Threonine deaminase mutant and application thereof

Abstract

The invention discloses a threonine deaminase mutant and a preparation method thereof. Compared with wild type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: the 256th site, the 257th site, the 262th site, the 484th site and the 488th site. The threonine deaminase mutant disclosed by the invention has relatively high enzymatic activity, good stability and low production cost, and is suitable for industrial production and preparation of 2-aminobutyric acid.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a threonine deaminase mutant and its use. Background Art

[0002] In the field of bioengineering, threonine deaminase (TD) is an important industrial enzyme widely used in the production of amino acids, organic acids and biopharmaceuticals. It participates in various metabolic pathways by catalyzing the conversion of threonine into α-ketobutyrate and ammonia. However, threonine deaminase existing in nature often has problems such as insufficient activity, poor stability and weak substrate specificity, which limits its scope and efficiency in industrial application.

[0003] Currently, most of the threonine deaminase used in the market is wild type, and its catalytic efficiency and stability often cannot meet the growing industrial demand. Therefore, it is urgent to develop a threonine deaminase mutant. Summary of the invention

[0004] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application provides a threonine deaminase mutant having a higher enzyme activity.

[0005] In the first aspect of the present application, the present application provides a threonine deaminase mutant. According to an embodiment of the present application, compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: position 256, position 257, position 262, position 484, and position 488. The threonine deaminase mutant of the present application has higher enzyme activity.

[0006] In the second aspect of the present application, the present application provides an isolated nucleic acid. According to an embodiment of the present application, the isolated nucleic acid encodes the threonine deaminase mutant described in the first aspect. The isolated nucleic acid of the present application can encode the obtained threonine deaminase mutant, and the threonine deaminase mutant has higher enzyme activity.

[0007] In the third aspect of the present application, the present application proposes an expression vector. According to an embodiment of the present application, the expression vector carries the isolated nucleic acid described in the second aspect. The isolated nucleic acid of the present application can express a threonine deaminase mutant having a higher enzyme activity.

[0008] In the fourth aspect of the present application, the present application proposes a host cell. According to an embodiment of the present application, the host cell carries the isolated nucleic acid described in the second aspect, or the expression vector described in the third aspect; or the host cell expresses the threonine deaminase mutant described in the first aspect. The isolated nucleic acid of the present application can express the obtained threonine deaminase mutant, and the threonine deaminase mutant has higher enzyme activity.

[0009] In the fifth aspect of the present application, the present application proposes a composition. According to an embodiment of the present application, the composition comprises: the threonine deaminase mutant described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, and the host cell described in the fourth aspect. As can be seen from the above, the threonine deaminase mutant described in the first aspect has a higher enzyme activity. Therefore, the composition containing the above threonine deaminase mutant can be used to prepare 2-aminobutyric acid.

[0010] In the sixth aspect of the present application, the present application proposes a method for improving the activity of threonine deaminase. According to an embodiment of the present application, the method comprises: mutating the following sites of the wild-type threonine deaminase: position 256, position 257, position 262, position 484, and position 488. The method of the present application can improve the enzyme activity of threonine deaminase.

[0011] In the seventh aspect of the present application, the present application proposes the use of the threonine deaminase mutant described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, or the composition described in the fifth aspect in the preparation of 2-aminobutyric acid.

[0012] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0014] Figure 1 The three-dimensional spatial structure of the protein of the wild-type threonine deaminase in Example 2 of the present application;

[0015] Figure 2 The SDS-PAGE gel electrophoresis diagram of each threonine deaminase mutant in Example 3 of the present application;

[0016] Figure 3 The relative enzyme activity data of H18, H19, and H21 at different culture temperatures in Example 5 of the present application;

[0017] Figure 4 The relative enzyme activity data of H18, H19, and H21 at different culture pH values ​​in Example 6 of the present application;

[0018] Figure 5 This is a schematic diagram of the enzymatic conversion to produce 2-aminobutyric acid in Example 10 of the present application. DETAILED DESCRIPTION

[0019] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0020] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0021] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0022] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0023] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0024] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0025] As used herein, the term "variant" or "mutant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0026] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred to a cell or host and / or between cells or hosts. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, the vector can produce a desired expression product by cultivating a suitable cell or host that contains the vector.

[0027] In this article, the term "cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention, and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid) and CoS cells.

[0028] The present application proposes a threonine deaminase mutant and its use, which will be described in detail below.

[0029] Threonine deaminase mutant and its use

[0030] In the first aspect of the present application, the present application provides a threonine deaminase mutant. According to an embodiment of the present application, compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: position 256, position 257, position 262, position 484, position 488.

[0031] The inventors modeled the amino acid sequence of the wild-type threonine deaminase using the Rosetta Fold2 online software tool (http: / / robetta.bakerlab.org / ) to obtain the three-dimensional spatial structure of the protein, and used multiple analysis software to analyze the conformation of the wild-type threonine deaminase, and found that the wild-type threonine deaminase had two loop regions with particularly high B factors (241-263, 482-497), and performed site-directed saturation mutagenesis virtual screening on the loop region to obtain a large number of threonine deaminase mutants. The results showed that based on the amino acid sequence of the wild-type threonine deaminase, at least one of the 256th, 257th, 262nd, 484th, and 488th positions was subjected to site-directed mutagenesis, and the obtained threonine deaminase mutant had high enzyme activity, good stability, low production cost, was suitable for industrial production, and was suitable for preparing 2-aminobutyric acid.

[0032] Herein, unless otherwise specified, the position of the mutation site in the threonine deaminase mutant is located based on the amino acid sequence of the wild-type threonine deaminase (the specific amino acid sequence is shown in SEQ ID NO: 1). For example, "position 256" is the 256th position numbered sequentially from the N-terminus to the C-terminus in the amino acid sequence shown in the wild-type threonine deaminase, and it should be understood that "position 256" also includes positions equivalent to 256, and the determination of equivalent positions can be determined by sequence alignment.

[0033] According to an embodiment of the present application, the above-mentioned threonine deaminase mutant may further include at least one of the following technical features:

[0034] According to an embodiment of the present application, the amino acid sequence of the wild-type threonine deaminase is shown in SEQ ID NO:1.

[0035] MMADSQPLSGAPEGAEYLRAVLRAPVYEAAQVTPLQKMEKLSSRLDNVILVKREDRQPVHSFKLRGAYAMMAGLTEEQKAHGVITASAGNHAQGVAFSSARLGVKALIVMPTATADIKVDAVRGFGG EVLLHGANFDEAKAKAIELSQQQGFTWVPPFDHPMVIAGQGTLALELLQQDAHLDRVFVPVGGGGLAAGVAVLIKQLMPQIKVIAVEAEDSACLKAALDAGHPVDLPRVGLFAEGVAVKRIGDETFRL CQ EYD DIITVDSDAICAAMKDLFEDVRAVAEPSGALALAGMKKYIALHNIRGERLAHILSGANVNFHGLRYVSERCELGEQREAALLAVTIPEEKGSFLKFCQLLGGRSVTEFNYRF ADAKNACIFVGVRLSRGLEERKEILQMLNDGGYSVVDLSDDEMAKLHVRYMVGGRPSHPLQERLYSFEFPESPGALLRFLNTLGTYWNISLFHYRSHGTDYGRVLAAFELG D HEP D FETRLNELGYDCHDETNNPAFRFFLAG (SEQ ID NO: 1).

[0036] It should be noted that in the present application, "the amino acid sequence is shown in SEQ ID NO: 1" includes the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of conservatively modified forms of SEQ ID NO: 1, all of which are within the protection scope of the present application.

[0037] As used herein, "conservatively modified amino acid sequences" refer to amino acid modifications that do not significantly affect or alter the activity and other properties of the threonine deaminase comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications can be introduced into the threonine deaminase of the present application by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Exemplarily, conservative modifications are not more than 80% of the total number, preferably not more than 90% of the total number. In this article, "conservatively modified amino acid sequences" also include amino acid modifications of natural mutations, and "natural mutations" refer to mutations caused by changes in alleles and the like caused by individual differences and other reasons in the natural mutation process of wild-type threonine deaminase.

[0038] According to an embodiment of the present application, compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: position 256 and position 488.

[0039] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is the 256th position.

[0040] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is position 488.

[0041] According to an embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is:

[0042] 1) C256M, Q257M, D262R, D262M, D262W, D262Y, D484M, D488T, D488R, or D488N; or

[0043] 2)C256L / M / G / K and D488R / M / K.

[0044] Herein, in the "letter-number-letter" of the mutation site, the number indicates the position of the mutation site in the wild-type threonine deaminase, the first letter indicates the amino acid of the wild-type threonine deaminase before mutation, and the second letter indicates the amino acid of the threonine deaminase mutant after mutation. For example, "C256M" means that the cysteine ​​C at position 256 of the wild-type threonine deaminase is mutated to methionine M; "C256L / M / G / K" means that the cysteine ​​C at position 256 of the wild-type threonine deaminase is mutated to one of methionine M, leucine L, glycine G, and lysine K.

[0045] In this article, there is no need for special explanation. When the threonine deaminase mutant has multiple mutation sites, the multiple mutation sites are located at different positions, that is, D262R, D262M, D262W, and D262Y mutations cannot exist at the same time, and D488T, D488R, and D488N mutations cannot exist at the same time.

[0046] According to an embodiment of the present application, compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: C256M, D488R, D488N. Exemplarily, the threonine deaminase mutant has a single mutation site, which is C256M, D488R, or D488N; the threonine deaminase mutant has multiple mutation sites, which are C256M and D488R, or C256M and D488N.

[0047] According to an embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is:

[0048] 1) C256M; or

[0049] 2) D488R; or

[0050] 3) D488N; or

[0051] 4) C256M and D488R; or

[0052] 5) C256G and D488M; or

[0053] 6) C256L and D488K; or

[0054] 7) C256L and D488R; or

[0055] 8)C256K and D488M.

[0056] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is C256M.

[0057] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is D488R.

[0058] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is D488N.

[0059] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation sites of the threonine deaminase mutant are C256M and D488R.

[0060] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation sites of the threonine deaminase mutant are C256G and D488M.

[0061] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation sites of the threonine deaminase mutant are C256L and D488 K.

[0062] Isolated nucleic acid, expression vector, host cell

[0063] In the second aspect of the present application, the present application provides an isolated nucleic acid. According to an embodiment of the present application, the isolated nucleic acid encodes the threonine deaminase mutant described in the first aspect.

[0064] According to an embodiment of the present application, the isolated nucleic acid is DNA.

[0065] It should be noted that, for the isolated nucleic acid mentioned herein, it should be understood by those skilled in the art that it actually includes any one or two of the complementary double strands. For convenience, in this article, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the isolated nucleic acid sequence in this application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0066] In the third aspect of the present application, the present application proposes an expression vector. According to an embodiment of the present application, the expression vector carries the isolated nucleic acid described in the second aspect. When the above-mentioned isolated nucleic acid is connected to the expression vector, the above-mentioned isolated nucleic acid can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the above-mentioned isolated nucleic acid. Of course, these control elements can come directly from the expression vector itself, or they can be exogenous, that is, not from the expression vector itself. Of course, the above-mentioned isolated nucleic acid can be operably connected to the control elements.

[0067] Herein, "operably linked" means linking the exogenous gene to the expression vector so that the control elements in the expression vector, such as transcription control sequences and translation control sequences, can play their intended function of regulating the transcription and translation of the exogenous gene. Commonly used expression vectors can be, for example, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present application are introduced into suitable recipient cells, the expression of the aforementioned threonine deaminase mutant can be effectively achieved under the mediation of the regulatory system, thereby achieving the in vitro acquisition of a large number of threonine deaminase mutants.

[0068] In some specific embodiments of the present application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0069] In some specific embodiments of the present application, the expression vector is a lentiviral vector.

[0070] In an optional embodiment of the present application, the expression vector is a plasmid expression vector.

[0071] In the fourth aspect of the present application, the present application provides a host cell. According to an embodiment of the present application, the host cell carries the isolated nucleic acid described in the second aspect, or the expression vector described in the third aspect; or the host cell expresses the threonine deaminase mutant described in the first aspect. The host cell can effectively express the aforementioned threonine deaminase mutant in the host cell under suitable conditions.

[0072] According to an embodiment of the present application, the host cell is obtained by introducing the vector described in the third aspect into a host cell.

[0073] It should be noted that the host cell of the present application is not particularly limited and can be a prokaryotic cell, a eukaryotic cell or a bacteriophage. The prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The aforementioned eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworm, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.

[0074] According to an embodiment of the present application, the host cell is a eukaryotic cell.

[0075] According to an embodiment of the present application, the host cell is a mammalian cell, including but not limited to BHK cells, CHO cells, NSO cells or COS cells, and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0076] It should be noted that the "suitable conditions" described in the present application refer to conditions suitable for the expression of the threonine deaminase mutants described in the present application. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the threonine deaminase mutants include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the threonine deaminase mutants according to the specific environment of the laboratory.

[0077] Composition

[0078] In the fifth aspect of the present application, the present application proposes a composition. According to an embodiment of the present application, the composition comprises: the threonine deaminase mutant described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, and the host cell described in the fourth aspect. As can be seen from the above, the threonine deaminase mutant described in the first aspect has a higher enzyme activity. Therefore, the composition containing the above threonine deaminase mutant can be used to prepare 2-aminobutyric acid.

[0079] According to an embodiment of the present application, the composition includes auxiliary materials.

[0080] Method for improving threonine deaminase activity

[0081] In the sixth aspect of the present application, the present application proposes a method for improving the activity of threonine deaminase. According to an embodiment of the present application, the method comprises: mutating the following sites of the wild-type threonine deaminase: position 256, position 257, position 262, position 484, and position 488. The method of the present application can improve the enzyme activity of threonine deaminase.

[0082] According to an embodiment of the present application, the amino acid sequence of the wild-type threonine deaminase is shown in SEQ ID NO:1.

[0083] According to an embodiment of the present application, the mutated site is:

[0084] 1) C256M, Q257M, D262R, D262M, D262W, D262Y, D484M, D488T, D488R, or D488N; or

[0085] 2)C256L / M / G / K and D488R / M / K.

[0086] According to an embodiment of the present application, the mutated site is:

[0087] 1) C256M; or

[0088] 2) D488R; or

[0089] 3) D488N; or

[0090] 4) C256M and D488R; or

[0091] 5) C256G and D488M; or

[0092] 6) C256L and D488K; or

[0093] 7) C256L and D488R; or

[0094] 8)C256K and D488M.

[0095] According to an embodiment of the present application, the mutated site is C256M, D488R, or D488N.

[0096] use

[0097] In the seventh aspect of the present application, the present application proposes the use of the threonine deaminase mutant described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, or the composition described in the fifth aspect in the preparation of 2-aminobutyric acid.

[0098] The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the literature in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0099] Materials and methods

[0100] 1) Materials: Plasmid pET28a was purchased from Changsha Abiwei Biotechnology Co., Ltd.; NdeI, XhoI and other restriction endonucleases, high-fidelity enzyme premix, and one-step rapid cloning kit were purchased from Shanghai Yisheng Biotechnology Co., Ltd.; synthetic primers, E. coli BL21 (DE3) competent cells, DNA marker, kanamycin, and isopropyl-β-d-thiogalactoside were all purchased from Shanghai Sangon Biotechnology Co., Ltd.; plasmid extraction kit, DNA gel recovery and purification kit, and DNA product purification kit were all purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; chemical reagents were all analytical grade. The plasmid extraction procedure was referred to the instructions of the plasmid extraction kit; the DNA gel recovery procedure was referred to the instructions of the DNA gel recovery and purification kit; the DNA fragment ligation procedure was referred to the instructions of the one-step rapid cloning kit.

[0101] 2) LB medium (g / L): peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, sterilized at 121°C for 20 min, and kanamycin was added to a final concentration of 50 μg / mL before use.

[0102] 3) Threonine deaminase substrate reaction solution: L-threonine 6 g / L, 0.1 M potassium phosphate buffer, pH = 7.5.

[0103] 4) Determination of enzyme activity: Accurately measure the supernatant of the ultrasonically disrupted bacterial solution and add it to the substrate reaction solution preheated to 30°C to make the final concentration of the bacteria 5g / L. Then stir and react in a 30°C water bath for 20 minutes. After diluting with water a certain multiple, use an enzyme marker to detect the absorbance value at 230nm. Calculate the concentration of the product 2-ketobutyric acid based on the standard curve of absorbance value and content.

[0104] 5) Definition of enzyme activity unit: Under the conditions of pH 7.5 and temperature 30°C, the amount of enzyme required to produce 1 μmol of 2-ketobutyric acid per minute is defined as 1 U.

[0105] Example 1: Construction of wild-type threonine deaminase (abbreviated as wild-type TD) genetically engineered bacteria

[0106] (1) Synthesis of wild-type td gene

[0107] According to the information disclosed in the NCBI database, threonine deaminase from Escherichia coli (GenBank: APK04219.1) was selected, and its amino acid sequence is shown in SEQ ID NO:1 in the sequence list, and its gene sequence is shown in SEQ ID NO:2 in the sequence list. The gene coding sequence was codon optimized according to the codon preference rule of Escherichia coli, and NdeI and

[0108] After the XhoI restriction site, it was sent to Nanjing GenScript Biotechnology Co., Ltd. for artificial synthesis (SEQ ID NO: 3).

[0109] The gene sequence of threonine deaminase TD is shown in SEQ ID NO: 2:

[0110]

[0111] The sequence of the td gene coding sequence after codon optimization is shown in SEQ ID NO: 3:

[0112] catatg ctcgag .

[0113] (2) Construction of wild-type TD pET28a vector

[0114] Design primers for constructing the vector. Use 1-F (5'-ctcgagcaccaccaccaccaccactga-3' (SEQ ID NO: 4)) / 1-R (5'-atgatgatgatgatggctgct-3' (SEQ ID NO: 5)) as primers, the purchased pET28a vector as template, 2×Hieff Plus PCR Master Mix (With Dye) high-fidelity enzyme premix PCR amplified the linearized vector; using 2-F (5'-agcagccatcatcatcatcatatgatggctgactctcaacca-3' (SEQ ID NO: 6)) / 2-R (5'-tggtggtggtggtgctcgagttaaccagccaagaagaatct-3' (SEQ ID NO: 7)) as primers and the td gene DNA fragment artificially synthesized by GenScript (nucleotide sequence as shown in SEQ ID NO: 3) as template, high-fidelity enzyme premix PCR amplified the td gene DNA fragment with 5' and 3' ends completely consistent with the two end sequences of the linearized vector; 1% agarose gel electrophoresis detection, and the amplified linearized vector (5.3kb) and td gene (1.6kb) DNA fragments were recovered using a DNA gel recovery and purification kit. Using Hieff Plus One Step Cloning Kit One-step rapid cloning kit performs homologous recombination between the linearized vector and td gene fragment recovered from gel. The recombination system is: 2.5μL of pET28a linearized vector, 2.5μL of td gene fragment, 2×Hieff Enzyme Premix 5 μL, and then react at 50°C for 30 min.

[0115] (3) Construction of wild-type TD genetically engineered bacteria

[0116] The homologous recombination product obtained in (2) was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and the transformation product was spread on LB plate containing 50 μg / mL kanamycin. After overnight culture at 37°C, positive transformants were selected and sent to Shanghai Shenggong Biotechnology Technology Service Co., Ltd. for sequencing. The transformant with correct sequencing was the plasmid vector TD-pET28a containing the wild-type td gene, and the transformant was also the wild-type TD genetic engineering bacteria.

[0117] Example 2: Determination of a single site-directed mutation site in threonine deaminase TD

[0118] The protein sequence of wild-type TD was modeled using the new artificial intelligence online software tool Rosetta Fold2 (http: / / robetta.bakerlab.org / ) to obtain its three-dimensional spatial structure (see Figure 1 ), and the model was visualized and processed using PyMol. The model was evaluated using PROCHECK and Verify 3D programs. Ramachandran plot was used to illustrate the two dihedral angles in the peptide plane ( The ratio of ψ to ψ) is used to indicate the allowed and unallowed conformations of amino acid residues, and the reliability of the protein model is evaluated by the Psi / Phi Ramachandran plot calculated by PROCHECK. The compatibility of the amino acid residues of the protein side chain is evaluated by Verify 3D software, and at least 80% of the amino acid residues pass the verification if the score is ≥0.2. The molecular docking program glide-dock in Schrodinger Suites software is used for molecular docking, and the B-factor distribution of the TD protein model is analyzed by Pymol. It is found that the B-factor of the two loop regions of the protein is particularly high (241st-263rd, 482nd-497th), so the loop region is subjected to site-directed saturation mutation virtual screening to obtain the mutation sites in Table 1. The binding free energy of the ligand and the protein is calculated for these mutation sites by Rosetta Flex ddG. The lower the binding free energy value, the higher the affinity between the receptor and the ligand, and the greater the possibility of increased enzyme activity.

[0119] Table 1:

[0120] Mutation site <![CDATA[ΔG (Binding) (Kcal / mol)]]> Mutation site <![CDATA[ΔG (Binding) (Kcal / mol)]]> WT -8.6 D484M --15.1 D262R -20.7 D488R -14.8 D488V -20.4 C256M -13.9 D262M -20.1 F253H -13.6 D262E -19.3 F253I -13.5 D488M -17.6 D262W -13.4 D488T -16.5 D262Y -13.1 Q257M -16.2 F253M -12.9 D262L -16.1 C256L -12.4 D488N -15.8 D262N -12.1

[0121] Example 3: Preparation and expression of single mutants of threonine deaminase

[0122] The original plasmid TD-pET28a was used as a template for site-directed mutagenesis to obtain a recombinant plasmid TD-X-pET28a carrying a mutant encoding threonine deaminase, wherein the site-directed mutagenesis method is as follows:

[0123] The primer sequences used to design the mutants are shown in Table 2 (the bold and underlined parts in the primer sequences are the bases corresponding to the mutation sites):

[0124] Table 2:

[0125]

[0126]

[0127] Using 1-F / F253H-R as primers and TD-pET28a plasmid as template, a large DNA fragment with F253H mutation site was amplified by PCR with high-fidelity enzyme premix. Using F253H-F / 2-R as primers and TD-pET28a plasmid as template, 2×Hieff Plus PCR Master Mix (With Dye) high-fidelity enzyme premix PCR amplified small DNA fragments with 5' and 3' ends that were completely consistent with the sequences at both ends of the large DNA fragment; 1% agarose gel electrophoresis was used to detect, and the amplified large and small DNA fragments were recovered using a DNA gel recovery and purification kit. Plus One Step Cloning Kit One-step rapid cloning kit performs homologous recombination on the large and small fragments of DNA recovered from the gel. The recombination system is: 2.5μL of large fragment DNA, 2.5μL of small fragment DNA, 2×Hieff Enzyme Premix 5μL, react at 50℃ for 30min.

[0128] The homologous recombination product obtained was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and the transformation product was spread on LB plate containing 50 μg / mL kanamycin sulfate. After overnight culture at 37°C, positive transformants were selected and sent to Shanghai Shenggong Biotechnology Technology Service Co., Ltd. for sequencing. The correctly sequenced transformant was the plasmid vector TD-F253H-pET28a containing the single mutant td gene, which was also the single mutant TD-F253H genetic engineering bacteria, numbered H1. In the same way, the engineered bacteria H2 with F253I mutation, H3 with F253M mutation, H4 with C256M mutation, H5 with C256L mutation, H6 with Q257M mutation, H7 with D262R mutation, H8 with D262M mutation, H9 with D262E mutation, H10 with D262L mutation, H11 with D262W mutation, H12 with D262Y mutation, H13 with D262N mutation, H14 with D484M mutation, H15 with D488V mutation, H16 with D488M mutation, H17 with D488T mutation, H18 with D488R mutation, and H19 with D488N mutation were constructed.

[0129] The correctly sequenced mutants H1-H19 genetically engineered bacteria were inoculated into test tubes containing 4 mL LB medium containing 50 μg / mL kanamycin, cultured overnight at 37°C and 200 rpm, and then transferred to 50 mL LB medium and cultured at 37°C and 200 rpm until OD 600The concentration of IPTG was 0.6-0.8, and IPTG was added to a final concentration of 0.2 mM. The culture was continued at 20°C and 200 rpm for 18 h. The fermentation broth was centrifuged at 9000 rpm for 5 min, and the supernatant was removed. The bacterial precipitate was retained and resuspended with water to a bacterial concentration of 50 g / L. Ultrasonic disruption was performed for 10 min, and the precipitate was removed by centrifugation. The supernatant was retained for SDS-PAGE gel electrophoresis analysis. The results are shown in Figure 2 The results showed that the mutant proteins of H3, H5, and H15 were not expressed normally; the mutant proteins of H7, H8, H10, H11, and H12 were poorly soluble; and the mutant proteins of H1, H2, H4, H6, H9, H13, H14, H16, H17, H18, and H19 were expressed normally and had good solubility.

[0130] Example 4: Enzyme activity assay of threonine deaminase mutants

[0131] Take 100 μL of enzyme solution supernatant and add it to a centrifuge tube containing 900 μL of substrate reaction solution, and place it at 30°C for 20 minutes. After the reaction is completed, dilute it with water to a certain multiple and centrifuge it, and take the supernatant for detection by ELISA.

[0132] The relative enzyme activity results of each single mutant are shown in Table 3:

[0133] Table 3:

[0134] name Mutation site Relative enzyme activity (%) WT - 100.00 H1 F253H 104.43 H2 F253I 102.42 H3 F253M 3.82 H4 C256M 119.07 H5 C256L 2.00 H6 Q257M 105.97 H7 D262R 105.52 H8 D262M 105.29 H9 D262E 103.77 H10 D262L 63.03 H11 D262W 105.64 H12 D262Y 109.83 H13 D262N 96.99 H14 D484M 107.11 H15 D488V 0 H16 D488M 100.32 H17 D488T 105.20 H18 D488R 145.95 H19 D488N 157.02

[0135] The results in Table 3 show that the threonine deaminase mutants with single point mutations of C256M, Q257M, D262R, D262M, D262W, D262Y, D484M, D488T, D488R, and D488N have significantly increased enzyme activities compared to the wild type. The threonine deaminase mutants with single point mutations of C256M (H4), D488R (H18), and D488N (H19) have higher enzyme activities, among which the mutant H19 with D488N has the largest increase in enzyme activity, which is 1.57 times that of the wild type.

[0136] Example 5: Construction and screening of TD mutation point library by iterative saturation mutation of amino acids at positions 256 and 488

[0137] The enzyme activity results of Example 4 show that the threonine deaminase mutants with single-point mutations C256M, D488R, and D488N have higher enzyme activities, indicating that the 256th and 488th amino acids have a greater influence on the enzyme activity of threonine deaminase TD. Therefore, in order to obtain mutants with better enzyme activity, the inventors used the wild-type TD as the starting strain and performed double-point iterative saturation mutations on the 256th and 488th amino acid sites. The specific method is as follows:

[0138] Design of double-site iterative saturation mutagenesis primer 3-F at amino acid positions 256 and 488: 5'-GGTGACGAGACTTTCAGATTG NNK CAAGAGTACTTGGACGACATC-3' (SEQ ID NO: 8) (the underlined part is a saturation mutation site, N is A or T or C or G, and K is G or T) and 3-R:

[0139] 5'-CTCGTTCAATCTAGTCTCGAA MNN TGGTTCGTGATCTCCCAATTC-3' (SEQ ID NO: 9) (the underline is a saturated mutation site, N is A or T or C or G, and M is A or C). Using the original plasmid TD-pET28a as a template, 3-F and 3-R were used as mutation primers to amplify a DNA fragment containing both the 256th and 488th mutation points. Using TD-pET28a as a template, 4-F (5'-TTCGAGACTAGATTGAACGAG-3', SEQ ID NO: 10) / 4-R

[0140] (5'-CAATCTGAAAGTCTCGTCACC-3', SEQ ID NO: 11) was used as primer, and PCR was performed to amplify the linearized vector DNA fragment with the 5' and 3' ends having sequences completely consistent with the two ends of the mutant DNA fragment; the two DNA fragments obtained above were subjected to electrophoresis and gel extraction and recovery, and the purified mutant DNA fragment was homologously recombined with the linearized vector DNA fragment. The product: vector was mixed at a molar ratio of 2:1, and 2×Hieff Enzyme Premix was reacted at 50°C for 30 minutes. Then, a recombinant genetic engineering bacterium was constructed according to the method shown in (3) of Example 1 to obtain a fully saturated mutant library containing positions 256 and 488.

[0141] From the mutant library, about 1000 single colonies were picked and inoculated into a 96-well deep-well culture plate containing 700uL LB medium, wherein the LB medium contained 50ug / mL kanamycin, and after culturing at 37°C for 6h, a final concentration of 0.2mM IPTG was added, the temperature was lowered to 20°C, and the induction culture was continued for 18h. After induction, the 96-well deep-well culture plate was centrifuged at 5000rpm for 10min, and the supernatant was discarded. The culture plate was then placed in an ultra-low temperature refrigerator at -80°C for 1h, taken out and placed at room temperature for 30min to thaw, and this freeze-thaw operation was repeated 3 times. After the freeze-thaw was completed, 200uL of water was added to resuspend the bacteria to obtain an enzyme solution, and then the enzyme activity was determined according to the method shown in Example 4, and mutants with higher enzyme activity than the wild type were screened.

[0142] The 30 mutants were screened by shaking flasks. The specific process was as follows: the mutants were inoculated into 50 mL LB medium and cultured at 37°C and 200 rpm until the OD 600 The concentration of IPTG was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM, and the culture was continued at 20°C and 200 rpm for 18 h. The fermentation broth was centrifuged at 9000 rpm for 5 min, the supernatant was removed, the bacterial precipitate was retained, and the bacterial precipitate was resuspended with water to a bacterial concentration of 50 g / L. Ultrasonic disruption was performed for 10 min, and the precipitate was removed by centrifugation. The enzyme supernatant was retained for enzyme activity determination. The determination method was the same as that in Example 4. Finally, 5 mutants with higher enzyme activity than the wild type were obtained. The specific data are shown in Table 4:

[0143] Table 4:

[0144] name Mutation site Relative enzyme activity (%) WT - 100.00 H20 C256L-D488R 107.9 H21 C256M-D488R 137.2 H22 C256G-D488M 118.9 H23 C256K-D488M 108.5 H24 C256L-D488K 113.4

[0145] The above five mutants (H20-H24) with higher activity than the wild type were sequenced, and the sequencing results showed that: the 256th amino acid of H20 mutated from C to L, and the 488th amino acid mutated from D to R, and the enzyme activity was increased by 1.07 times; the 256th amino acid of H21 mutated from C to M, and the 488th amino acid mutated from D to R, and the enzyme activity was increased by 1.37 times; the 256th amino acid of H22 mutated from C to G, and the 488th amino acid mutated from D to M, and the enzyme activity was increased by 1.18 times; the 256th amino acid of H23 mutated from C to K, and the 488th amino acid mutated from D to M, and the enzyme activity was increased by 1.08 times; the 256th amino acid of H24 mutated from C to L, and the 488th amino acid mutated from D to K, and the enzyme activity was increased by 1.13 times.

[0146] Example 6: Optimal Temperature of Threonine Deaminase Mutants with Improved Enzyme Activity

[0147] The optimal reaction temperature was determined using the supernatant of the lysate of the three mutants with higher enzyme activity obtained in Examples 4 and 5 above. After the obtained supernatant of the lysate was mixed with the enzyme reaction solution, it was placed at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and 60°C for reaction for 20 minutes. After the reaction was completed, water was added to dilute it to a certain multiple, centrifuged, and the supernatant was taken for detection by an ELISA instrument. The results showed that the mutants all showed higher enzyme activity at 30-40°C. This example exemplifies some mutants, and the specific relative enzyme activity data are as follows Figure 3 shown.

[0148] Figure 3 The results showed that the three high enzyme activity mutants (H18, H19, H21) all showed high enzyme activity at 30-40°C. The optimum reaction temperature of H18 was 30°C, that of H19 was 35°C, and that of H21 was 35°C.

[0149] Example 7: Optimal pH of threonine deaminase TD mutant with improved enzyme activity

[0150] The optimal reaction pH was determined using the supernatant of the lysate of the three highly active mutants (H18, H19, and H21) obtained in the above examples. Enzyme reaction solutions with different pH values ​​(6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5) were prepared using different buffer solutions. The enzyme reaction solutions of different pH values ​​were mixed with the supernatant of the mutant lysate, and then placed at 35°C for reaction for 20 minutes. After the reaction was completed, water was added to dilute it to a certain multiple, and then centrifuged. The supernatant was taken for detection by an enzyme marker. The results showed that the mutants exhibited higher enzyme activity at pH values ​​between 7.0 and 8.5. This example exemplifies some mutants, and the specific relative enzyme activity data are shown in Figure 2. Figure 4 shown.

[0151] Figure 4 The results showed that the three high enzyme activity mutants (H18, H19, H21) all showed high enzyme activity at pH values ​​between 7.0 and 8.5. The optimum pH for H18, H19, and H21 was 7.5.

[0152] Example 8: Obtaining a genetically engineered bacterium (H25) that co-expresses leucine dehydrogenase and formate dehydrogenase

[0153] In this example, two strains were used to react together to produce 2-aminobutyric acid, one of which was a genetically engineered bacterium that produced the threonine deaminase mutant H19 (see Example 3 for details, hereinafter referred to as genetically engineered bacterium H19), and the other was a genetically engineered bacterium that co-expressed leucine dehydrogenase and formate dehydrogenase, numbered H25.

[0154] According to the information disclosed in the NCBI database, leucine dehydrogenase LDH (amino acid sequence shown in SEQ ID NO: 12) from Bacillus (GenBank: WP_000171355.1) and formate dehydrogenase FDH (amino acid sequence shown in SEQ ID NO: 13) from Campylobacter aquatica (GenBank: AB091484.1) were selected. The gene coding sequences of LDH and FDH were codon optimized according to the codon preference rule of Escherichia coli, and NdeI and XhoI restriction sites were designed at both ends of the coding sequence of LDH, and HindIII and NotI restriction sites were designed at both ends of the coding sequence of FDH, and then sent to Nanjing GenScript Biotechnology Co., Ltd. for artificial synthesis (SEQ ID NO: 14 and SEQ ID NO: 15).

[0155] The amino acid sequence of the leucine dehydrogenase is shown in SEQ ID NO: 12:

[0156] MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPALGGTRMWTYDSEEAAIEDALRLAKGMTYKNAAAGLNLGGAKTVIIGDPRKDKSEAMFRALGRYIQGLNGRYITAEDVGTTVDDMDIIHEETDFVTGISPSFGSSGNPSPVTAYGVYRGMKAAAKEAFGTDNLEGKVIAVQGVGNVAYHLCKHLHAEGAKLIVTDINKEAVQRAVEEFGASAVEPNEIYGVECDIYAPCALGATVNDETIPQLKAKVIAGSANNQLKEDRHGDIIHEMGIVYAPDYVINAGGVINVADELYGYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKNSRSTYLRNGHDIISRR(SEQ ID NO:12);

[0157] The amino acid sequence of the formate dehydrogenase is as shown in SEQ ID NO:13:

[0158] MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGPDSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMILGLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHASPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTLAGYAGDVWFPQPAPADHPWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKAG(SEQ ID NO:13);

[0159] The sequence of the LDH coding sequence after codon optimization is as shown in SEQ ID NO:14:

[0160] catatg ctcgag ;

[0161] The sequence of the FDH coding sequence after codon optimization is shown in SEQ ID NO: 15:

[0162] aagctt gcggccgc .

[0163] Primers were designed to construct a vector for co-expression of LDH and FDH. 1-F (5'-acgtcgttaactcgagtctggtaaagaaacc-3' (SEQ ID NO: 16)) / 1-R (5'-tctctagtgtcatatgtatatctccttcttatac-3' (SEQ ID NO: 17)) was used as primers, the purchased pRSFDuet-1 vector was used as template, and 2×Hieff Plus PCR Master Mix (With Dye) high-fidelity enzyme premix PCR amplified the linearized vector; 2-F (5'-aggagatatacatatgacactagagatatttgaa-3' (SEQ ID NO: 18)) / 2-R (5'-ctttaccagactcgagttaacgacgtgagataatgtca-3' (SEQ ID NO: 19)) was used as primers, and the LDH gene DNA fragment synthesized by GenScript (nucleotide sequence as shown in SEQ ID NO: 14) was used as a template, and the LDH gene DNA fragment was amplified by high-fidelity enzyme premix PCR; 1% agarose gel electrophoresis was used to detect, and the amplified linearized vector (3.8kb) and LDH gene (1.1kb) DNA fragments were recovered using a DNA gel recovery and purification kit. The two recovered DNA fragments were double-digested with NdeI and XhoI, purified with a DNA purification kit, and then connected with T4 DNA ligase. The connection system is: vector DNA 5μL, LDH gene fragment 5μL, 10×Ligase Buffer 2μL, Gold T4 DNA Ligase 1μL, ultrapure water 7μL. React at 16℃ overnight.

[0164] The overnight ligation product was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and the transformation product was spread on LB plate containing 50 μg / mL kanamycin. After overnight culture at 37°C, positive transformants were selected and sent to Shanghai Shenggong Biotechnology Technology Service Co., Ltd. for sequencing. The correctly sequenced transformant was the plasmid vector pRSFDuet-LDH containing the LDH gene, which was also the LDH genetic engineering bacteria, numbered H20.

[0165] The LDH genetically engineered bacteria H20 were inoculated into LB medium containing 50ug / mL Kana, cultured at 37°C for 12h, and the bacteria were collected. The plasmid DNA (pRSFDuet-LDH) was extracted using a bacterial plasmid extraction kit. 3-F (5'-agctgggtaagcggccgcataatgcttaagtcg-3' (SEQ ID NO: 20)) / 3-R (5'-ctttagccataagcttgtcgacctgcaggcg-3' (SEQ ID NO: 21)) were used as primers, and the pRSFDuet-LDH vector was used as a template. 2×Hieff Plus PCRMaster Mix (With Dye) high-fidelity enzyme premix PCR amplified the vector DNA; 4-F (5'-gcaggtcgacaagcttatggctaaagtacta-3' (SEQ ID NO: 22)) / 4-R (5'-taagcattatgcggccgcttacccagctttt-3' (SEQ ID NO: 23)) was used as primers, and the FDH gene DNA fragment synthesized by GenScript (nucleotide sequence as shown in SEQ ID NO: 15) was used as a template, and the FDH gene DNA fragment was amplified by high-fidelity enzyme premix PCR; 1% agarose gel electrophoresis was performed, and the amplified vector DNA (4.9kb) and FDH gene (1.2kb) DNA fragments were recovered using a DNA gel recovery and purification kit. The two recovered DNA fragments were double-digested with HindIII and NotI, purified with a DNA purification kit, and then connected with T4 DNA ligase. The connection system is: vector DNA 5μL, FDH gene fragment 5μL, 10×Ligase Buffer 2μL, Gold T4 DNA Ligase 1μL, ultrapure water 7μL. React at 16℃ overnight.

[0166] The overnight ligation product was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and the transformation product was spread on LB plate containing 50 μg / mL kanamycin. After overnight culture at 37°C, positive transformants were selected and sent to Shanghai Shenggong Biotechnology Service Co., Ltd. for sequencing. The correctly sequenced transformant was the plasmid vector pRSFDuet-LDH-FDH containing FDH and LDH genes. This transformant is a genetically engineered bacterium that co-expresses LDH and FDH, numbered H25.

[0167] Example 9: Bacteria fermentation culture

[0168] 1 mL of the genetically engineered bacteria H19 for producing threonine deaminase mutants prepared in Example 3 and the genetically engineered bacteria H25 for co-expressing leucine dehydrogenase / formate dehydrogenase prepared in Example 8 were activated on LB slant medium and cultured at 37° C. for 16 h; an inoculated loop of the bacterial lawn was transferred from the slant medium to 100 mL of LB seed medium and cultured at 37° C. and 160 rpm for 10 to 16 h. The seeds were inoculated at a volume ratio of 8% into a 5L fermentation tank containing 3L fermentation medium. The temperature was controlled at 37°C±1°C, the pH was maintained at 7.0±0.1 during the fermentation process, the ventilation volume was controlled at 0.5vvm~3.0vvm, the rotation speed was controlled at 400rpm~600rpm, the dissolved oxygen was maintained at 30% by adjusting the rotation speed and ventilation volume, and after fermentation for 7~8h, the temperature was lowered to a stable level of 20~30°C, and IPTG was added with a final concentration of 0.2mM to induce for about 18h. When the OD of the fermentation no longer increased, the fermentation was terminated, and the fermentation yield of the two strains was ≥6%.

[0169] The fermentation liquid was centrifuged at 6000 rpm for 20 min, the supernatant was discarded to obtain wet bacteria, the bacteria were resuspended in water to a final concentration of 100 g / L, the bacterial liquid was subjected to high-pressure homogenization to obtain cell lysate, and the lysate supernatant obtained after centrifugation was obtained to obtain the crude enzyme solution.

[0170] The crude enzyme solutions of H19 and H25 were analyzed by SDS-PAGE gel electrophoresis, and the results showed that all proteins were expressed normally.

[0171] Example 10: Enzymatic conversion to produce 2-aminobutyric acid

[0172] The reaction substrates were weighed sequentially into 1L of water, so that the final concentrations of ammonium formate, NAD, and L-threonine in the reaction vessel were 60g / L, 0.6g / L, and 10g / L, respectively. After adjusting the pH to 7.5 with aqueous ammonia, the H19 crude enzyme solution with a final concentration of 3g / L and the H25 crude enzyme solution with a final concentration of 40g / L prepared in Example 9 were added. The reaction was carried out at 35°C and 150rpm. During the reaction, 10g / L of threonine was added every 0.5-1h (a total of 9 times), and formic acid was used to maintain the pH at 7.5. The reaction was terminated after 10h. The schematic diagram of the enzymatic conversion to produce 2-aminobutyric acid is shown in Figure 5 . The sample was taken to detect the content of 2-aminobutyric acid, and the results showed that the 2-aminobutyric acid yield was 82.94g / L and the conversion rate was 95.81%. The reaction results of the wild-type WT crude enzyme solution operated in the same way showed that the 2-aminobutyric acid yield was 43.68g / L and the conversion rate was 50.46%. The co-reaction of H19 crude enzyme solution and H25 crude enzyme solution increased the conversion rate by 45.35% compared with the co-reaction of WT crude enzyme solution and H25 crude enzyme solution.

[0173] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0174] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A threonine deaminase mutant, characterized in that: Compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one mutation site as follows: No. 256, No. 257, No. 262, No. 484, No.

488.

2. The threonine deaminase mutant according to claim 1, characterized in that Compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is the 256th and / or 488th position.

3. The threonine deaminase mutant according to claim 1, characterized in that Compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is: 1) C256M, Q257M, D262R, D262M, D262W, D262Y, D484M, D488T, D488R, or D488N; or 2)C256L / M / G / K and D488R / M / K.

4. The threonine deaminase mutant according to claim 1, characterized in that Compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is: 1) C256M; or 2) D488R; or 3) D488N; or 4) C256M and D488R; or 5) C256G and D488M; or 6) C256L and D488K; or 7) C256L and D488R; or 8)C256K and D488M.

5. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the threonine deaminase mutant according to any one of claims 1 to 4.

6. An expression vector, characterized in that: The expression vector carries the isolated nucleic acid of claim 5.

7. A host cell, characterized in that The host cell carries the isolated nucleic acid of claim 5 or the expression vector of claim 6; or The host cell expresses the threonine deaminase mutant according to any one of claims 1 to 4.

8. A composition, characterized in that include: The threonine deaminase mutant according to any one of claims 1 to 4, the isolated nucleic acid according to claim 5, the expression vector according to claim 6, the host cell according to claim 7; and optionally, auxiliary materials.

9. A method for increasing the activity of threonine deaminase, characterized in that: include: The following positions of wild-type threonine deaminase were mutated: No. 256, No. 257, No. 262, No. 484, No. 488; Optionally, the mutated site is: 1) C256M, Q257M, D262R, D262M, D262W, D262Y, D484M, D488T, D488R, or D488N; or 2) C256L / M / G / K and D488R / M / K; Optionally, the mutated site is: 1) C256M; or 2) D488R; or 3) D488N; or 4) C256M and D488R; or 5) C256G and D488M; or 6) C256L and D488K; or 7) C256L and D488R; or 8)C256K and D488M.

10. Use of the threonine deaminase mutant according to any one of claims 1 to 4, the isolated nucleic acid according to claim 5, the expression vector according to claim 6, the host cell according to claim 7, or the composition according to claim 8 in the preparation of 2-aminobutyric acid.

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