Methanol dehydrogenase mutant with improved catalytic activity and use thereof

Abstract

The application discloses a methanol dehydrogenase mutant with improved catalytic activity. The application is based on a methanol dehydrogenase Mdh Bs derived from Bacillus stearothermophilus, and the mutant Mdh Bs with improved catalytic activity in methanol is obtained by constructing a full gene random mutation library, screening and sequencing. V79A The obtained mutant has a good application prospect in methanol bio-utilization.

Description

[0001] This application is a divisional application of the invention patent application with the application number "202310131430.6" and the application date "2023-02-17", and the invention name "Methanol dehydrogenase mutant with improved catalytic activity and its application". TECHNICAL FIELD

[0002] The present application belongs to the field of molecular biology and bioengineering, and specifically relates to a methanol dehydrogenase mutant with improved catalytic activity and its application. BACKGROUND

[0003] Biomanufacturing is an important way to promote the evolution of industrial manufacturing towards a sustainable development model. However, biomanufacturing is highly dependent on food raw materials at present. Methanol is a primary platform product of coal chemical industry, shale gas chemical industry and industrial and agricultural waste gasification, and is a natural one-carbon compound. It has the advantages of wide sources, low price, high conversion rate, etc. Compared with sugars, it has lower and stable price, and higher average carbon atom reduction degree, which can provide more reducing power for biosynthesis process, and has obvious advantages in biological transformation. Therefore, methanol is an ideal raw material for biomanufacturing bulk chemicals, and has great development potential and broad development prospects in the future. Research on the biological transformation of methanol has great economic and social significance.

[0004] In the process of microbial one-carbon metabolism, methanol is first catalyzed by methanol dehydrogenase (Mdh) to generate formaldehyde. Subsequently, formaldehyde is further synthesized into energy and carbon skeleton required for cell growth and metabolism through different assimilation pathways such as ribulose monophosphate pathway (RuMP), xylulose monophosphate pathway (XuMP) and serine pathway, or is oxidized to 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 a key reaction limiting the efficiency of methanol biological transformation. Therefore, there is an urgent need in the art to direct the evolution of Mdh to obtain Mdh with higher activity, so as to make up for the kinetic defects of methanol biological transformation and help to achieve efficient methanol biological transformation. SUMMARY

[0006] The purpose of the present application is to provide a methanol dehydrogenase mutant with improved catalytic activity. The present application is based on a methanol dehydrogenase Mdh derived from Bacillus stearothermophilus (B. stearothermophilus) DSM 2334. Bacillus stearothermophilus DSM 2334 Bs(hereinafter referred to as wild-type methanol dehydrogenase), by constructing methanol dehydrogenase Mdh Bs A full gene random mutation library was screened and sequenced to obtain three mutants of 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 To explore the single-site activity; further use the site-directed saturation mutagenesis strategy to construct a T153 saturation mutation library and verify it through sequencing, and screen out mutants Mdh with improved catalytic activity T153S and Mdh T153V These mutants have good application prospects in methanol bioutilization.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions:

[0008] 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:

[0009] (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

[0010] The amino acid residues are mutated at positions 153, 72, 79, 214, and 309; and the catalytic activity is improved compared to the methanol dehydrogenase shown in SEQ ID NO: 1;

[0011] or

[0012] (b) The methanol dehydrogenase mutant has 95%, preferably 98%, and more preferably 99% of the amino acid sequence of (a).

[0013] 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 described in (a);

[0014] or

[0015] (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).

[0016] 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:

[0017] 153: Pro or Ser or Val;

[0018] 72nd position: Thr;

[0019] 79th: Ala;

[0020] 214th position: Asp;

[0021] 309th position: Lys.

[0022] In a specific embodiment, the amino acid sequence of the methanol dehydrogenase mutant is shown in any one of SEQ ID NOs: 3-9.

[0023] In a second aspect, the present invention provides a polynucleotide encoding the methanol dehydrogenase mutant according to the first aspect.

[0024] In a third aspect, the present invention provides an expression vector comprising the polynucleotide according to the second aspect of the present invention.

[0025] In a fourth aspect, the present invention provides a host cell, comprising the expression vector according to the third aspect of the present invention or a polynucleotide having the methanol dehydrogenase mutant according to the second aspect of the present invention integrated into its genome.

[0026] In a preferred embodiment, the host cell is a bacterium; more preferably, the host cell is Escherichia coli

[0027] ( Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), or Bacillus subtilis ( Bacillus subtilis ); Most preferably, the host cell is Corynebacterium glutamicum ( Corynebacterium glutamic ).

[0028] 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.

[0029] Beneficial effects of the present invention

[0030] 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 MDH were both 2.5 times higher than those of wild-type MDH. T153P and Mdh T153S Mutant unit OD 600nm The 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 the foundation for the realization of industrial-scale methanol bioconversion. DETAILED DESCRIPTION

[0031] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative 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 encompassed within the scope of protection that the present invention is intended to protect.

[0032] Unless otherwise noted, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under 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.

[0033] Definition and explanation:

[0034] 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.

[0035] In this article, the terms "methanol bioconversion" and "bioconversion of 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 through the methanol metabolic pathway in microorganisms, or the conversion of methanol into products such as bacteria, amino acids, organic acids, and polyols.

[0036] As used herein, the terms "methanol dehydrogenase," "Methanol dehydrogenase," and "Mdh" have the meanings commonly understood by those skilled in the art, and refer to an enzyme that can catalyze the oxidation of methanol to produce formaldehyde, the amino acid sequence of which is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO: 2.

[0037] As used herein, the terms "wild-type" and "naturally occurring" refer to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism, can be isolated from a source in nature, and has not been intentionally modified by humans in the laboratory is naturally occurring.

[0038] As used herein, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of an individual fragment or a component of a larger nucleotide sequence structure, derived from a nucleotide sequence isolated at least once in quantity or concentration, and capable of identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard molecular biology methods (e.g., using cloning vectors). 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) where "U" replaces "T." In other words, a "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (either individual fragments or entire fragments), or can be a component or constituent of a larger nucleotide structure, such as an expression vector or polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0039] As used herein, 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).

[0040] In the present text, the term "mutant" refers to a polynucleotide or polypeptide that comprises an alteration (i.e., a substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to a "wild type", or "compared to", polynucleotide or polypeptide, wherein a substitution refers to the replacement of 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 after the nucleotide or amino acid occupying a position, adjacent and immediately following. It is well known to those skilled in the art that if one wishes to mutate an enzyme in order to obtain a mutant with improved activity, the key is to find the site(s) where mutation can improve the activity. In the present application, the wild type methanol dehydrogenase with the amino acid sequence shown in SEQ ID NO: 1 is mutated at specific positions to obtain a methanol dehydrogenase mutant with significantly improved activity.

[0041] In a specific embodiment, the present inventors have found that mutating the methanol dehydrogenase at one or more of the following positions of the amino acid sequence shown in SEQ ID NO: 1 can significantly improve the catalytic activity of the methanol dehydrogenase: position 153, position 72, position 79, position 214, and / or position 309.

[0042] In the present text, the terms "methanol dehydrogenase" or "methanol dehydrogenase of the present application" or "methanol dehydrogenase mutant of the present application" have the same meaning and can be used interchangeably herein, and refer to a methanol dehydrogenase with significantly improved catalytic activity for methanol oxidation, obtained by mutating the wild type methanol dehydrogenase with the amino acid sequence shown in SEQ ID NO: 1 at one or more of the above-mentioned positions.

[0043] In view of the teachings of the present application and the prior art, those skilled in the art will further appreciate that the "methanol dehydrogenase of the present application" should also include variants thereof that have the same or similar function as the "methanol dehydrogenase of the present application", but have a slightly different amino acid sequence from the methanol dehydrogenase of the examples of the present application. Such variants include, but are not limited to, deletion, insertion, and / or substitution of one or more (typically 1-30, preferably 1-10, more preferably 1-6, even more preferably 1-3, most preferably 1) amino acids, and addition of one or more (typically 30 or fewer, preferably 10 or fewer, more preferably 6 or 3 or fewer) amino acids at the C-terminus and / or N-terminus. For example, it is well known to those skilled in the art that substitution with a similar or identical amino acid, for example, substitution of isoleucine with leucine, does not change the function of the resulting protein. For another example, addition of one or several amino acids at the C-terminus and / or N-terminus, for example, a 6xHis tag added for ease of isolation, does not change the function of the resulting protein.

[0044] Those skilled in the art should also understand that the variant form of the "methanol dehydrogenase of the present invention" described herein does not include the case where it is mutated and reverted to the wild-type methanol dehydrogenase; in other words, the variant form of the methanol dehydrogenase of the present invention is obtained by further mutation based on the methanol dehydrogenase obtained in the examples of the present invention, but the amino acid residues corresponding to positions 153, 72, 79, 214, and / or 309 of the amino acid sequence shown in SEQ ID NO: 1 are the same as those in the amino acid sequence of the methanol dehydrogenase obtained in the examples of the present invention.

[0045] As used herein, the term "corresponding to" has the meaning commonly understood by those skilled in the art. Specifically, "corresponding to" refers to a position in one sequence that corresponds to a specified position in the other sequence after alignment for homology or sequence identity. Thus, for example, with respect to "the amino acid residue corresponding to position 79 of the amino acid sequence set forth in SEQ ID NO: 1," if a 6×His tag is added to one end of the amino acid sequence set forth in SEQ ID NO: 1, then position 79 in the resulting mutant corresponding to amino acid position 85 of the amino acid sequence set forth in SEQ ID NO: 1 may be the amino acid residue corresponding to position 79 of the amino acid sequence set forth in SEQ ID NO: 1.

[0046] 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%.

[0047] Methods for determining sequence homology or identity that are well 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, eds., 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., eds., M Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Biology. Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining 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 BLASTX program is publicly available 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.

[0048] Variant forms of polypeptides include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and proteins encoded by DNA that can hybridize with the DNA encoding the "methanol dehydrogenase of the present invention" under high or low stringency conditions. The present invention also includes other polypeptides, such as fusion proteins comprising the "methanol dehydrogenase of the present invention" or fragments thereof. In addition to substantially full-length polypeptides, the present invention also encompasses active fragments of the "methanol dehydrogenase of the present invention." Typically, such fragments have at least about 20 consecutive amino acids of the amino acid sequence of the "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.

[0049] The present invention also provides analogs of "methanol dehydrogenase". The differences between these analogs and the natural "methanol dehydrogenase of the present invention" may be differences in amino acid sequence, or differences in modified forms that do 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), as well as 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.

[0050] Modifications (generally without altering the primary structure) include chemical derivatization of peptides in vivo or in vitro, such as acetylation or carboxylation, and glycosylation. Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine). Proteins modified to enhance resistance to proteolysis or optimize solubility are also included.

[0051] In the present invention, the conservative variant polypeptide of "methanol dehydrogenase" refers to a polypeptide in which at most 20, preferably at most 10, more preferably at most 5, and most preferably at most 3 amino acids are replaced by 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.

[0052] In the present invention, the abbreviations of amino acids are shown in Table 1:

[0053] Table 1 Amino acid abbreviations

[0054]

[0055] In view of the teachings of the present application and the prior art, those skilled in the art can produce mutants of conservative variation by amino acid substitution according to the following exemplary examples: substitution of Ala for Ser or Thr, substitution of Arg for Gin, His or Lys, substitution of Asn for Glu, Gin, Lys, His or Asp, substitution of Asp for Asn, Glu or Gin, substitution of Cys for Ser or Ala, substitution of Gin for Asn, Glu, Lys, His, Asp or Arg, substitution of Glu for Gly, Asn, Gin, Lys or Asp, substitution of Gly for Pro, substitution of His for Asn, Lys, Gin, Arg or Tyr, substitution of He for Leu, Met, Val or Phe, substitution of Leu for He, Met, Val or Phe, substitution of Lys for Asn, Glu, Gin, His or Arg, substitution of Met for He, Leu, Val or Phe, substitution of Phe for Trp, Tyr, Met, He or Leu, substitution of Ser for Thr or Ala, substitution of Thr for Ser or Ala, substitution of Trp for Phe or Tyr, substitution of Tyr for His, Phe or Trp, and substitution of Val for Met, He or Leu. In addition, conservative mutations also include naturally occurring mutations due to differences in individuals, strains, species, etc. of gene origin.

[0056] In view of the above, in a specific embodiment, the amino acid sequence of the methanol dehydrogenase of the present application has the amino acid residue shown below at one or more positions selected from the group consisting of:

[0057] 153: Pro or Ser or Val;

[0058] 72: Thr;

[0059] 79: Ala;

[0060] 214: Asp;

[0061] 309: Lys.

[0062] In a specific embodiment, the amino acid sequence of the methanol dehydrogenase mutant is as shown in any one of SEQ ID NOs: 3-9.

[0063] In the present application, the polynucleotide sequence encoding the "methanol dehydrogenase" can be inserted into a recombinant expression vector or genome.

[0064] 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 an origin of replication, a promoter, a marker gene, and translation control elements.

[0065] Those skilled in the art can construct expression vectors containing a DNA sequence encoding methanol dehydrogenase and appropriate transcriptional / translational control signals using well-known methods, including in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0066] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of 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.

[0067] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0068] The host cells described herein include host cells containing the aforementioned expression vectors or having the coding sequence of the "methanol dehydrogenase" of the present invention integrated into their genome. The host cells or strains of the present invention are capable of efficiently expressing the novel methanol dehydrogenase with high catalytic performance, thereby achieving efficient methanol bioconversion.

[0069] 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 ).

[0070] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0071] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out 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, after the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0072] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing 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 a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, high-pressure homogenization, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0073] In view of the teachings of the present invention and the prior art, those skilled in the art will understand that the methanol dehydrogenase of the present invention, its coding sequence, expression vector, and host cell can be used for methanol bioconversion.

[0074] The term "transformation" as used herein has a meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The methods of transformation 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.

[0075] 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, and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

[0076] As used herein, the terms "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0077] 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.

[0078] 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".

[0079] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0080] The culture medium used in the embodiment of the present invention is:

[0081] 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; additionally supplemented with 2 mM MgSO4, 2 mM CaCl2, 10 mg / L VB1 and trace metal elements to make M9 + culture medium.

[0082] LB medium: 10 g / L tryptone, 10 g / L NaCl, and 5 g / L yeast powder.

[0083] The steps for protein purification and enzyme property detection are as follows: (1) Frozen bacteria (OD 600nm=40) and resuspended in PBS buffer (pH=7.4). (2) Ultrasonic cell disruption: 40% power, 120W, 1 s for each disruption, 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 level by SDS-PAGE electrophoresis and quantify it using a BCA protein quantification kit. (7) Enzyme activity determination: The determination reaction was carried out in a PBS buffer system (pH=7.4) 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 aspirated and added to 100 μL of enzyme solution. The DDL formation kinetic curve was measured at 412 nm using a microplate reader to determine the formaldehyde content. (8) K M Determination of the value: The concentration gradient of the reaction substrate methanol was set to 10 mM, 20 mM, 40 mM, 100 mM, 250 mM, and 500 mM. The other reactants remained unchanged. The specific enzyme activity of the wild type and mutant at different methanol concentrations was measured. Michaelis-Menten curves were fitted using GraphPad Prism 5.0 software, and the changes in substrate affinity were analyzed by correlation processing. (9) Calculation of catalytic efficiency: The values ​​obtained in the previous determination were V max 、 K M , enzyme activity data were processed to calculate the activity of wild type and mutant K cat / K M values ​​and compare their catalytic efficiencies.

[0084] Example 1: Construction of a random mutation library of the methanol dehydrogenase gene

[0085] 1. Construction of methanol dehydrogenase Mdh Bs Whole-genome random mutation library

[0086] The methanol dehydrogenase described in this example is derived from Bacillus stearothermophilus ( Bacillus stearothermophilus DSM 2334), which is called Mdh BsThe amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2. The primers used for library construction are shown in Table 2. 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. 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 cloned product targeting oh my Bs A random mutation library with a full gene length of 1020 bp was prepared. The library quality was analyzed and evaluated by colony PCR and Sanger sequencing. The colonies on the plate were then scraped and plasmids were extracted to obtain Mdh. Bs mixed plasmid library.

[0087] Table 2 Primers for constructing methanol dehydrogenase random mutation library

[0088]

[0089] 2. Methanol dehydrogenase Mdh Bs Screening and sequencing of whole-genome random mutation libraries

[0090] To screen for Mdh BsTo prepare the mutant library, 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 was first electroporated 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 was transferred to M9 medium supplemented with 10 g / L glucose. + Culture in the culture medium at 37°C with shaking at 220 rpm for 12 h. 600nm = 0.05, second transfer to M9 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 an OD of 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 incubated upside down in a 37°C incubator overnight.

[0091] Single colonies grown on the plates were inoculated into 96-deep-well plates containing 200 μL of LB medium per well and cultured at 37°C, 800 rpm, for 12 h. The cultures were then transferred to 96-deep-well plates containing 200 μL of M9 medium (containing 2 g / L glucose) per well. 0.1 mM IPTG was added for induction and cultured at 37°C, 800 rpm, for 8 h. After 8 h, 400 μL of the bacterial suspension, 320 μL of Nash's reagent (T. Nash, 1953. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Journal of Biological Chemistry. 55, 416-421), and 80 μL of 5 M methanol were added. Samples were collected at 40 min and 4 h after the reaction and centrifuged. The supernatant was then measured for absorbance at 412 nm. The principle of this assay is that in the presence of ammonium acetate, the acetylacetone reagent can react with formaldehyde produced by methanol oxidation 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 by 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 of the samples were all higher than 1 mM, indicating that Mdh mutants with improved methanol catalytic activity in vivo can be screened from this library.

[0092] 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 OD 600nm The formaldehyde concentrations were higher than those in the wild type.

[0093] Table 3 Mutation information and unit OD of mutants 600nm Formaldehyde concentration

[0094]

[0095] Example 2. Expression of methanol dehydrogenase mutants and detection of enzymatic properties

[0096] Restriction endonucleases Yes I. XhoI. The pET21a plasmid was double-digested to obtain the vector backbone, and then the target gene of the mutant obtained in Example 1 was inserted into the oh my V79A 、 oh my N214D+E309K 、 oh my K72T+T153P and 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 K72T+T153P , split out Mdh K72T and Mdh T153P The activity of single sites was further investigated.

[0097] The above recombinant bacterial colonies were inoculated into 5 mL LB medium containing ampicillin resistance, cultured at 37°C, 220 rpm for 12 h, and 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 in a -20 ° C refrigerator for subsequent enzymatic property detection. V max The enzyme activities are shown in Table 5.

[0098] Table 4 Concentration of components in the methanol dehydrogenase activity assay reaction system

[0099]

[0100] Table 5 Mutants V max and enzyme activity

[0101]

[0102] 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 main functional mutation is T153P point mutation, Mdh T153P The specific enzyme activity is 1.7 times that of the wild type.

[0103] Example 3. Construction of T153 site-directed saturation mutagenesis library

[0104] The recombinant plasmid pTrc99A- oh my As a template, a pair of primers with mutation sites were used to perform PCR amplification with a high-fidelity enzyme to obtain a recombinant plasmid with the specified mutation site. Dpn The plasmid template was degraded by treatment with restriction endonuclease I at 37°C for 1 hour. The digested product was ligated into the pTrc99A plasmid vector to construct a T153 site-saturation mutagenesis library. The subsequent screening process was the same as described in Example 1.

[0105] The library was sorted by flow cytometry and Sanger sequencing to obtain 19 additional mutants with altered amino acid types at position T153. The formaldehyde production catalyzed by these mutants in whole cells was measured using Nash reagent, and the results are shown in Table 6.

[0106] 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 other 16 mutants was also higher than that of the wild type, while the OD 600nm The formaldehyde concentrations of the Mdh were comparable to or significantly lower than those of the wild type. 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.

[0107] Table 6 Mutant information and unit OD 600nm Formaldehyde concentration

[0108]

[0109] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A methanol dehydrogenase mutant, characterized in that The amino acid sequence is obtained by mutating position 79 of the sequence shown in SEQ ID NO: 1 to Ala.

2. The methanol dehydrogenase mutant according to claim 1, characterized in that The amino acid sequence of the methanol dehydrogenase mutant is shown in SEQ ID NO:

3.

3. A polynucleotide encoding the methanol dehydrogenase mutant according to claim 1 or 2.

4. An expression vector, characterized in that The expression vector comprises the polynucleotide according to claim 3.

5. A host cell, characterized in that The host cell comprises the expression vector according to claim 4 or a polynucleotide having the methanol dehydrogenase mutant according to claim 3 integrated into its genome; the host cell is a non-plant variety or an animal variety.

6. The host cell according to claim 5, characterized in that The host cell is a bacterium.

7. The host cell according to claim 6, characterized in that The host cell is Escherichia coli ( Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), or Bacillus subtilis ( Bacillus subtilis ).

8. The host cell according to claim 7, characterized in that The host cell is Corynebacterium glutamicum ( Corynebacterium glutamicum ).

9. Use of the methanol dehydrogenase mutant according to claim 1 or 2, the polynucleotide according to claim 3, the expression vector according to claim 4, or the host cell according to any one of 5 to 8 in methanol bioconversion.

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