adenine ring and may affect the activity of the mutant, so they were also selected as modification targets. Among them, D195, I196, and K200 are considered to affect the activity of NADP. + The key amino acid residues of affinity, and D195, N240, A243 are relevant with the recovery of enzyme activity.Therefore, above-mentioned amino acid residues are divided into two classes of cofactor change (D195, I196, K200) and activity recovery (D195, N240, K242, A243, Q246).Being limited to huge storage capacity (table 2) in library construction process, the library construction of mutant plasmid is divided into two rounds by the present embodiment.The library construction in the first round is intended to obtain the MDH mutant of successful reversal cofactor, in order to avoid mutant activity lower and produce false negative and be convenient to primer design and fragment recombination.First, construct the mutation library comprising these five sites of D195, I196, K200, N240 and A243.

[0143] Table 2. Design of mutant libraries with altered cofactor preferences

[0144]

[0145] Among them, R=A or G, V=A, C or G, N=A, C, G or T, K=G or T.

[0146] Using the plasmid pTrc99A-mdh (containing the sequence set forth in SEQ ID NO: 1) as a plasmid template, amino acid mutations were introduced at positions 195, 196, 200, 240, and 243 using primer pair SM1-F / SM1-R, respectively. The plasmid backbone was amplified using primer pair SM2-F / SM2-R using pTrc99A-mdh as a template. The resulting amplified product was digested with DpnI enzyme, and the DNA fragments containing the amino acid mutations and the amplified plasmid backbone were purified and recovered using a standard DNA product purification kit purchased from Beijing Tiangen Biochemical Technology Co., Ltd. The purified DNA fragments were seamlessly cloned using the ClonExpress MultiS One Step Cloning Kit C112 from Novozymes. The recombinant strains were then transformed into fully qualified Gold Trans1-T1 cells. The next day, single colonies were picked and subjected to colony PCR sequencing to evaluate the plasmid library. Colonies were then scraped from the plates with ddH2O for plasmid library extraction. After completing the first round of screening, the plasmid containing the mutants obtained from the screening was used as a template to introduce mutations at positions 242 and 246 using primer pairs 242N-F / 242N-R and 246N-F / 246N-R to complete the construction of the mutant library (primers used for amplification are shown in Table 3).

[0147] Table 3. Primers used in the methanol dehydrogenase site-saturation mutagenesis library

[0148]

[0149] Among them, R=A or G, V=A, C or G, N=A, C, G or T, M=A or C, K=G or T.

[0150] 2. Screening of the methanol dehydrogenase site-directed saturation mutagenesis library

[0151] In this example, the screening of the MDH site-saturation mutagenesis library was based on NADPH-deficient E. coli (NADPH aux , which is disclosed in the literature Lindner SN, et al. NADPH-Auxotrophic E. coli: A Sensor Strain for Testing in Vivo Regeneration of NADPH. ACS synthetic biology. 2018; 7(12): 2742-2749., wherein NADPH aux (SIJ488ΔzwfΔmaeBΔpntABΔsthAΔicd::kan), which is incorporated herein by reference) and used as an in vivo screening platform for mutant screening. Similarly, in this strain, all NADPH production genes were knocked out. When the strain was cultured in a minimal medium containing methanol, the strain could only grow if the mutants contained in the strain could use methanol to produce NADPH. Therefore, the difference in strain growth can be used to reflect the different MDH mutants using NADP + Different enzyme activities exhibited by cofactors.

[0152] In this example, the plasmid library was first electroporated into NADPH aux After 50 min of recovery at 37°C and 200 rpm, the bacterial suspension was inoculated into M9 medium supplemented with 10 mM sodium gluconate, 5 mM α-ketoglutaric acid and ampicillin antibiotics, and the initial OD was controlled. 600 The OD value was 0.1, and the cells were cultured at 37°C and 220 rpm. 600 To 0.4-0.6, add 0.1mM IPTG to induce for 6h. After the induction expression is completed, wash the bacteria three times with M9 medium and transfer to M9 medium supplemented with 18mM glycerol, 3mM α-ketoglutaric acid, 500mM methanol and ampicillin antibiotics, and control the initial OD 600is 0.1, and cultured at 37°C and 220rpm. Depending on the growth of the strain, continuous subculture is performed to enrich the MDH mutants with significantly improved catalytic activity. When there is no obvious acceleration in the growth of the strain, the bacterial solution is diluted and applied to a solid culture medium containing the same components. The solid plate is cultured in a 37°C incubator. After a single clone grows out, colony PCR is performed, and sequencing is performed to identify the genotype changes of the mutants obtained. After two rounds of screening and evolution, the MDH mutants obtained are shown in Table 4, among which the mutations I85V and T310A that occurred in SM6 were mutations randomly introduced during the above-mentioned PCR introduction of amino acid mutations.

[0153] Table 4. Methanol dehydrogenase mutants obtained by growth-based screening

[0154]

[0155] Example 2: Expression of mutant proteins and enzyme activity determination

[0156] In this example, the pET21a plasmid was digested with restriction endonucleases NdeI and XhoI to obtain a plasmid backbone, and then the gene sequence encoding the MDH mutant in Example 1 was ligated to the pET21a plasmid backbone. The recombinant expression plasmid was then transformed into E. coli BL21 (DE3) to obtain 6 strains of NADP-producing strains. + Recombinant strain for MDH mutant with improved cofactor catalytic activity: BL21(DE3)-pET21a-mdh SM1 , BL21(DE3)-pET21a-mdh SM2 , BL21(DE3)-pET21a-mdh SM3 , BL21(DE3)-pET21a-mdh SM4 , BL21(DE3)-pET21a-mdh SM5 , BL21(DE3)-pET21a-mdh SM6 And a recombinant strain BL21 (DE3)-pET21a-mdh carrying wild-type MDH. Then the strain carrying the expression plasmid was inoculated into LB liquid medium and cultured overnight. Then the initial OD 600 The strain was transferred to 100 mL of LB liquid medium containing ampicillin antibiotics at 0.05 and cultured at 37°C and 220 rpm. 600 When the p-value reached approximately 0.6, IPTG was added to a final concentration of 0.1 mM and expression was induced at 16°C for 16 hours. The cells were then harvested by centrifugation at 4°C and resuspended in pre-chilled PBS buffer (pH 7.4). After cell disruption by sonication, the protein was purified using a nickel column. The purified protein was used for subsequent enzymatic characterization.

[0157] In the NADP + In terms of affinity modification, the enzyme activities of the mutants SM1-SM6 obtained in the present invention with NADP+ as the cofactor were higher than those with NAD+ as the cofactor ( Figure 1 ). In these mutants, SM2 is not sensitive to NADP + The enzyme activity is high and the effect on NAD + The enzyme activity exhibited is low. In the present invention, the mutation V37A that was previously identified to improve catalytic activity was introduced into SM2 to obtain a new mutant SM7, the amino acid sequence of which is shown in SEQ ID NO: 9. + The activity of methanol as a cofactor was further improved and exceeded that of wild-type MDH with NAD + is the enzyme activity exhibited by the cofactor towards methanol. See Table 5 for details.

[0158] Table 5. Enzyme parameters of MDH mutants for different cofactors

[0159]

[0160] Example 3: 195,196 mutations to NADP + The influence of affinity

[0161] To investigate the effects of D195S, I196R and their combination on NADP + To investigate the effect of affinity, in this example, plasmid pTrc99A-mdh (containing the sequence set forth in SEQ ID NO: 1) was used as a plasmid template. Primer pairs 195-F / qR and qF / SM2-R, 196-F / qR and qF / SM2-R, and 1956-F / qR and qF / SM2-R were used, respectively, to mutate aspartic acid at position 195 to serine (mutant SM8), isoleucine at position 196 to arginine (mutant SM9), and aspartic acid at position 195 to serine and isoleucine at position 196 to arginine (mutant SM10). After digesting the plasmid template pTrc99A-mdh with DpnI, the amplified and digested DNA fragments were purified and recovered using a standard DNA product purification kit purchased from Beijing Tiangen Biochemical Technology Co., Ltd. The purified DNA fragments were seamlessly cloned using the ClonExpress MultiS One-Step Cloning Kit C112 from Novozymes. The recombinant fragments were then transformed into fully qualified Gold Trans1-T1 cells. The following day, single colonies grown on the plates were inoculated into liquid culture and cultured overnight. The next day, plasmids were extracted and sequenced.

[0162] Table 6. Primers used for reconstruction of MDH mutants

[0163]

[0164] The plasmid pTrc99A-mdh was sequenced correctly D195S 、pTrc99A-mdh I196R 、pTrc99A-mdh D195S+I196R As a template, the primer pair erBs-F / erBs-R was used to amplify the mdh gene fragment. After obtaining the MDH mutant gene fragment, it was ligated into the pET21a plasmid backbone described in Example 2. The recombinant expression plasmid was then transformed into E. coli BL21 (DE3) to obtain three strains of NADP-producing strains. + Recombinant strain for MDH mutant with improved cofactor catalytic activity: BL21(DE3)-pET21a-mdh SM8 , BL21(DE3)-pET21a-mdh SM9 , BL21(DE3)-pET21a-mdh SM10 Then the strain carrying the expression plasmid was inoculated into LB liquid medium and cultured overnight. Then the initial OD 600 The strain was transferred to 100 mL of LB liquid medium containing ampicillin antibiotics at 0.05 and cultured at 37°C and 220 rpm. 600 When the p-value reached approximately 0.6, IPTG was added to a final concentration of 0.1 mM and expression was induced at 16°C for 16 hours. The cells were then harvested by centrifugation at 4°C and resuspended in pre-chilled PBS buffer (pH 7.4). After cell disruption by sonication, the protein was purified using a nickel column. The purified protein was used for subsequent enzymatic characterization.

[0165] The experimental results show that ( Figure 2 ), when aspartic acid at position 195 was mutated to serine, mutant SM8 used NADP + The specific enzyme activity exhibited by NAD as a cofactor is higher than that of + As a cofactor, the enzyme activity was exhibited. + The specific enzyme activity was improved with NAD as cofactor, but the mutant + The enzyme activity exhibited by the cofactor is still higher than that of NADP + When amino acid residues 195 and 196 were mutated simultaneously, mutant SM10 was synthesized with NADP + and NAD + The specific enzyme activities of the cofactors decreased to a certain extent, but NADP + The specific enzyme activity with NAD as cofactor was significantly higher than that with + The specific enzyme activity exhibited by the cofactor.

[0166] Table 7. Reconstructed methanol dehydrogenase mutants

[0167]

[0168] SEQ ID NO: 1

[0169]

[0170] SEQ ID NO:2

[0171] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIFIDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0172] SEQ ID NO:3

[0173] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVSRSDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKNFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0174] SEQ ID NO:4

[0175] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVSRSDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKIAFEQAYQSVKRGGTLVVVGLPNADLPIFIDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0176] SEQ ID NO:5

[0177] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVSRSDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKNFEIAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0178] SEQ ID NO:6

[0179] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVSRSDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKYNFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0180] SEQ ID NO:7

[0181] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVSRSDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKFNFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0182] SEQ ID NO:8

[0183] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGVPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVSRSDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKNFEQAYQSVKRGGTLVVVGLPNADLPIFIDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVEAAELEEINEVFERMEKGKINGRIVLKLKED*

[0184] SEQ ID NO:9

[0185] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIECAGACHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVSRSDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKIAFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0186] SEQ ID NO:10

[0187] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVSISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0188] SEQ ID NO:11

[0189] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVDRSDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIFIDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0190] SEQ ID NO:12

[0191] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAI YGIGGLGHIALQYAKAMGLNVVAVSRSDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0192] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0193] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. NADP + A methanol dehydrogenase mutant with improved cofactor catalytic activity, wherein The methanol dehydrogenase mutant is selected from any one of the following groups consisting of (I)-(V): (I) the methanol dehydrogenase mutant comprises a mutation at at least one of positions 195 and 196 corresponding to the sequence shown in SEQ ID NO: 2, compared to the sequence shown in SEQ ID NO: 2; preferably, the mutant comprises a mutated amino acid residue at at least one of positions D195S and I196R corresponding to the sequence shown in SEQ ID NO: 2; (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO: 2; (III) A mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions: (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (I); (b) the full-length complementary polynucleotide of (a); (IV) a fragment of the mutant shown by any one of (I), (II) or (III), wherein the fragment still has methanol dehydrogenase activity; (V) A polypeptide having an amino acid sequence as shown in (I), (II), (III) or (IV) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus.

2. The methanol dehydrogenase mutant according to claim 1, wherein The methanol dehydrogenase mutant comprises a mutation at at least one of positions 85, 242, 243, 246, and 310 of the sequence shown in SEQ ID NO: 2, and, optionally, comprises a mutation at position 37; Preferably, the methanol dehydrogenase mutant has a mutated amino acid residue at at least one of the following positions corresponding to the sequence shown in SEQ ID NO: 2: I85V, K242I, A243N, Q246I, K242Y, K242F, T310A, and, optionally, V37A.

3. The methanol dehydrogenase mutant according to claim 1 or 2, wherein The methanol dehydrogenase mutant corresponds to the sequence shown in SEQ ID NO: 2 and has the following (m1) to (m 10 ) is a mutation shown in any one of: (m1)D195S; (m2)I196R; (m3)D195S, I196R; (m4)D195S, I196R, A243N; (m5)D195S, I196R, K242I; (m6)D195S, I196R, A243N, Q246I; (m7)D195S, I196R, K242Y, A243N; (m8)D195S, I196R, K242F, A243N; (m9)I85V, D195S, I196R, A243N, T310A; (m 10 )V37A、D195S、I196R、K242I。 4. An isolated polynucleotide encoding the methanol dehydrogenase mutant according to any one of claims 1 to 3.

5. A recombinant expression vector, wherein: The expression vector comprises the polynucleotide according to claim 4.

6. A recombinant host cell, wherein The recombinant host cell comprises the methanol dehydrogenase mutant according to any one of claims 1 to 3, the polynucleotide according to claim 4, or the recombinant expression vector according to claim 5; Optionally, the polynucleotide of claim 4 is integrated into the genome of the recombinant host cell; Optionally, the host cell is a bacterium; preferably, the host cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, or Bacillus methanolicus; more preferably, the host cell is Escherichia coli.

7. A cell culture comprising the recombinant host cell of claim 6.

8. A product comprising the methanol dehydrogenase mutant according to any one of claims 1 to 3, the polynucleotide according to claim 4, the recombinant expression vector according to claim 5, the recombinant host cell according to claim 6, or the cell culture according to claim 7.

9. A method for bioutilization of methanol, comprising the step of utilizing the methanol dehydrogenase mutant according to any one of claims 1 to 3, the isolated polynucleotide according to claim 4, the recombinant expression vector according to claim 5, the recombinant host cell according to claim 6, the cell culture according to claim 7, or the product according to claim 8; Optionally, products of methanol bioutilization include amino acids, amino acid derivatives, natural secondary metabolites, aldehydes, alcohols, and / or organic acids; Optionally, the methanol bioutilization comprises catalytic oxidation of methanol, the product of which is formaldehyde and / or its derivatives; Optionally, in a system for methanol bioutilization, NADP is included. + .

10. Use of the methanol dehydrogenase mutant according to any one of claims 1 to 3, the polynucleotide according to claim 4, the recombinant expression vector according to claim 5, the recombinant host cell according to claim 6, the cell culture according to claim 7, or the product according to claim 8 in methanol bioconversion; Optionally, the products of the methanol bioconversion include amino acids, amino acid derivatives, natural secondary metabolites, alcohols, aldehydes and / or organic acids; Optionally, the products of the methanol bioconversion include formaldehyde and its derivatives.

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