A methanol dehydrogenase mutant with improved catalytic activity and application thereof

Abstract

The application discloses a methanol dehydrogenase mutant with improved catalytic activity. The application is based on a methanol dehydrogenase Mdh from Bacillus stearothermophilus Bs , and the mutant Mdh Bs with improved catalytic activity in methanol is obtained by constructing a full gene random mutation library, screening and sequencing. N241D , Mdh E309K and Mdh N241D+E309K , and the obtained mutant has a good application prospect in methanol bioavailability.

Description

[0001] This application is a divisional application of the invention patent application with the application number "202310131430.6", the application date of February 17, 2023, and the invention name of "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, at present, biomanufacturing is highly dependent on food raw materials. According to the statistics of the National Bureau of Statistics, about 200 million tons of food for fermentation are used in China, which is equivalent to about 30% of the total grain output. The supply of raw materials 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, and is a natural one-carbon compound. It has the advantages of wide source, low price, high conversion rate, etc. Compared with sugars, it has lower and more stable prices, 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 of bulk chemicals, and has great development potential and broad development prospects in the future. Therefore, the study of methanol biological transformation 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 that limits 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 mutant derived from Bacillus stearothermophilus (Bacillus stearothermophilus Methanol dehydrogenase Mdh of DSM 2334 Bs (hereinafter referred to as wild-type methanol dehydrogenase), by constructing a methanol dehydrogenase Mdh Bs Full gene random mutation library, 3 mutants Mdh with improved in vivo methanol catalytic activity were obtained by screening and sequencing V79A , Mdh N214D+E309K , Mdh K72T+T153P ; at the same time, the unit point activity of the optimal mutant Mdh K72T+T153P , Mdh K72T , and Mdh T153P was explored; further, a T153 saturation mutation library was constructed by using site-directed saturation mutation strategy and verified by sequencing, and mutants Mdh T153S , and Mdh T153V with improved catalytic activity were screened. These mutants have good application prospect in methanol bioavailability.

[0007] To achieve the above object, the technical scheme of the present application is as follows:

[0008] In the first aspect, the present application provides a methanol dehydrogenase mutant, which is selected from any one of the following:

[0009] (a) the amino acid sequence thereof is mutated from the sequence shown in SEQ ID NO: 1, and is mutated at one or more amino acid residue sites selected from the group consisting of positions 153, 72, 79, 214, and 309; and has improved catalytic activity compared with the methanol dehydrogenase shown in SEQ ID NO: 1;

[0010]

[0011] or

[0012] (b) the methanol dehydrogenase mutant has 95%, preferably 98%, more preferably 99% sequence identity to the amino acid sequence of (a), and has the function of the methanol dehydrogenase mutant of (a), wherein 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 in the amino acid sequence of (a);

[0013]

[0014] or

[0015] ​​(c) the methanol dehydrogenase mutant consists of 1-30, more preferably 1-10, still more preferably 1-6, most preferably 1-3 amino acid residues added or deleted at the C-terminus and / or N-terminus of the amino acid sequence of (a) and has the function of the methanol dehydrogenase mutant of (a), wherein the amino acid residue corresponding to position 153, 72, 79, 214, and / or 309 of the amino acid sequence shown in SEQ ID NO: 1 is identical to that in the amino acid sequence of (a).

[0016] According to an embodiment of the present application, the amino acid sequence of the methanol dehydrogenase mutant is mutated at one or more positions selected from the group consisting of:

[0017] 153: Pro or Ser or Val;

[0018] 72: Thr;

[0019] 79: Ala;

[0020] 214: Asp;

[0021] 309: Lys.

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

[0023] In a second aspect, the present application provides a polynucleotide encoding the methanol dehydrogenase mutant of the first aspect.

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

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

[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 glutamicum

[0028] ​In a fifth aspect, the present application provides use of the methanol dehydrogenase mutant of the first aspect of the present application, the polynucleotide of the second aspect of the present application, the expression vector of the third aspect of the present application or the host cell of the fourth aspect of the present application in methanol bioconversion.

[0029] Advantages of the present application

[0030] The present application obtains a series of methanol dehydrogenase mutants with significantly improved catalytic activity. In enzyme activity determination, the specific activity of each mutant is V max and the specific activity is up to 2.5 times of the wild-type methanol dehydrogenase. In methanol bioconversion, Mdh T153P and Mdh T153S The mutant unit OD 600nm The concentration of formaldehyde is 1.6-2.4 times of the wild-type control. The mutant of the present application has higher catalytic activity, which is beneficial to accelerate the bioconversion of methanol, thereby having significant economic value and social value, and laying a foundation for realizing industrial large-scale methanol bioconversion. DETAILED DESCRIPTION

[0031] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope intended to be protected by the present application.

[0032] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods not specified in the following examples are generally carried out according to conventional conditions, such as the conditions 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] Definitions and explanations:

[0034] In this paper, the "one-carbon compound", "C1 compound" refers to the 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 paper, the "methanol bioconversion", "bioconversion of methanol" has the same meaning, which refers to the conversion of methanol to harmless products (such as carbon dioxide) through a series of enzyme-catalyzed reactions in the methanol metabolic pathway of microorganisms, or the conversion of methanol to bacterial cells, amino acids, organic acids, polyols, etc.

[0036] In the present text, the "methanol dehydrogenase", "Methanol dehydrogenase", "Mdh" has the meaning commonly understood by a person skilled in the art and refers to an enzyme capable of catalyzing the oxidation of methanol to formaldehyde, the amino acid sequence of which is shown in SEQ ID NO: 1, the nucleotide sequence of the encoding gene of which is shown in SEQ ID NO: 2.

[0037] In the present text, the "wild type", "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.

[0038] In the present text, the "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a single fragment or can be a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence that has been isolated at least once in quantity or concentration, which can be identified, manipulated and the sequence and its component nucleotide sequences recovered 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), wherein "U" replaces "T". In other words, "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (single fragments or entire fragments) or can 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.

[0039] In the present text, the "polypeptide", "peptide" and "protein" are used interchangeably herein and are amino acid polymers of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also includes the amino acid polymer which has 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] It will also be understood by those skilled in the art that the variant of the methanol dehydrogenase enzyme of the present application as described herein does not include the case where the mutation is reverted to the wild-type methanol dehydrogenase enzyme; in other words, the variant of the methanol dehydrogenase enzyme of the present application is obtained by further mutating the methanol dehydrogenase enzyme obtained in the embodiments of the present application, but the amino acid residue corresponding to position 153, 72, 79, 214, and / or 309 of the amino acid sequence shown in SEQ ID NO: 1 is the same as in the amino acid sequence of the methanol dehydrogenase enzyme obtained in the embodiments of the present application.

[0045] The term "corresponding to" as used herein has the meaning commonly understood by those of ordinary skill in the art. In particular, "corresponding to" means that, after homology or sequence identity alignment, a position in one sequence corresponds to a specified position in another sequence. Thus, for example, in the case of "the amino acid residue corresponding to position 79 of the amino acid sequence shown in SEQ ID NO: 1", if a 6xHis tag is added at 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 can be position 85.

[0046] In a particular embodiment, the homology or sequence identity can be 90% or more, preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology.

[0047] Methods to determine sequence homology or identity known to those of ordinary skill in the art include, but are not limited to, Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods of determining identity are those that result in the greatest match between the sequences tested. Methods of 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 suite of programs (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S. F. 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] Variants of the polypeptides include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to the "methanol dehydrogenase of the present invention" under conditions of high or low stringency. 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 polypeptides that are nearly full length, the present invention also includes active fragments of the "methanol dehydrogenase of the present invention". Typically, the fragments have at least about 20 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the amino acid sequence of the "methanol dehydrogenase of the present invention".

[0049] The present invention also provides analogs of the "methanol dehydrogenase". These analogs can differ from the naturally occurring "methanol dehydrogenase of the present invention" by differences in the amino acid sequence, by modifications that do not affect the sequence, or by both. These polypeptides include naturally or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, by site-directed mutagenesis, or by other known molecular biology techniques. Analogues also include those with residues other than naturally occurring L-amino acids (e.g., D-amino acids), as well as analogues with non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It is understood that the proteins of the present invention are not limited to the representative proteins listed above.

[0050] Modifications (which generally do not change the primary structure) include chemical derivatization of the polypeptide in vivo or in vitro, such as acetylation or carboxylation, and modifications include glycosylation. Modifications also include sequences with phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Proteins modified to increase their resistance to proteolysis or to optimize solubility are also included.

[0051] In the present invention, a conservative variant of the "methanol dehydrogenase" is a polypeptide in which up to 20, preferably up to 10, more preferably up to 5, and most preferably up to 3 amino acids are replaced by similar or nearly similar amino acids, but the conservative variant polypeptide still has the same or similar activity as the "methanol dehydrogenase of the present invention", i.e., significantly increased activity in catalyzing the oxidation of methanol.

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

[0053] Table 1 Abbreviations for Amino Acids

[0054]

[0055] In view of the teachings of the present invention and the prior art, those skilled in the art can generate conservative variants 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, 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 due to differences in individuals, strains, species of gene origin.

[0056] 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:

[0057] 153: Pro or Ser or Val;

[0058] 72nd position: Thr;

[0059] 79th: Ala;

[0060] 214th position: Asp;

[0061] 309th position: Lys.

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

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

[0064] The term "recombinant expression vector" refers to a nucleic acid construct that has been generated by recombinant DNA technology, and includes a bacterial plasmid, bacteriophage, yeast plasmid, plant cell viral vector, mammalian cell viral vector, or other vector. In general, any plasmid or vector can be used as long as it is replicable and 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 a translational control sequence.

[0065] Those skilled in the art can use well-known methods to construct an expression vector containing a "methanol dehydrogenase" encoding DNA sequence and suitable transcription / translation control signals, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be operably linked to a suitable 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 for eukaryotic cell culture, neomycin resistance for eukaryotic cells, and green fluorescent protein (GFP), or kanamycin or ampicillin resistance for E. coli.

[0067] The vector containing the appropriate DNA sequence described above and a suitable 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 expression vector described above or the coding sequence of the "methanol dehydrogenase" of the present application integrated on the genome. The host cells or strains of the present application can efficiently express a novel methanol dehydrogenase with high catalytic performance, thereby achieving efficient biological conversion of methanol.

[0069] The host cells of the present application can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells. In specific embodiments, the strains include, but are not limited to: E. coli (Escherichia coli) Escherichia coli ), Corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum ), Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ). In a preferred embodiment, the strain is Corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum

[0070] ​Transformation of host cells with recombinant DNA can be performed using conventional techniques appropriate to the host cell used. Where the host is a prokaryote, such as E. coli, a competent cell capable of taking up and replicating the DNA is harvested after the exponential growth phase, treated with CaCl2, and the steps used are well known in the art. Alternatively, MgCl2can be used. If desired, transformation can be performed by electroporation. Where the host is a eukaryote, DNA can be introduced using methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.

[0071] The resulting transformants can be cultured in conventional nutrient media using conventional techniques, the expressed polypeptide of the application being encoded by the gene. The culture conditions, such as temperature, pH, and the like, can be readily determined by those of ordinary skill in the art. The recombinant polypeptide in the above method can be constitutively expressed or conditionally expressed, for example, after the host cell has grown to an appropriate cell density, the selected promoter is induced by a suitable method (e.g., temperature shift or chemical induction), and the cells are cultured for an additional period of time.

[0072] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted from the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those of ordinary skill 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 lysis, ultrasonication, 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.

[0073] In view of the teachings of the present application and the prior art, those of ordinary skill in the art will appreciate that the methanol dehydrogenase of the present application and its encoding sequence, expression vector, host cell can be used for methanol bioconversion.

[0074] The term "transformation" herein is generally understood by those of ordinary skill in the art to mean the process of introducing foreign DNA into a host. The methods of transformation include 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.

[0075] The culture of the host cell herein can be performed 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 of the culture medium can be appropriately adjusted according to the actual situation.

[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 ± one standard deviation of the error in the apparatus or method used to determine the value.

[0078] As used herein, the definition of "or" is the inclusive, not the exclusive sense unless explicitly indicated otherwise or indicated in the context that only alternatives are intended. The term "or" as used herein means either or both of the listed alternatives.

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

[0080] Culture medium used in the embodiments of the present application:

[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; and additionally adding 2 mM MgSO4, 2 mM CaCl2, 10 mg / L VB1 and trace metal elements to prepare M9 + Culture medium.

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

[0083] Protein purification and enzymatic property detection steps are as follows: (1) the 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 Nde I. Eco RI restriction endonuclease site. Nde I. Eco RI to pTrc99A- ccdB The plasmid was double-digested and the digestion product a was purified and recovered. mdh 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 Norwegian to construct a mdh 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 growing on the plates were picked and inoculated into 96-deep well plates containing 200 μL LB medium per well, and incubated at 37°C, 800 rpm for 12 h. Then the culture was transferred into 96-deep well plates containing 200 μL M9 medium (containing 2 g / L glucose) per well, and 0.1 mM IPTG was added to induce the expression of the enzyme. The plates were incubated at 37°C, 800 rpm for 8 h. After 8 h, 400 μL of the culture was mixed with 320 μL Nash 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 5M methanol. The mixture was incubated for 40 min and 4 h, and then centrifuged to collect the supernatant. The absorbance of the supernatant was measured at 412 nm. The principle of the assay is that acetylacetone reagent can react with formaldehyde to form 3,5-diacetyl-l,4-dihydro-2,6-dimethylpyridine (DDL) in the presence of ammonium acetate. The DDL has a characteristic absorption peak at 412 nm, and the concentration of formaldehyde can be indirectly reflected by the absorbance. The standard curve was measured using 0-2 mM formaldehyde standard solution. The concentration of formaldehyde generated in each sample was calculated according to the standard curve. The results showed that the formaldehyde concentration of the wild-type Mdh unit OD 600nm was less than 1 mM, and the formaldehyde concentration of about 30%-40% of the clones in the mutant library was higher than 1 mM, indicating that Mdh mutants with improved catalytic activity of methanol in vivo could be screened from the library. 600nm

[0092] The mutants with improved formaldehyde concentration unit OD 600nm were subjected to Sanger sequencing analysis. The sequencing results are shown in Table 3. In addition to the same mutant clones, three mutants were finally obtained, and the formaldehyde concentration unit OD 600nm of each of them was higher than that of the wild type.

[0093] Table 3. Mutant mutation information and formaldehyde concentration unit OD 600nm

[0094]

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

[0096] The restriction endonuclease Nde I, Xho ​​I. 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 mdh V79A 、 mdh N214D+E309K 、 mdh K72T+T153P and wild type mdh The genes were connected to the pET21a protein expression vector. Subsequently, the successfully constructed expression vector was transformed into the protein expression host E. coli BL21(DE3)-pET21a- mdh , BL21(DE3)-pET21a- mdh V79A , BL21(DE3)-pET21a- mdh N214D+E309K , BL21(DE3)-pET21a- mdh 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 them in a -20 ° C refrigerator for subsequent enzymatic property detection. V max The relative 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- mdh 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 application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, and the like made within the spirit and principle of the present application should be included in the scope of the present application.

Claims

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

4.

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

3. An expression vector, characterized in that The expression vector comprises the polynucleotide according to claim 2.

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

5. The host cell according to claim 4, characterized in that The host cell is a bacterium.

6. The host cell according to claim 5, characterized in that The host cell is Escherichia coli ( Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), or Bacillus subtilis ( Bacillus subtilis ).

7. Use of the methanol dehydrogenase mutant according to claim 1, the polynucleotide according to claim 2, the expression vector according to claim 3, or the host cell according to 5 or 6 in methanol bioconversion.

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