Methanol dehydrogenase mutant with improved catalytic activity and application thereof

Abstract

The invention discloses a methanol dehydrogenase mutant with improved catalytic activity, and belongs to the technical field of enzyme directed evolution and protein engineering. The methanol dehydrogenase is subjected to random mutation by means of directed evolution. After the construction of the plasmid library is completed, the methanol dehydrogenase with higher activity is screened by using a high-throughput and simple in-vivo screening system. Two methanol dehydrogenase mutants S1 (V37A) and S2 (I58T / M292T) with significantly improved methanol catalytic activity are obtained, V37 is subjected to saturation mutation on the basis, and mutants with improved formaldehyde generation rate are further screened. The methanol dehydrogenase mutant obtained by the invention can provide a good catalytic element for bioconversion of methanol, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to a methanol dehydrogenase mutant with improved catalytic activity, belonging to the technical fields of enzyme directed evolution and protein engineering. Background Art

[0002] In recent years, with the continuous increase in global population and the deteriorating climate, the search for new, green, sustainable carbon sources to replace the commonly used sugar-based carbon sources has attracted considerable attention. Methanol, as a widely available, inexpensive, and high-energy-density organic one-carbon compound, is considered a particularly 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 a key, rate-limiting enzyme in the bioutilization of methanol. However, MDH's relatively mediocre catalytic activity and low affinity for methanol severely restrict the bioconversion of methanol. During microbial one-carbon metabolism, methanol is first catalyzed by MDH to formaldehyde. Subsequently, formaldehyde is further converted to energy and carbon skeletons required for cell growth and metabolism through different assimilation pathways, such as the ribulose monophosphate pathway (RuMP), the xylulose monophosphate pathway (XuMP), and the serine cycle, or oxidized to CO2 through dissimilation pathways.

[0004] However, in existing technologies, methanol oxidation is the key reaction limiting the efficiency of methanol bioconversion due to the suboptimal enzymatic properties of MDH. Therefore, there is an urgent need for strategies to improve the catalytic activity of MDH through directed evolution or semi-rational design, thereby obtaining more active MDHs to overcome the kinetic deficiencies of methanol bioconversion. This is of great significance for building an efficient methanol bioconversion platform. Summary of the Invention

[0005] Problems to be solved by the invention

[0006] In the bioconversion of methanol, the key rate-limiting enzyme MDH has low affinity for the substrate methanol and low catalytic efficiency, which restricts the bioconversion of methanol.

[0007] Solutions for solving problems

[0008] In order to make up for the deficiency of low methanol activity of methanol dehydrogenase from Bacillus stearothermophilus DSM 2334, the object of the present invention is to provide a methanol dehydrogenase mutant with improved catalytic activity.

[0009] The purpose of the present invention is achieved by the following technical solutions:

[0010] [1]. A methanol dehydrogenase mutant, wherein the methanol dehydrogenase mutant comprises any one of the following groups (i) to (v):

[0011] (i) the methanol dehydrogenase mutant comprises a mutation at one or more positions corresponding to at least position 37, position 58 or position 292 of the sequence shown in SEQ ID NO: 2, compared to the sequence shown in SEQ ID NO: 2;

[0012] (ii) having 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), excluding mutants of the sequence shown in SEQ ID NO: 2;

[0013] (iii) a mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions:

[0014] (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (i);

[0015] (b) the full-length complementary polynucleotide of (a);

[0016] (iv) a fragment of the mutant shown in any one of (i), (ii) or (iii), wherein the fragment still has methanol dehydrogenase activity;

[0017] (v) A polypeptide having an amino acid sequence as shown in (i), (ii), (iii) or (iv) wherein one or more amino acids are added to or deleted from at least one of the N-terminus and the C-terminus.

[0018] [2] The methanol dehydrogenase mutant according to [1], wherein the methanol dehydrogenase mutant corresponds to the sequence shown in SEQ ID NO: 2 and has a mutated amino acid at at least one of the following positions:

[0019] V37A, V37C, V37T, V37I, V37M, V37L, I58T, M292T.

[0020] [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 a mutation as shown in (m1) or (m9):

[0021] (m1)V37A;

[0022] (m2)V37C;

[0023] (m3)V37T;

[0024] (m4)V37I;

[0025] (m5)V37M;

[0026] (m6)V37L;

[0027] (m7)I58T;

[0028] (m8)M292T;

[0029] (m9)I58T, M292T.

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

[0031] [5]. An expression vector comprising the isolated polynucleotide described in [4].

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

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

[0034] Optionally, the selected recombinant host cell is a bacterium;

[0035] Preferably, the recombinant host cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis or Bacillus methanolicus;

[0036] More preferably, the recombinant host cell is Escherichia coli.

[0037] [7]. A cell culture comprising the recombinant host cell according to [6].

[0038] [8]. A product comprising the methanol dehydrogenase mutant according to any one of [1] to [3], the isolated polynucleotide according to [4], the expression vector according to [5], the recombinant host cell according to [6], or the cell culture according to [7].

[0039] [9] A method for methanol bioconversion, comprising the step of utilizing the methanol dehydrogenase mutant according to any one of [1] to [3], the isolated polynucleotide according to [4], the expression vector according to [5], the recombinant host cell according to [6], the cell culture according to [7], or the product according to [8];

[0040] Optionally, the products of the methanol bioconversion include amino acids, amino acid derivatives, natural secondary metabolites, alcohols, aldehydes and / or organic acids;

[0041] Optionally, methanol is used as a substrate to convert and produce formaldehyde and its derivatives;

[0042] In the methanol bioconversion step, the reaction temperature is 30-60° C. and the pH is 5.5-8.5.

[0043]

[10] Use of the methanol dehydrogenase mutant according to any one of [1] to [3], the isolated polynucleotide according to [4], the 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;

[0044] Optionally, the products of the methanol bioconversion include amino acids, amino acid derivatives, natural secondary metabolites, alcohols, aldehydes and / or organic acids.

[0045] Effects of the Invention

[0046] (1) The two MDH mutants MDH S1 (V37A) and MDH S2 (I58T / M292T) provided by the present invention have significantly improved catalytic efficiency compared with the wild type, and their corresponding specific enzyme activities are 17.17mU / mg and 23.26mU / mg, which are 1.7 and 2.3 times that of the wild type, respectively. cat / K m ) were 8.2 and 20 times that of the wild type, respectively.

[0047] (2) The mutants at position 37 of MDH provided by the present invention, V37C, V37T, V37I, V37M, V37A, V37L, I58T, M292T, and I58T / M292T, significantly increased the formaldehyde production rate compared to the wild type. In particular, the formaldehyde production rates of I58T / M292T, V37A, and V37L increased by 48.44%, 57.22%, and 130%, respectively, compared to the wild type. This indicates that amino acid position 37 is a key amino acid residue affecting the catalytic activity of MDH. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The unit OD of cells expressing different MDH mutants is shown 600 Cellular formaldehyde production rate. DETAILED DESCRIPTION

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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).

[0057] 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.

[0058] 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.

[0059] 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."

[0060] 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.

[0061] 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).

[0062] 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 1 or 2) 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.

[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 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 easily transformed, transfected, transduced, etc. with a mutant polypeptide, a polynucleotide encoding a mutant polypeptide, or a recombinant expression vector comprising the present invention. The term "recombinant host cell" encompasses a host cell that is different from the parent cell after the polynucleotide or recombinant expression vector encoding the 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, specifically, the host cell is derived from a microorganism of the genus Escherichia, the genus Bacillus, or the genus Corynebacterium. In some preferred embodiments, the host cell is derived from the genus Escherichia, more preferably 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, amino acid derivatives, natural secondary metabolites, alcohols, aldehydes, 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, a methanol dehydrogenase mutant is provided, wherein the methanol dehydrogenase mutant comprises any one of the following groups consisting of (i) to (v):

[0077] (i) the methanol dehydrogenase mutant comprises a mutation at one or more positions corresponding to at least position 37, position 58 or position 292 of the sequence shown in SEQ ID NO: 2, compared to the sequence shown in SEQ ID NO: 2;

[0078] (ii) having 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), excluding mutants of the sequence shown in SEQ ID NO: 2;

[0079] (iii) a mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions:

[0080] (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (i);

[0081] (b) the full-length complementary polynucleotide of (a);

[0082] (iv) a fragment of the mutant shown in any one of (i), (ii) or (iii), wherein the fragment still has methanol dehydrogenase activity;

[0083] (v) A polypeptide having an amino acid sequence as shown in (i), (ii), (iii) or (iv) wherein one or more amino acids are added to or deleted from at least one of the N-terminus and the C-terminus.

[0084] In some specific embodiments, the methanol dehydrogenase mutant corresponds to the sequence shown in SEQ ID NO: 2, and has a mutated amino acid at at least one of the following positions: V37A, V37C, V37T, V37I, V37M, V37L, I58T, M292T.

[0085] Furthermore, the methanol dehydrogenase mutant corresponds to the sequence shown in SEQ ID NO: 2, and has mutations as shown in (m1) to (m9):

[0086] (m1) V37A (S1 in the embodiment of the present invention);

[0087] (m2)V37C;

[0088] (m3)V37T;

[0089] (m4)V37I;

[0090] (m5)V37M;

[0091] (m6)V37L;

[0092] (m7)I58T;

[0093] (m8)M292T;

[0094] (m9)I58T, M292T (S2 in the embodiment of the present invention).

[0095] 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.

[0096] <Second Aspect>

[0097] In the second aspect of the present invention, an isolated polynucleotide is provided, wherein the polynucleotide encodes the methanol dehydrogenase mutant as described in the first aspect of the present disclosure.

[0098] 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.

[0099] 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.

[0100] <Third Aspect>

[0101] In the third aspect of the present invention, an expression vector is provided, wherein the expression vector comprises the polynucleotide described in the second aspect of the present invention.

[0102] 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.

[0103] <Fourth Aspect>

[0104] 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 expression vector described in the third aspect of the present invention.

[0105] 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.

[0106] 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.

[0107] In some embodiments, the recombinant host cell is a bacterium; further, the recombinant host cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis or Bacillus methanolicus; more preferably, the recombinant host cell is Escherichia coli.

[0108] <Fifth Aspect>

[0109] 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.

[0110] 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.

[0111] <Sixth Aspect>

[0112] 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, and the cell culture described in the fifth aspect.

[0113] In some optional embodiments, the product may include enzyme preparations, bacterial preparations, kits, etc.

[0114] <Seventh Aspect>

[0115] In the seventh aspect of the present invention, a method for preparing formaldehyde is provided, which comprises the steps of utilizing the methanol dehydrogenase mutant as described in the first aspect of the present invention, the polynucleotide as described in the second aspect, the expression vector as described in the third aspect, the recombinant host cell as described in the fourth aspect, the cell culture as described in the fifth aspect, or the product as described in the sixth aspect.

[0116] 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.

[0117] In some optional embodiments, the method uses methanol as a substrate to convert and produce formaldehyde and its derivatives.

[0118] In some exemplary embodiments, the method comprises contacting the methanol dehydrogenase mutant, recombinant host cell and / or cell culture with methanol.

[0119] In the methanol bioconversion step, the reaction temperature is 30-60°C, preferably 30-55°C, more preferably 30-37°C; the pH is 5.5-8.5, preferably 6.5-8.0, more preferably 6.8-7.5.

[0120] <Eighth Aspect>

[0121] 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.

[0122] 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.

[0123] In some exemplary embodiments, the product of the methanol bioconversion includes formaldehyde and its derivatives.

[0124] Example

[0125] 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.

[0126] The culture medium involved in the following examples is as follows:

[0127] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0128] 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.

[0129] MDH enzyme activity detection:

[0130] 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.

[0131] Table 1. Enzyme activity detection system

[0132]

[0133] Definition and explanation:

[0134] 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).

[0135] In the following examples, the concentrations of antibiotics added were: kanamycin, 50 μg / mL; ampicillin, 100 μg / mL.

[0136] Example 1: Construction and screening of a methanol dehydrogenase random mutation library

[0137] 1. Construction of recombinant vector containing mutants

[0138] In this example, the MDH is derived from Bacillus stearothermophilus DSM 2334, and its nucleotide sequence is shown in SEQ ID NO: 1. Using the recombinant plasmid pTrc99A-mdh containing the nucleotide sequence shown in SEQ ID NO: 1 as a plasmid template, mdh was amplified using the primer pair erBS-F / erBS-R (Table 3). To reduce the introduction of incorrect bases during amplification, a DNA polymerase with lower fidelity was used and 0.05, 0.1, and 0.2 mM MnCl2 were added to the PCR system. The specific PCR reaction system is shown in Table 2:

[0139] Table 2. Error-prone PCR reaction system

[0140]

[0141] Note: The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; cycle settings: denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, for a total of 30 cycles; followed by extension at 72°C for 5 min.

[0142] After amplification, the pTrc99A-mdh plasmid template was digested with DpnI enzyme. To obtain the recombinant plasmid backbone, the pTrc99A plasmid was digested with restriction endonucleases NdeI and EcoRI. The above DNA fragments were purified and recovered using a common DNA product purification kit purchased from Beijing Tiangen Biochemical Technology Co., Ltd. The purified and recovered DNA fragments were seamlessly cloned using the recombination kit ClonExpressMultiS One Step Cloning Kit C112 from Novozymes. The recombinant system was then transformed into full-strength gold Trans1-T1 competent cells, and single clones were picked the next day for colony PCR and sequencing to evaluate the plasmid library. The colonies on the plate were then scraped off with ddH2O, and the plasmid library was extracted.

[0143] Table 3. Primers for constructing methanol dehydrogenase random mutation library

[0144]

[0145] 2. Methanol dehydrogenase random mutation library screening

[0146] In this embodiment, the screening of the MDH random mutation library is based on the energy-deficient E. coli and uses it as an in vivo screening platform to screen mutants. In this energy-deficient strain, the key gene lpd is knocked out, making it an NADH-deficient E. coli (NADH aux , which is disclosed in the literature Wenk S., et al. An "energy-auxotroph" Escherichia coli provides an in vivo platform for assessing NADH regeneration systems. Biotechnology and bioengineering, 2020, 117(11): 3422-3434., wherein the Δlpd strain (SIJ488Δlpd::cap) is incorporated herein by reference). By expressing MDH in the strain and placing it in a minimal medium supplemented with methanol and sodium acetate, strain growth can be achieved and coupled with MDH activity.

[0147] First, the plasmid library obtained in the above steps was electroporated into NADH aux After 50 min of recovery at 37°C and 200 rpm, the bacterial suspension was inoculated into M9 medium containing kanamycin supplemented with 20 mM sodium acetate, 20 mM sodium succinate, and 20 mM glycerol, and the initial OD was controlled. 600 The OD value was 0.25, and the cells were cultured at 37°C and 220 rpm.600 To 0.4-0.6, add 0.1mM IPTG (isopropylthiogalactoside) to induce for 6h. After the induction expression is completed, the bacterial liquid is transferred to M9 medium supplemented with 20mM sodium acetate, 500mM methanol, 0.5g / L yeast extract and kanamycin, and the initial OD is controlled. 600 The concentration of the culture medium was set to 0.1 and cultured at 37°C and 220 rpm. Depending on the growth of the strain, serial passages were performed to enrich for strains containing MDH mutants with significantly enhanced catalytic activity. When the strain growth was no longer significantly accelerated, the bacterial suspension was diluted and plated onto solid culture medium containing the same ingredients. The solid plates were incubated at 37°C. Once a single colony emerged, colony PCR was performed and sequencing was performed to identify the genotypic changes of the mutants.

[0148] 3. Mutant formaldehyde production rate test

[0149] To further evaluate the formaldehyde production rates of these mutants, plasmids were extracted from strains containing the MDH mutants identified above with significantly enhanced catalytic activity. These plasmids were then transformed into E. coli MG1655. These strains carrying the various mutants were then inoculated into LB liquid medium containing kanamycin. After overnight culture, the cells were transferred to 96-well plates. Each well contained 400 μL of M9 medium supplemented with 2 g / L glucose, 1 g / L casein hydrolysate, and kanamycin. 0.1 mM IPTG was also added, and expression was induced at 37°C and 800 rpm for 6 hours. Following induction, 320 μL of Nash reagent and 80 μL of 5 M methanol were added to each well. Under the catalysis of MDH, methanol is oxidized to formaldehyde (FD), which then combines with Nash's reagent to form 3,5-diacetyl-1,4-dihydro-2,6-dimethylpyridine (DDL). DDL exhibits a characteristic absorption peak at 412 nm, and changes in absorbance reflect formaldehyde concentration. Through whole-cell catalytic experiments, two mutants with significantly accelerated formaldehyde production rates were successfully identified, as detailed in Table 4.

[0150] Table 4. Mutant information and unit OD 600 Cellular formaldehyde production rate

[0151]

[0152] Example 2: Expression of mutant proteins and enzyme activity detection

[0153] In this example, the pET21a plasmid was digested with restriction endonucleases NdeI and XhoI to obtain a plasmid backbone, and the MDH mutant genes described in Examples 1 and 2 were subsequently ligated to the pET21a plasmid backbone. The recombinant expression plasmids were then transferred into E. coli BL21(DE3) to obtain two recombinant strains carrying MDH mutants with enhanced activity: BL21(DE3)-pET21a-mdh S1 , BL21(DE3)-pET21a-mdh S2 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 After the pH 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.

[0154] Through enzyme activity detection, the present invention successfully obtained two MDH mutants with improved activity. Among them, the specific enzyme activities of MDH mutants S1 and S2 were 1.7 times and 2.3 times that of the wild type, respectively. The affinity of the two MDH mutants obtained for methanol was improved to a certain extent, and the mutation of V37A significantly reduced the K of MDH for methanol. m In addition, the catalytic efficiency of the two mutants for methanol was 8.2 and 20 times that of the wild type, respectively. Specific enzymatic parameters are shown in Table 5.

[0155] Table 5. Enzymatic parameters of MDH mutants with enhanced activity

[0156]

[0157] Example 3: Investigation of the effects of residues at positions 37, 58, and 292 on the catalytic performance of MDH

[0158] 1. Construction of recombinant vector containing mutants

[0159] In this example, the effects of these mutation sites on the catalytic activity of MDH were further explored. On the one hand, the effects of the I58T and M292T single mutations on the catalytic activity of MDH were explored. On the other hand, a V37 site-directed saturation mutation library was constructed to explore the effects of other mutations at this site on the catalytic activity of MDH. The recombinant plasmid pTrc99A-mdh containing the nucleotide sequence shown in SEQ ID NO: 1 was used as a plasmid template, and the primer pairs shown in Table 6 were used to construct the MDH mutant plasmid.

[0160] Table 6. Primers for construction of mutant plasmids

[0161]

[0162] The specific PCR reaction system is shown in Table 7 (taking V37L as an example):

[0163] Table 7. Fragment amplification PCR reaction system

[0164]

[0165] Note: The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; cycle settings: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, for a total of 30 cycles; followed by extension at 72°C for 5 min.

[0166] After amplification, the pTrc99A-mdh plasmid template was digested with DpnI enzyme. The DNA fragments were then purified and recovered using a standard DNA product purification kit purchased from Beijing Tiangen Biochemical Technology Co., Ltd. The purified and recovered DNA fragments were seamlessly cloned using the ClonExpress MultiS One Step Cloning Kit C112, a recombination kit from Norwegian. The recombinant system was then systematically transformed into full-strength gold Trans1-T1 competent cells. The next day, single clones were picked, colony PCR was performed, and sequencing was performed. The mutant plasmids that had been sequenced correctly were then electroporated into E. coli MG1655. Finally, the whole-cell catalysis experiment was performed according to the steps in Example 1.

[0167] Experimental results (Table 5 and Figure 1 ) showed that I58T and M292T had a synergistic effect on improving the catalytic efficiency of MDH, and the effect of the combination of these two mutations was greater than that of each of them alone. Residue 37 seems to be the key amino acid residue affecting the catalytic efficiency of MDH. When the valine at position 37 mutated into amino acids such as histidine and tryptophan, the catalytic activity of MDH decreased significantly. However, when the valine at position 37 mutated into leucine, the catalytic activity of MDH was significantly improved, and the formaldehyde generation rate of V37L increased by 130% compared with the wild type ( Figure 1 ).

[0168] SEQ ID NO:1

[0169]

[0170] SEQ ID NO:2

[0171] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIFIDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0172] SEQ ID NO:3

[0173] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIECAGACHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIFIDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0174] SEQ ID NO:4

[0175] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLTPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIFIDTVLNGVSVKGSIVGTRKDTQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0176] SEQ ID NO:5

[0177] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLTPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0178] SEQ ID NO:6

[0179] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAI YGIGGLGHIALQYAKAMGLNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDTQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0180] SEQ ID NO:7

[0181] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGXCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAI YGIGGLGHIALQYAKAMGLNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0182] Wherein, X represents histidine H, tryptophan W, aspartic acid D, arginine R, glutamic acid E, glutamine Q, proline P, arginine K, asparagine N, glycine G, tyrosine Y, phenylalanine F, serine S, cysteine C, isoleucine I, methionine M, and leucine L.

[0183] SEQ ID NO:35

[0184] VKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGTCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAI YGIGGLGHIALQYAKAMGLNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED*

[0185] 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.

[0186] 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. A methanol dehydrogenase mutant, wherein: The methanol dehydrogenase mutant comprises any one of the following groups consisting of (i) to (v): (i) the methanol dehydrogenase mutant comprises a mutation at one or more positions corresponding to at least position 37, position 58 or position 292 of the sequence shown in SEQ ID NO: 2, compared to the sequence shown in SEQ ID NO: 2; (ii) having 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), excluding 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 in 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) wherein one or more amino acids are added to or deleted from 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 corresponds to the sequence shown in SEQ ID NO: 2, and has a mutated amino acid at at least one of the following positions: V37A, V37C, V37T, V37I, V37M, V37L, I58T, M292T.

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 mutations as shown in (m1) to (m9): (m1)V37A; (m2)V37C; (m3)V37T; (m4)V37I; (m5)V37M; (m6)V37L; (m7)I58T; (m8)M292T; (m9)I58T, M292T.

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

5. An expression vector, wherein: The expression vector comprises the isolated polynucleotide of 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 isolated polynucleotide according to claim 4, or the expression vector according to claim 5; Optionally, the isolated polynucleotide according to claim 4 is integrated into the genome of the recombinant host cell; Optionally, the selected recombinant host cell is a bacterium; Preferably, the recombinant host cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis or Bacillus methanolicus; More preferably, the recombinant 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 isolated polynucleotide according to claim 4, the 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 methanol bioconversion, 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 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, the products of the methanol bioconversion include amino acids, amino acid derivatives, natural secondary metabolites, alcohols, aldehydes and / or organic acids; Optionally, methanol is used as a substrate to convert and produce formaldehyde and its derivatives; In the methanol bioconversion step, the reaction temperature is 30-60° C. and the pH is 5.5-8.

5.

10. Use of the methanol dehydrogenase mutant according to any one of claims 1 to 3, the isolated polynucleotide according to claim 4, the 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.

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