Methanol dehydrogenase mutant with improved catalytic activity and application thereof

Abstract

The invention discloses a methanol dehydrogenase mutant with improved catalytic activity. Based on methanol dehydrogenase MdhBs from bacillus stearothermophilus, a methanol dehydrogenase MdhBs whole-genome random mutation library is constructed, and a mutant MdhV79A with improved in-vivo methanol catalytic activity is obtained through screening and sequencing. The obtained mutant has a good application prospect in methanol biological utilization.

Description

[0001] This application is a divisional application of an invention patent application with an application date of February 17, 2023, application number "202310131430.6", and invention name "A methanol dehydrogenase mutant with improved catalytic activity and its application". Technical Field

[0002] The invention belongs to the fields of molecular biology and bioengineering, and in particular relates to a methanol dehydrogenase mutant with improved catalytic activity and application thereof. Background Art

[0003] Biomanufacturing is an important way to promote the evolution of industrial manufacturing to a sustainable development model, but currently biomanufacturing is highly dependent on grain raw materials. According to the National Bureau of Statistics, my country uses about 200 million tons of grain for fermentation, which is equivalent to about 30% of the total grain output. Raw material supply has become one of the bottlenecks of biomanufacturing. Methanol is a primary platform product of coal chemical industry, shale gas chemical industry and industrial and agricultural waste gasification. It is a natural one-carbon compound with the advantages of wide sources, low price and high conversion rate. Compared with sugars, its price is lower and more stable, and it also has the advantages of high average carbon atom reduction degree, which can provide more reducing power for the biosynthesis process and has obvious advantages in participating in biotransformation. Therefore, methanol is an ideal raw material for biomanufacturing bulk chemicals, and has huge development potential and broad development prospects in the future. Research on the biotransformation of methanol has great economic and sociological significance.

[0004] In the process of microbial one-carbon metabolism, methanol is first catalyzed by methanol dehydrogenase (Mdh) to formaldehyde. Subsequently, formaldehyde is further synthesized into energy and carbon skeletons required for cell growth metabolism through different assimilation pathways such as ribulose monophosphate pathway (RuMP), xylulose monophosphate pathway (XuMP) and serine cycle (Serine pathway), or oxidized to produce CO2 through dissimilation pathway.

[0005] However, in the prior art, due to the poor enzymatic properties of Mdh and the fact that the reverse reaction catalyzed by Mdh is more likely to occur, methanol oxidation becomes the key reaction that limits the efficiency of methanol bioconversion. Therefore, there is an urgent need to conduct directed evolution of Mdh in this field to obtain Mdh with higher activity, thereby compensating for the kinetic defects of methanol bioconversion and helping to achieve efficient methanol bioconversion. Summary of the invention

[0006] The object of the present invention is to provide a methanol dehydrogenase mutant with improved catalytic activity. The present invention is based on the mutant derived from Bacillus stearothermophilus (Bacillus stearothermophilus Methanol dehydrogenase Mdh Bs (hereinafter referred to as wild-type methanol dehydrogenase), by constructing methanol dehydrogenase Mdh Bs The whole gene random mutation library was screened and sequenced to obtain three mutants Mdh with improved methanol catalytic activity in vivo V79A , Mdh N214D+E309K , Mdh K72T+T153P ; At the same time, the optimal mutant Mdh K72T+T153P Split Mdh K72T and Mdh T153P The single-site activity was explored; a site-directed saturation mutagenesis strategy was further used to construct a T153 saturation mutation library and verified by sequencing, and mutants Mdh with improved catalytic activity were screened. T153S and Mdh T153V These mutants have good application prospects in methanol bioutilization.

[0007] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a methanol dehydrogenase mutant, wherein the methanol dehydrogenase mutant is selected from any one of the following: (a) the amino acid sequence is derived from a mutation of the sequence shown in SEQ ID NO: 1, wherein one or more amino acids selected from the group consisting of The amino acid residues are mutated at positions 153, 72, 79, 214 and 309; and compared with the methanol dehydrogenase shown in SEQ ID NO: 1, the catalytic activity is improved; or (b) the methanol dehydrogenase mutant has 95%, preferably 98%, and more preferably 99% of the amino acid sequence of (a). and has the function of the methanol dehydrogenase mutant as described in (a), wherein the amino acid residues at positions 153, 72, 79, 214, and / or 309 corresponding to the amino acid sequence shown in SEQ ID NO: 1 are the same as those in the amino acid sequence as described in (a); or (c) The methanol dehydrogenase mutant is composed of 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues added or deleted at the C-terminus and / or N-terminus of the amino acid sequence described in (a), and has the function of the methanol dehydrogenase mutant described in (a), wherein the amino acid residues at positions 153, 72, 79, 214, and / or 309 corresponding to the amino acid sequence shown in SEQ ID NO: 1 are the same as those in the amino acid sequence described in (a).

[0008] According to an embodiment of the present invention, the amino acid sequence of the methanol dehydrogenase mutant is mutated to the following amino acid residues at one or more sites selected from the group consisting of: 153: Pro or Ser or Val; 72nd: Thr; 79th: Ala; 214th: Asp; 309th: Lys.

[0009] In a specific embodiment, the amino acid sequence of the methanol dehydrogenase mutant is shown in any one of SEQ ID NOs: 3-9.

[0010] In a second aspect, the present invention provides a polynucleotide encoding the methanol dehydrogenase mutant according to the first aspect.

[0011] In a third aspect, the present invention provides an expression vector comprising the polynucleotide described in the second aspect of the present invention.

[0012] In a fourth aspect, the present invention provides a host cell, wherein the host cell comprises the expression vector described in the third aspect of the present invention or a polynucleotide having the methanol dehydrogenase mutant described in the second aspect of the present invention integrated into its genome.

[0013] In a preferred embodiment, the host cell is a bacterium; more preferably, the host cell is Escherichia coli ( Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), or Bacillus subtilis ( Bacillus subtilis ); Most preferably, the host cell is Corynebacterium glutamicum ( Corynebacterium glutamic ).

[0014] In a fifth aspect, the present invention provides use of the methanol dehydrogenase mutant described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention in methanol bioconversion.

[0015] Beneficial effects of the present invention The present invention obtains a series of methanol dehydrogenase mutants with significantly improved catalytic activity. In the enzyme activity assay, the mutants V max The enzyme activity and specific activity of methanol dehydrogenase were both up to 2.5 times higher than those of wild-type methanol dehydrogenase. T153P and Mdh T153S Mutant unit OD 600nmThe formaldehyde concentration is 1.6-2.4 times that of the wild type control. The mutant of the present invention has higher catalytic activity, which is conducive to accelerating the bioconversion of methanol, thus having significant economic and social value, and laying a foundation for realizing industrial large-scale bioconversion of methanol. DETAILED DESCRIPTION

[0016] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0017] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0018] Definition and explanation: In this article, the "one carbon compound" or "C1 compound" refers to a compound containing one carbon atom in the molecule, such as methanol, methane, etc., which is the primary platform product of coal chemical industry, shale gas chemical industry and industrial and agricultural waste gasification.

[0019] In this article, the terms "methanol bioconversion" and "bioconversion methanol" have the same meaning, and both refer to the conversion of methanol into environmentally friendly products (such as carbon dioxide) through a series of enzymatic reactions via the methanol metabolic pathway in microorganisms, or the conversion of methanol into products such as bacteria, amino acids, organic acids, polyols, etc.

[0020] As used herein, the term “methanol dehydrogenase”, “Methanol dehydrogenase”, or “Mdh” has the meaning commonly understood by those skilled in the art, and refers to an enzyme that can catalyze the oxidation of methanol to produce formaldehyde, and its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence of the encoding gene is shown in SEQ ID NO: 2.

[0021] As used herein, the term "wild-type" or "naturally occurring" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a source in nature, and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0022] As used herein, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide may be in the form of a separate fragment or may be a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence separated at least once in quantity or concentration, and can be identified, manipulated, and restored by standard molecular biology methods (e.g., using a cloning vector) and its component nucleotide sequences. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), in which "U" replaces "T". In other words, a "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (a separate fragment or an entire fragment), or may be a component or ingredient of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0023] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0024] As used herein, the term "mutant" refers to a polynucleotide or polypeptide that contains changes (i.e., substitutions, insertions and / or deletions) at one or more (e.g., several) positions relative to a "wild type" or "compared" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to the removal of a nucleotide or amino acid occupying a position. Insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position. Those skilled in the art know that if an enzyme is to be mutated in order to obtain a mutant with improved activity, the key is to find sites that can improve activity after mutation. In the present invention, a specific site of the wild-type methanol dehydrogenase whose amino acid sequence is shown in SEQ ID NO: 1 is mutated to obtain a methanol dehydrogenase mutant with significantly improved activity.

[0025] In a specific embodiment, the methanol dehydrogenase obtained by the inventors by mutating one or more of the following sites of the amino acid sequence shown in SEQ ID NO: 1 can significantly improve the catalytic activity of the methanol dehydrogenase.

[0026] In this article, the terms "methanol dehydrogenase" or "methanol dehydrogenase of the present invention" or "methanol dehydrogenase mutant of the present invention" have the same meaning and can be used interchangeably herein, all referring to a methanol dehydrogenase having significantly improved catalytic methanol oxidation activity obtained by mutating one or more of the above-mentioned sites based on the wild-type methanol dehydrogenase with an amino acid sequence as shown in SEQ ID NO:1.

[0027] In view of the teachings of the present invention and the prior art, those skilled in the art should also understand that "methanol dehydrogenase of the present invention" should also include variant forms thereof, which have the same or similar functions as "methanol dehydrogenase of the present invention", but whose amino acid sequence is slightly different from the amino acid sequence of methanol dehydrogenase in the embodiment of the present invention. These variant forms include (but are not limited to): one or more (usually 1-30, preferably 1-10, more preferably 1-6, more preferably 1-3, and most preferably 1) amino acid deletion, insertion, and / or substitution, and one or more (usually within 30, preferably within 10, more preferably within 6 or 3) amino acids added to the C-terminus and / or N-terminus. For example, it is well known to those skilled in the art that substitution with amino acids with similar or similar properties, such as substitution of isoleucine with leucine, will not change the function of the resulting protein. For another example, adding one or more amino acids to the C-terminus and / or N-terminus, such as a 6×His tag added for easy separation, usually does not change the function of the resulting protein.

[0028] Those skilled in the art should also understand that the variant form of "methanol dehydrogenase of the present invention" described herein does not include the case where it is mutated and restored to the wild-type methanol dehydrogenase; in other words, the variant form of methanol dehydrogenase of the present invention is obtained by further mutation on the basis of the methanol dehydrogenase obtained in the embodiment of the present invention, but the amino acid residues at positions 153, 72, 79, 214, and / or 309 corresponding to the amino acid sequence shown in SEQ ID NO: 1 are the same as those in the amino acid sequence of methanol dehydrogenase obtained in the embodiment of the present invention.

[0029] The term "corresponding to" as used herein has the meaning generally understood by those of ordinary skill in the art. Specifically, "corresponding to" means that after two sequences are aligned for homology or sequence identity, a position in one sequence corresponds to a specified position in another sequence. Thus, for example, with respect to "the amino acid residue corresponding to position 79 of the amino acid sequence shown in SEQ ID NO. 1", if a 6×His tag is added to one end of the amino acid sequence shown in SEQ ID NO. 1, then the position corresponding to position 79 of the amino acid sequence shown in SEQ ID NO. 1 in the resulting mutant may be position 85.

[0030] In a specific embodiment, the homology or sequence identity can be greater than 90%, preferably greater than 95%, more preferably 96%, 97%, 98%, or 99% homology.

[0031] Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., M Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method for determining identity is to obtain the maximum match between the sequences tested. The method for determining identity is compiled in publicly available computer programs. Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN and FASTA (Altschul, SF et al., 1990). The public can obtain the BLASTX program from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

[0032] Variant forms of polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of "methanol dehydrogenase of the present invention" under high or low stringency conditions. The present invention also includes other polypeptides, such as fusion proteins comprising "methanol dehydrogenase of the present invention" or fragments thereof. In addition to almost full-length polypeptides, the present invention should also include active fragments of "methanol dehydrogenase of the present invention". Usually, the fragment has at least about 20 consecutive amino acids of the amino acid sequence of "methanol dehydrogenase of the present invention", usually at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0033] The present invention also provides analogs of "methanol dehydrogenase". The difference between these analogs and the natural "methanol dehydrogenase of the present invention" may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins listed above.

[0034] Modifications (usually without changing the primary structure) include: chemical derivatization of polypeptides in vivo or in vitro such as acetylation or carboxylation, and modifications also include glycosylation. Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes proteins that have been modified to improve their resistance to proteolysis or optimize their solubility.

[0035] In the present invention, the conservative variant polypeptide of "methanol dehydrogenase" refers to a polypeptide formed by replacing at most 20, preferably at most 10, more preferably at most 5, and most preferably at most 3 amino acids with similar or similar properties compared with the amino acid sequence of the methanol dehydrogenase in the examples of the present invention, but the conservative variant polypeptide still has the same or similar activity as the methanol dehydrogenase in the examples of the present invention, that is, the activity of catalyzing methanol oxidation is significantly improved.

[0036] In the present invention, the abbreviations of amino acids are shown in Table 1: Table 1 Amino acid abbreviations

[0037] In view of the teachings of the present invention and the prior art, those skilled in the art can generate conservative variant mutants by amino acid substitution as shown in the following examples: substitution of Ala to Ser or Thr, substitution of Arg to Gln, His or Lys, substitution of Asn to Glu, Gln, Lys, His or Asp, substitution of Asp to Asn, Glu or Gln, substitution of Cys to Ser or Ala, substitution of Gln to Asn, Glu, Lys, His, Asp or Arg, substitution of Glu to Gly, Asn, Gln, Lys or Asp, substitution of Gly to Pro, substitution of His to Asn , Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Lys to Asn, Glu, Gln, His or Arg substitution, Met to Ile, Leu, Val or Phe substitution, Phe to Trp, Tyr, Met, Ile or Leu substitution, Ser to Thr or Ala substitution, Thr to Ser or Ala substitution, Trp to Phe or Tyr substitution, Tyr to His, Phe or Trp substitution, and Val to Met, Ile or Leu substitution. In addition, conservative mutations also include naturally occurring mutations resulting from differences in individuals, strains, and species of gene sources.

[0038] In view of this, in a specific embodiment, the amino acid sequence of the methanol dehydrogenase of the present invention has the following amino acid residues at one or more sites selected from the group consisting of: 153: Pro or Ser or Val; 72nd: Thr; 79th: Ala; 214th: Asp; 309th: Lys.

[0039] In a specific embodiment, the amino acid sequence of the methanol dehydrogenase mutant is shown in any one of SEQ ID NOs: 3-9.

[0040] In the present invention, the polynucleotide sequence encoding "methanol dehydrogenase" can be inserted into a recombinant expression vector or genome.

[0041] The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0042] Those skilled in the art can use well-known methods to construct expression vectors containing "methanol dehydrogenase" encoding DNA sequences and appropriate transcription / translation control signals, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0043] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or kanamycin or ampicillin resistance for Escherichia coli.

[0044] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0045] The host cells described herein include host cells containing the above expression vector or the coding sequence of the "methanol dehydrogenase" of the present invention integrated into the genome. The host cells or strains of the present invention can efficiently express a novel methanol dehydrogenase with high catalytic performance, thereby achieving efficient methanol bioconversion.

[0046] The host cell of the present invention can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell. In a specific embodiment, the strain includes but is not limited to: Escherichia coli ( Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Bacillus subtilis ( Bacillus subtilis In a preferred embodiment, the strain is Corynebacterium glutamicum ( Corynebacterium glutamicum ).

[0047] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0048] The transformant obtained can be cultivated with conventional methods to express the polypeptide encoded by the gene of the present invention. According to the host cell used, the culture medium used in the cultivation can be selected from various conventional culture media. Cultivate under conditions suitable for the growth of the host cells. The recombinant polypeptide in the above method can be constitutively expressed or conditionally expressed, for example, when the host cells grow to a suitable cell density, induce the promoter selected with a suitable method (such as temperature conversion or chemical induction), and the cells are cultivated for a period of time.

[0049] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, high pressure homogenization, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0050] In view of the teachings of the present invention and the prior art, a person skilled in the art will understand that the methanol dehydrogenase of the present invention and its coding sequence, expression vector, and host cell can be used for methanol bioconversion.

[0051] The term "transformation" herein has a meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The method of transformation includes any method of introducing nucleic acid into a cell, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0052] The host cells herein can be cultured according to conventional methods in the art, 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.

[0053] As used herein, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0054] As used herein, "about" means that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0055] As used herein, the definition of "or" is only alternatives and "and / or", but unless it is explicitly stated that there are only alternatives or the alternatives are mutually exclusive, the term "or" in a claim means "and / or".

[0056] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0057] The culture medium used in the embodiment of the present invention is: M9 medium: 17.1 g / L Na2HPO4·12 H2O, 3 g / L KH2PO4, 1 g / L NH4Cl, 1 g / L casein hydrolysate, 0.5 g / L NaCl; and additionally 2 mM MgSO4, 2 mM CaCl2, 10 mg / L VB1 and trace metal elements are added to make M9 + Culture medium.

[0058] LB medium: 10 g / L tryptone, 10 g / L NaCl, and 5 g / L yeast powder.

[0059] The steps for protein purification and enzyme property detection are as follows: (1) freeze-dried bacteria (OD 600nm =40) and resuspended in PBS buffer (pH=7.4). (2) Ultrasonic cell disruption: 40% power, 120W, 1 s for each disruption and 3 s for disruption, 15 min. (3) Centrifuge the cell disruption solution obtained in the previous step at high speed, 13000 rpm, 30 min, and collect the supernatant. (4) Purify the target protein using nickel ion affinity chromatography, and add gradient concentrations of imidazole to elute the impurities. (5) Desalt and concentrate the target protein using a 10 kDa ultrafiltration tube to obtain purified wild-type and mutant proteins. (6) Observe the protein expression by SDS-PAGE electrophoresis, and quantify it using a BCA protein quantification kit. (7) Enzyme activity determination: The assay reaction was carried out in a PBS buffer (pH=7.4) system with a total volume of 200 μL. The components in the enzyme reaction system are shown in Table 4. After incubation at 37°C for 10 min, 100 μL of the reactant was added to 100 μL of enzyme solution. The DDL generation kinetic curve was measured at 412 nm using an ELISA reader to determine the formaldehyde content. (8) K MValue determination: Set the concentration gradient of the reaction substrate methanol: 10 mM, 20 mM, 40 mM, 100 mM, 250 mM, 500 mM, and keep the other reactants unchanged. Determine the specific enzyme activity of the wild type and mutant under different methanol concentrations. Use GraphPad Prism 5.0 software to fit the Michaelis-Menten curve and perform correlation processing to analyze the change of substrate affinity. (9) Catalytic efficiency calculation: The catalytic efficiency obtained in the previous measurement was V max , K M , enzyme activity data were processed to calculate the wild type and mutant K cat / K M values ​​and compare their catalytic efficiencies.

[0060] Example 1: Construction of a random mutation library of the methanol dehydrogenase gene 1. Construction of methanol dehydrogenase Mdh Bs Whole-genome random mutation library The methanol dehydrogenase described in this example is derived from Bacillus stearothermophilus ( Bacillus stearothermophilus DSM 2334), which is called Mdh Bs , whose amino acid sequence is shown in SEQ ID NO: 1, and whose nucleotide sequence is shown in SEQ ID NO: 2. The primers used for library construction are shown in Table 2, and the pTrc99A plasmid (Egon Amann, Jürgen Brosius, 1985. 'ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli . Gene. 40, 183-190) as a template, using primers pTrc-F / pTrc-R and ccdB-F / ccdB-R, to construct pTrc99A- ccdB Plasmid, and at the same time introduced into the plasmid through primers Yes I. Echo RI restriction endonuclease site. Yes I. Echo RI to pTrc99A- ccdB The plasmid was double-digested, and the digestion product a was purified and recovered. oh my Bs The gene was used as a template, and primers mdhclear-F / mdhclear-R were designed. At the same time, 0.05 mM or 0.1 mM Mn 2+PCR amplification was performed and the PCR amplification product b was purified and recovered. The recovered product a and the recovered product b were cloned and connected using the ClonExpressMultiS One Step Cloning Kit (C113) of Novozymes to construct a oh my Bs A random mutation library with a gene full length of 1020 bp was prepared. The quality of the library was analyzed and evaluated by colony PCR and Sanger sequencing verification. The colonies on the plate were then scraped and plasmids were extracted to obtain Mdh Bs Mixed plasmid library.

[0061] Table 2 Primers for constructing methanol dehydrogenase random mutation library

[0062] 2. Methanol dehydrogenase Mdh Bs Screening and sequencing of the whole-genome random mutation library To screen for Mdh Bs Mutant library, first electrophoretically transform the formaldehyde biosensor plasmid pSB4K5-sensor (Benjamin M. Woolston, et al., 2018. Development of aformaldehyde biosensor with application to synthetic methylotrophy. Biotechnology Bioengineering. 115, 206-215) based on the transcriptional regulator FrmR into Escherichia coli E. coli MG1655(Frederick R. Blattner, et al., 1997. The complete genome sequence of Escherichia coli K-12. Science. 277(5331), 1453) competent cells. After confirming that the sensor plasmid was successfully transferred into the cells, the Mdh mixed plasmid library obtained above was electroporated into Escherichia coli E. coil MG1655 (pSB4K5-sensor) competent cells were incubated at 37°C and 220 rpm for 50 min. Subsequently, the whole tube of bacterial suspension after incubation was transferred to M9 supplemented with 10 g / L glucose. + The culture medium was incubated at 37°C with shaking at 220 rpm for 12 h. 600nm = 0.05, second transfer to M9 supplemented with 2 g / L glucose + Culture medium until OD 600nm=0.4, add 10 μg / L tetracycline for 4 h, then add 0.001 mM IPTG for 2 h. After induction, take 144 μL M9 + The culture medium, 36 μL bacterial solution and 20 μL 5M methanol were added to a 96-well plate and cultured at 37°C with shaking at 800 rpm for 40 min. The cultured bacterial solution was diluted with M9 medium to OD 600nm =0.05, the fluorescence signal generated by the library was analyzed by flow cytometry, and the high fluorescence area was sorted at a ratio of 0.01%. The sorted plate was inverted and cultured overnight in a 37°C incubator.

[0063] The monoclonal colonies grown on the plate were picked and inoculated into 96-deep-well plates containing 200 μL LB medium in each well, and cultured at 37°C, 800 rpm for 12 h. Then they were transferred to 96-deep-well plates containing 200 μL M9 medium (containing 2 g / L glucose) in each well, and 0.1 mM IPTG was added for induction, and cultured at 37°C, 800 rpm for 8 h. After 8 h, 400 μL of bacterial solution, 320 μL Nash reagent (T. Nash, 1953. The colorimetric estimation of formaldehyde by meansof the Hantzsch reaction. Journal of Biological Chemistry. 55, 416-421) and 80 μL 5M methanol were taken, and samples were centrifuged at two time points of 40 min and 4 h to obtain the supernatant, and the absorbance was measured at 412 nm. The principle of this assay is that in the presence of ammonium acetate, the acetylacetone reagent can react with the formaldehyde produced by the oxidation of methanol to generate 3,5-diacetyl-1,4-dihydro-2,6-dimethylpyridine (DDL), which has a characteristic absorption peak at 412 nm and can indirectly reflect the formaldehyde concentration through absorbance quantification. At the same time, a standard curve was determined using a 0-2 mM formaldehyde standard solution, and the formaldehyde concentration generated in each sample was calculated based on the drawn standard curve. The results showed that the wild-type Mdh unit OD 600nm The formaldehyde concentration was lower than 1 mM, and about 30%-40% of the clones in the mutant library had an OD 600nm The formaldehyde concentrations were all higher than 1 mM, indicating that Mdh mutants with improved methanol catalytic activity in vivo can be screened from this library.

[0064] The above-screened unit OD 600nm The mutants with increased formaldehyde concentration were subjected to Sanger sequencing analysis. The sequencing results are shown in Table 3. In addition to the clones with the same mutation, three mutants were finally obtained. Their unit OD600nm The formaldehyde concentrations of the strains were higher than those of the wild type.

[0065] Table 3 Mutation information and unit OD of mutants 600nm Formaldehyde concentration

[0066] Example 2. Expression of methanol dehydrogenase mutants and detection of enzymatic properties Restriction endonucleases Yes I. Xho I double-digested the pET21a plasmid to obtain the vector backbone, and then the target gene of the mutant obtained in Example 1 was oh my V79A , oh my N214D+E309K , oh my K72T+T153P As well as wild type oh my The genes were connected to the pET21a protein expression vector. Subsequently, the successfully constructed expression vector was transformed into the protein expression host E. to be cultivated BL21(DE3)-pET21a- oh my , BL21(DE3)-pET21a- oh my V79A , BL21(DE3)-pET21a- oh my N214D+E309K , BL21(DE3)-pET21a- oh my K72T+T153P , used for the expression and enzymatic property detection of different enzymes. Among them, the double-site mutant Mdh with the highest catalytic activity in vivo, the highest catalytic efficiency in vitro, and the best soluble expression was K72T+T153P , split Mdh K72T and Mdh T153P The activity of single sites was further investigated.

[0067] The above recombinant bacterial colonies were inoculated into 5 mL LB medium containing ampicillin resistance and cultured at 37°C, 220 rpm for 12 h. They were then transferred to 100 mL LB liquid medium and cultured at 37°C, 220 rpm until OD 600nm =0.6-0.8, add IPTG with a final concentration of 0.1 mM, induction culture at 16°C for 16 h, collect the cells, and store them in a -20°C refrigerator for subsequent enzymatic property detection. The enzyme activity of each mutant was detected according to the enzymatic property detection method of the present invention. V max The relative enzyme activities are shown in Table 5.

[0068] Table 4 Concentration of components in the methanol dehydrogenase activity assay reaction system

[0069] Table 5 Mutants V max and enzyme activity

[0070] It can be seen that compared with wild-type Mdh, Mdh V79A , Mdh N214D+E309K , Mdh K72T+T153P The specific enzyme activities of the mutants were all improved, which were 1.2 times, 2.1 times and 2.5 times that of the wild type, respectively, indicating that the above mutants have good application prospects for the bioconversion and utilization of methanol. K72T+T153P mutants, in which the mutation that plays a major role is the T153P point mutation, Mdh T153P The specific enzyme activity is 1.7 times that of the wild type.

[0071] Example 3. Construction of T153 site-directed saturation mutation library The recombinant plasmid pTrc99A- oh my As a template, a pair of primers with mutation sites were used for PCR amplification with a high-fidelity enzyme to obtain a recombinant plasmid with the specified mutation site. Dpn I restriction endonuclease was treated at 37°C for 1 h to degrade the plasmid template. The digestion product was ligated to the pTrc99A plasmid vector to construct a T153 site-saturation mutation library. The subsequent screening process was the same as described in Example 1.

[0072] The library was sorted by flow cytometry and Sanger sequencing was performed to obtain 19 other mutants with altered amino acid types at position T153. The amount of formaldehyde generated by the whole cells of these mutants was determined using Nash reagent, and the results are shown in Table 6.

[0073] It can be seen that Mdh T153P and Mdh T153S Mutant unit OD 600nm The formaldehyde concentrations of Mdh were 2.4 and 1.6 times that of the wild type, respectively. T153V Mutant unit OD 600nm The formaldehyde concentration of the mutants was also higher than that of the wild type, while the OD 600nmThe formaldehyde concentrations of the wild type were comparable or significantly lower. It can be seen that when the amino acid at position 153 mutated to S, P, or V, the activity of Mdh increased; when the amino acid at position 153 mutated to M, the activity of Mdh was basically the same as that of the wild type; when the amino acid at position 153 mutated to the other 14 amino acids, the activity of Mdh decreased. This indicates that Mdh T153P , Mdh T153S and Mdh T153V The mutant has good application prospects in methanol bioutilization.

[0074] Table 6 Mutant information and unit OD 600nm Formaldehyde concentration

[0075] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A methanol dehydrogenase mutant, characterized in that The methanol dehydrogenase mutant is selected from any one of the following: (a) the amino acid sequence of which is derived from the sequence shown in SEQ ID NO: 1, wherein the amino acid residue at position 79 is mutated; and compared with the methanol dehydrogenase shown in SEQ ID NO: 1, the catalytic activity is improved; or (b) the methanol dehydrogenase mutant has 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence of (a), and has the function of the methanol dehydrogenase mutant of (a), wherein position 79 corresponding to the amino acid sequence of SEQ ID NO: 1 is the same as that in the amino acid sequence of (a); or (c) The methanol dehydrogenase mutant is formed by adding or deleting 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues at the C-terminus and / or N-terminus of the amino acid sequence described in (a), and has the function of the methanol dehydrogenase mutant described in (a), wherein position 79 corresponding to the amino acid sequence shown in SEQ ID NO: 1 is the same as that in the amino acid sequence described in (a).

2. The methanol dehydrogenase mutant according to claim 1, characterized in that The amino acid sequence of the methanol dehydrogenase mutant has an Ala mutation at position 79.

3. The methanol dehydrogenase mutant according to claim 2, characterized in that The amino acid sequence of the methanol dehydrogenase mutant is shown in SEQ ID NO:

3.

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

5. An expression vector, characterized in that: The expression vector comprises the polynucleotide according to claim 4.

6. A host cell, characterized in that The host cell comprises the expression vector of claim 5 or a polynucleotide having the methanol dehydrogenase mutant of claim 4 integrated into its genome.

7. The host cell according to claim 6, characterized in that The host cell is a bacterium; more preferably, the host cell is Escherichia coli ( Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), or Bacillus subtilis ( Bacillus subtilis ); Most preferably, the host cell is Corynebacterium glutamicum ( Corynebacterium glutamicum ).

8. Use of the methanol dehydrogenase mutant according to any one of claims 1 to 3, the polynucleotide according to claim 4, the expression vector according to claim 5, or the host cell according to 6 or 7 in methanol bioconversion.

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