Dihydroxyacetone phosphate transaminase CJ1361 mutant and application of dihydroxyacetone phosphate transaminase CJ1361 mutant in fermentation synthesis of serinol

Abstract

The invention relates to a dihydroxyacetone phosphate transaminase CJ1361 mutant with improved stability, and a gene recombinant bacterium containing the dihydroxyacetone phosphate transaminase CJ1361 mutant and an enzyme liquid of the gene recombinant bacterium as biocatalysts, which are used for synthesizing serinol by a microbial fermentation method. Compared with the wild type dihydroxyacetone phosphate transaminase CJ1361, the yield of serinol produced by the dihydroxyacetone phosphate transaminase CJ1361 mutant provided by the invention is remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of industrial biotechnology, specifically relating to a CJ1361 mutant of Campylobacter jejuni phosphate transaminase and its application in the fermentation synthesis of serine. Background Technology

[0002] Serine alcohol, also known as 2-amino-1,3-propanediol, is an amino alcohol and a key intermediate in the synthesis of the nonionic contrast agent iopamidol. Currently, chemical synthesis of serine alcohol is the main method for large-scale production. Most amino alcohols or their precursors are derived from petrochemical or highly explosive reaction reagents. Common precursors include 2-nitro-1,3-propanediol, nitromethane, dihydroxyacetone (DHA), dihydroxyacetone oxime, or 5-amino-1,3-dioxane. However, these chemical synthesis processes exhibit some drawbacks, such as low yields, the formation of hazardous byproducts, or the use of fossil fuels as raw materials. The high cost of some reactants and required equipment makes industrial applications unsatisfactory. Furthermore, some chemical synthesis processes produce 1-amino-2,3-propanediol, which is difficult to separate from serine alcohol, increasing the difficulty of post-processing. Compared with traditional chemical methods, biosynthesis offers advantages such as milder reaction conditions, better selectivity, simpler post-processing, and environmental friendliness. Therefore, research on non-natural biosynthesis technology of serine is of great significance. Summary of the Invention

[0003] The purpose of this invention is to address the problems of high pollution, high energy consumption, and difficulty in separation and purification in existing chemical synthesis methods of serine. This invention provides a stable CJ1361 mutant dihydroxyacetone transaminase and a recombinant strain of this CJ1361 mutant gene, along with its enzyme solution, as a biocatalyst for the microbial fermentation synthesis of serine. When glucose is the substrate, the serine yield obtained by the mutant strain after 40 hours of fermentation increased from 4.3 g / L to 5.6 g / L and 6.3 g / L, respectively, representing increases of 30% and 47% compared to the wild-type CjTA. This provides a novel biocatalyst for the microbial fermentation synthesis of the iopamidol intermediate 2-amino-1,3-propanediol.

[0004] The present invention provides a CJ1361 mutant of dihydroxyacetone transaminase, which is obtained by single-point mutation or combination mutation at positions 156, 223, 270 and 279 of the amino acid sequence shown in SEQ ID NO.2.

[0005] According to an embodiment of the present invention, the CJ1361 mutant of dihydroxyacetone phosphate transaminase is obtained by making one, two or more single-point mutations or combined mutations at positions 156, 223, 270 and 279 of the amino acid sequence shown in SEQ ID NO.2.

[0006] According to an embodiment of the present invention, the dihydroxyacetone transaminase CJ1361 is derived from Campylobacter jejuni. According to an embodiment of the present invention, the amino acid sequence of the dihydroxyacetone transaminase CJ1361 is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0007] According to an embodiment of the present invention, the CJ1361 mutant of dihydroxyacetone phosphate transaminase is formed by performing one, two, or more of the following mutations on the amino acid sequence shown in SEQ ID NO. 2:

[0008] (1) The aspartic acid at position 156 is mutated to lysine (D156K);

[0009] (2) The propionic acid at position 223 is mutated to glycine (A223G);

[0010] (3) The aspartic acid at position 270 is mutated to tryptophan (D270W);

[0011] (4) Serine at position 279 is mutated to alanine (S279A).

[0012] According to an embodiment of the present invention, the CJ1361 mutant of dihydroxyacetone phosphate transaminase is obtained by mutating propionic acid at position 223 of the amino acid sequence shown in SEQ ID NO.2 to glycine, and optionally by making one, two or more single-point mutations or combination mutations at positions 156, 270 and 279.

[0013] According to an embodiment of the present invention, the CJ1361 mutant of dihydroxyacetone phosphate transaminase is formed by mutating propionic acid at position 223 of the amino acid sequence shown in SEQ ID NO. 2 to glycine (A223G), and optionally further performing any one, two or more of the following mutations:

[0014] (1) The aspartic acid at position 156 is mutated to lysine;

[0015] (3) The 270th aspartic acid molecule was mutated to tryptophan;

[0016] (4) The serine at position 279 is mutated to alanine.

[0017] According to an embodiment of the present invention, the CJ1361 mutant of dihydroxyacetone transaminase is formed by mutating propionic acid at position 223 of the amino acid sequence shown in SEQ ID NO. 2 to glycine (A223G), aspartic acid at position 156 to lysine (D156K), and aspartic acid at position 270 to tryptophan (D270W). According to an embodiment of the present invention, the CJ1361 mutant of dihydroxyacetone transaminase is CJ1361-D156K / A223G / D270W, denoted as CjTA. M1 The amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0018] According to an embodiment of the present invention, the CJ1361 mutant of dihydroxyacetone phosphate transaminase is formed by mutating propionic acid at position 223 of the amino acid sequence shown in SEQ ID NO. 2 to glycine (A223G) and serine at position 279 to alanine (S279A). According to an embodiment of the present invention, the CJ1361 mutant of dihydroxyacetone phosphate transaminase is CJ1361-A223G / S279A, denoted as CjTA. M2 The amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.

[0019] The full-length base sequences of dihydroxyacetone transaminase CJ1361 and its mutant are both 1077 bp, with the start codon being ATG and the stop codon being TAA, from the first base to the 1077th base.

[0020] This invention also provides biological materials (expression plasmid vectors or combinations of expression plasmid vectors) related to the above-mentioned dihydroxyacetone transaminase CJ1361 mutant, said biological materials being any one of E1) to E5) below:

[0021] E1) encodes the nucleic acid molecule (Group 1 polynucleotide) of the CJ1361 mutant of the above-mentioned dihydroxyacetone transaminase;

[0022] E2) contains an expression cassette containing the nucleic acid molecules described in E1;

[0023] E3) A recombinant vector containing the nucleic acid molecule described in E1), or a recombinant vector containing the expression cassette described in E2;

[0024] E4) Recombinant microorganisms containing the nucleic acid molecules described in E1), or recombinant microorganisms containing the expression cassette described in E2), or recombinant microorganisms containing the recombinant vector described in E3);

[0025] E5) A transgenic cell line containing the nucleic acid molecule described in E1) or a transgenic cell line containing the expression cassette described in E2).

[0026] According to an embodiment of the present invention, the recombinant vector uses pzElac as the expression vector.

[0027] According to an embodiment of the invention, the biological material optionally further comprises a second group of polynucleotides, the second group of polynucleotides comprising at least one polynucleotide encoding a phosphatase. According to an embodiment of the invention, the polynucleotide encoding the phosphatase is a polynucleotide encoding histamine phosphatase, such as histamine phosphatase hisB. Preferably, the protein sequence accession number of histamine phosphatase hisB is CAA31814, or it is an amino acid sequence having at least 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by CAA31814.

[0028] According to an embodiment of the invention, the biological material optionally further comprises a third group of polynucleotides; the third group of polynucleotides comprises at least one polynucleotide encoding an amino acid donor. According to an embodiment of the invention, the amino acid donor is an amino acid, preferably glutamic acid. According to an embodiment of the invention, ammonia can be provided by action of a dehydrogenase on the amino acid. According to an embodiment of the invention, ammonia can be provided by action of glutamate dehydrogenase on glutamate. According to an embodiment of the invention, the polynucleotide encoding the amino acid donor is a polynucleotide encoding glutamate dehydrogenase, for example, a polynucleotide encoding glutamate dehydrogenase gdhA. According to an embodiment of the invention, the glutamate dehydrogenase gdhA is the amino acid sequence of protein sequence accession number AAC74831, or an amino acid sequence having at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by AAC74831.

[0029] According to an embodiment of the present invention, the biological material optionally further comprises a fourth group of polynucleotides; the fourth group of polynucleotides comprises at least one polynucleotide encoding a protein as follows:

[0030] Serine transporters (e.g., serine transporter gene yedA, protein sequence accession number AAC75025), O-acetylserine transporters (e.g., O-acetylserine transporter eamA, protein sequence accession number AAC74606), cysteine / O-acetylserine efflux proteins (e.g., cysteine / O-acetylserine efflux protein eamB, protein sequence accession number AAC75631), threonine transporters (e.g., threonine transporter thrE, protein sequence accession number AAC75025), threonine transporters (e.g., threonine transporter thrE, protein sequence accession number AAC75025), threonine transporters (e.g., threonine transporter thrE, protein sequence accession number AAC75025), threonine transporters (e.g., threonine transporter gene yedA ... Accession number AAK61331; for example, threonine transporter rhtA, protein sequence accession number AAC73900), homoserine and homoserine lactone efflux transporters (e.g., homoserine and homoserine lactone efflux transporter rhtB, protein sequence accession number AAT48223), threonine efflux proteins (e.g., threonine efflux protein, protein sequence accession number AAT48222), and threonine intima transporters (threonine intima transporter, protein sequence accession number AAC75025). Preferably, the biological material comprises a polynucleotide encoding a serine transporter, such as serine transporter yedA.

[0031] According to embodiments of the present invention, the expression of any one, two, or more of the following genes in the biological material is optionally inhibited, weakened, or eliminated: pyruvaldehyde synthase encoding genes (e.g., pyruvaldehyde synthase encoding gene mgsA, protein sequence accession number CAA72119), glycerol-3-phosphate dehydrogenase encoding genes (e.g., glycerol-3-phosphate dehydrogenase encoding gene gpsA, protein sequence accession number AAC76632), triose phosphate isomerase encoding genes (e.g., triose phosphate isomerase encoding gene tpiA, protein sequence accession number AAC76901), and glyceraldehyde-3-phosphate dehydrogenase encoding genes (e.g., glyceraldehyde-3-phosphate dehydrogenase encoding gene gapA, protein sequence accession number AAC74849). According to embodiments of the present invention, the expression of acetone aldehyde synthase encoding genes (e.g., acetone aldehyde synthase encoding gene mgsA, protein sequence accession number CAA72119) and glyceraldehyde-3-phosphate dehydrogenase encoding genes (e.g., glyceraldehyde-3-phosphate dehydrogenase encoding gene gapA, protein sequence accession number AAC74849) in the genetically engineered strain is inhibited, weakened, or eliminated. According to embodiments of the present invention, the expression of gene gapA is inhibited, weakened, or eliminated by inserting a Pflic promoter before the glyceraldehyde-3-phosphate dehydrogenase encoding gene gapA. According to embodiments of the present invention, the biological material uses E. coli W3110 Pflic-gapAΔmgsA as the host bacterium.

[0032] According to embodiments of the present invention, the biomaterial may optionally further comprise polynucleotides encoding the following enzymes: hexokinase, phosphohexokinase, 6-phosphofructokinase, and aldolase.

[0033] The present invention also provides a method for preparing the above-mentioned dihydroxyacetone transaminase CJ1361 mutant, wherein the dihydroxyacetone transaminase CJ1361 mutant is prepared by site-directed saturation mutagenesis.

[0034] According to an embodiment of the present invention, the preparation method is as follows:

[0035] The starting strain E. coli W3110 Pflic-gapAΔmgsA / pzElac-CjTA was activated and the plasmid pzElac-CjTA was extracted and stored at -20℃.

[0036] Site-directed and combined mutations were performed at the target sites to construct a mutation library;

[0037] The formation of the product was detected by liquid chromatography, and the CJ1361 mutation of dihydroxyacetone phosphate transaminase was obtained.

[0038] The present invention also provides the application of the CJ1361 mutant of dihydroxyacetone phosphate transaminase, or the above-mentioned biological material, in the production of serine.

[0039] According to an embodiment of the present invention, the production is the fermentation of glucose to produce serine. According to an embodiment of the present invention, the final concentration of the glucose substrate is 1 g / L to 40 g / L, for example, 20 g / L.

[0040] According to an embodiment of the present invention, the production method maintains the L-glutamate concentration in the culture medium. According to an embodiment of the present invention, the L-glutamate concentration in the culture medium is maintained at 5 g / L.

[0041] According to an embodiment of the present invention, the production method is as follows: engineered bacteria containing the above-mentioned dihydroxyacetone transaminase CJ1361 mutant gene or the above-mentioned recombinant microorganisms are induced to undergo shake-flask fermentation culture, using glucose as a substrate, and cultured at 30°C for 40 hours. The fermentation broth is then separated and purified to obtain serine.

[0042] According to an embodiment of the present invention, the production method is as follows: the CJ1361 gene of dihydroxyacetone transaminase (SEQ ID NO.1) is mutated using site-directed saturation mutagenesis technology, the obtained mutant plasmid is transferred into competent cells (e.g., E. coli W3110 Pflic-gapAΔmgsA competent cells) by heat shock, the obtained strain is inoculated, transferred, induced and fermented, and serine is produced by glucose fermentation.

[0043] According to an embodiment of the present invention, the fermentation medium further comprises: 8 g / L ammonium sulfate, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 2 g / L betaine, 2 g / L magnesium sulfate heptahydrate, 0.2 g / L nicotinic acid, and 0.02 g / L vitamin B1. After adjusting the pH to 6.8-7.2, it is sterilized at 121°C for 20 minutes, placed at 30-40°C, and then sterilized glucose is added at a concentration of approximately 20 g / L.

[0044] According to an embodiment of the present invention, the engineered bacteria containing the above-mentioned CJ1361 mutant gene of dihydroxyacetone transaminase are further cultured as follows: the engineered bacteria containing the CJ1361 mutant gene of dihydroxyacetone transaminase are inoculated into LB liquid medium containing a final concentration of 50 μg / mL spectinomycin and cultured at 37°C for 10 h to obtain seed culture; the seed culture is inoculated into fresh fermentation medium containing a final concentration of 50 μg / mL spectinomycin at an inoculation rate of 1.0% (v / v) and cultured at 37°C and 200 rpm for 2 h (OD200). 600 =0.6~0.8), add isopropyl β-D-thiogalactoside (IPTG) to the culture medium to a final concentration of 0.15mM, culture at 30℃ for 40h, centrifuge at 4℃ and 8000rpm for 10min, take the supernatant fermentation broth to separate and purify, and obtain serine alcohol.

[0045] According to an embodiment of the present invention, the engineered bacteria containing the above-mentioned dihydroxyacetone transaminase CJ1361 mutant gene are constructed as follows: using Escherichia coli W3110 as the starting strain, W3110Pflic-gapAΔmgsA is constructed as the host strain using the CRISPR-Cas9 system; the above-mentioned dihydroxyacetone transaminase CJ1361 mutant is synthesized by codon optimization targeting Escherichia coli according to the protein sequence corresponding to wild-type CJ1361 (SEQ ID NO:2); then, the phosphatase gene and dehydrogenase gene are cloned from the genome of Escherichia coli MG1655, and the obtained DNA fragments are combined and linked to the lac promoter of plasmid pZElac in different ways using Gibson assembly to obtain a recombinant plasmid, which is then transformed into the host bacteria.

[0046] According to an embodiment of the present invention, the phosphatase is further described as histamine phosphatase, such as histamine phosphatase hisB. Histamine phosphatase hisB is the amino acid sequence of protein accession number CAA31814, or an amino acid sequence having at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by CAA31814.

[0047] According to an embodiment of the present invention, the dehydrogenase is a glutamate dehydrogenase, such as glutamate dehydrogenase gdhA. The glutamate dehydrogenase gdhA is the amino acid sequence of protein accession number AAC74831, or an amino acid sequence having at least 96%, 97%, 98%, or at least 99% sequence identity with the amino acid sequence represented by AAC74831.

[0048] This invention relates to the inoculation, transfer, induction, and cell recovery of the CJ1361 mutant genetically engineered bacterium containing dihydroxyacetone transaminase (DHT). The culture medium can be any medium in the art that can promote bacterial growth and the production of serinel, preferably LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in distilled water, and adjusted to pH 7.5. There are no particular limitations on the culture method and conditions; these can be optimized based on factors such as host type and culture method.

[0049] A mutant is a gene that has at least one base (nucleotide) alteration, deletion, or insertion relative to the natural or wild-type gene. The mutation (one or more nucleotide alterations, deletions, and / or insertions) can occur in the coding region of the gene or in introns, the 3'UTR, the 5'UTR, or the promoter region. As a non-limiting example, a mutant gene can be a gene with an insertion in the promoter region that can increase or decrease gene expression; it can be a gene with a deletion that results in the production of a non-functional protein, a truncated protein, a dominant-inactive protein, or no protein; or it can be a gene with one or more point mutations that result in a change in the amino acids encoding the protein or in abnormal splicing of the gene transcript.

[0050] Wild-type: refers to the form found in nature. For example, naturally occurring or wild-type polypeptide or polynucleotide sequences are sequences that exist in organisms and have not been intentionally modified by human intervention.

[0051] Beneficial effects

[0052] The CJ1361 mutant dihydroxyacetone transaminase constructed in this invention significantly increases the yield of serine compared to wild-type CJ1361, providing a novel biocatalyst and production process for the microbial fermentation synthesis of serine. In the fermentation system, the mutant inoculum size is 1%, the glucose substrate feed rate can reach 20 g / L, the product concentration gradually increases over time, the reaction is completed within 40 hours, and the substrate conversion rate is greater than 30%. Detailed Implementation

[0053] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0054] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0055] LB medium: 10 g / L peptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0056] Fermentation medium: 8 g / L ammonium sulfate, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 2 g / L betaine, 2 g / L magnesium sulfate heptahydrate, 0.2 g / L nicotinic acid, 0.02 g / L vitamin B1. Adjust the pH to 6.8-7.2, then sterilize at 121℃ for 20 minutes. Let it stand at 30-40℃ and add sterilized glucose at a concentration of about 20 g / L.

[0057] Example 1: Construction of a CJ1361 mutant library of dihydroxyacetone phosphate transaminase

[0058] (1) Starting strain

[0059] (2) Using *E. coli* W3110 Pflic-gapAΔmgsA / pzElac-CjTA as the starting strain, denoted as strain CjTA, the plasmid pzElac-CjTA was activated and extracted. The amino acid sequence of dihydroxyacetone transaminase CJ1361 is shown in SEQ ID NO.2. The starting strain contains polynucleotides encoding hexokinase, phosphohexokinase, 6-phosphofructokinase, and aldolase. The root plasmid pzElac-CjTA contains polynucleotides encoding hisitamine phosphatase hisB and glutamate dehydrogenase gdhA. Construction of the mutant library

[0060] The preparation of the CJ1361 mutant library of dihydroxyacetone transaminase was first achieved through site-directed saturation mutagenesis and combinatorial mutagenesis. Using the vector pzElac-CjTA in strain CjTA as a template, the primers in Table 1 were used to perform polymerase chain reaction (PCR) according to the following procedure.

[0061] PCR reaction system (25 μL): 1 μL upstream primer (100 μM), 1 μL downstream primer (100 μM), 12.5 μL 2×Phanta buffer, 0.5 μL dNTP mixture (10 mM each), 1 μL plasmid template, 0.5 μL DNA polymerase and 8.5 μL ultrapure water.

[0062] The PCR amplification procedure is as follows:

[0063] Step 1: 98℃ for 3 minutes;

[0064] Step 2: 98℃ for 15 seconds;

[0065] Step 3: 60℃ for 15 seconds;

[0066] Step 4: 72℃2000bp / 1min (skip to step 2, 30cycles);

[0067] Step 5: 72℃ for 10 minutes;

[0068] Step 6: 16℃ for 15 minutes;

[0069] Step 7: Store at 4℃ or immediately sample for agarose gel electrophoresis.

[0070] The recombinant plasmid purified using a Clean Up purification kit (Axygen Scientific, Int, catalog number AP-PCR-250) was transferred into E. coli W3110 Pflic-gapAΔmgsA competent cells, and the clones were seeded into 10 mL LB agar plates and cultured at 37°C for 12 h. Combinatorial mutations were performed using CjTA. M2 For example, pzElac-CjTA-A223G was used as a template, with S279A-F as the upstream primer and S279A-R as the downstream primer, to perform polymerase chain reaction (PCR). The recombinant plasmid purified by the Cleanup purification kit (Axygen Scientific, Int, catalog number AP-PCR-250) was transferred to E. coli BL21(DE3) competent cells, and the clones were seeded into 10 mL LB agar plates and cultured at 37°C for 12 h.

[0071] Table 1 Primer design for CJ1361 mutant dihydroxyacetone transaminase (DHT)

[0072]

[0073]

[0074] Example 2: Screening of the CJ1361 mutant library of dihydroxyacetone phosphate transaminase

[0075] (1) Culture, induction and expression of strains

[0076] Positive clones of strain CjTA were randomly selected from plates and inoculated into 10 mL of LB liquid medium (containing 50 μg / mL spectinomycin). The culture was incubated at 37°C for 12 h to obtain a seed culture. 2 mL of the seed culture was sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing. After confirming the sequencing results were correct, 1% (v / v) of the seed culture was inoculated into 10 mL of fermentation medium (containing 50 μg / mL spectinomycin). The culture was incubated at 37°C with shaking at 200 rpm for 2 h. Then, IPTG (final concentration 0.15 mM) was added, and the culture was incubated at 30°C for 40 h. During the fermentation process, the L-glutamate concentration in the medium was maintained at 5 g / L. The resulting fermentation broth was purified to obtain serine.

[0077] (2) Screening method for CJ1361 mutant of dihydroxyacetone transaminase (DHT)

[0078] Take 2 mL of the fermentation broth after 40 h of fermentation, centrifuge at 12000 rpm for 2 min. Dilute the supernatant 50 times, shake thoroughly, and filter through a 0.22 μm filter membrane. Take 200 μL for liquid chromatography to determine the content of the product. Serine alcohol concentration determination: All standards used were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). The high-performance liquid chromatograph was a Shimadzu LC-40D; the column was a Welch Ultimate AQ-C18, 4.6 × 250 mm; the column oven was set to 40 degrees Celsius; the detector was a UV detector (set to constant temperature at 40 degrees Celsius), with a detection wavelength of 338 nm; the mobile phase was sodium acetate aqueous solution and methanol, with gradient elution. Using serine alcohol yield as a screening index, the CJ1361 mutant CjTA of dihydroxyacetone transaminase was obtained. M1 With CjTA M2 .

[0079] Table 2 Data on serine shake-flask fermentation

[0080]

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

[0082] Sequence information

[0083]

[0084]

[0085]

Claims

1. A CJ1361 mutant of dihydroxyacetone transaminase, wherein the CJ1361 mutant of dihydroxyacetone transaminase is obtained by making one, two or more single-point mutations or combined mutations at positions 156, 223, 270 and 279 of the amino acid sequence shown in SEQ ID NO.

2.

2. The mutant according to claim 1, characterized in that, The CJ1361 mutant of dihydroxyacetone phosphate transaminase is obtained by performing one, two, or more of the following mutations on the amino acid sequence shown in SEQ ID NO.2: (1) The aspartic acid at position 156 is mutated to lysine (D156K); (2) The propionic acid at position 223 is mutated to glycine (A223G); (3) The aspartic acid at position 270 is mutated to tryptophan (D270W); (4) Serine at position 279 is mutated to alanine (S279A).

3. The mutant according to claim 1 or 2, characterized in that, The CJ1361 mutant of dihydroxyacetone phosphate transaminase is obtained by mutating propionic acid at position 223 of the amino acid sequence shown in SEQ ID NO.2 to glycine, and optionally by making one, two or more single-point mutations or combination mutations at positions 156, 270 and 279.

4. The mutant according to any one of claims 1-3, characterized in that, The CJ1361 mutant of dihydroxyacetone phosphate transaminase is formed by mutating propionic acid at position 223 of the amino acid sequence shown in SEQ ID NO.2 to glycine (A223G), and optionally further performing any one, two or more of the following mutations: (1) The aspartic acid at position 156 is mutated to lysine; (3) The 270th aspartic acid molecule was mutated to tryptophan; (4) The serine at position 279 is mutated to alanine.

5. The mutant according to any one of claims 1-4, characterized in that, The CJ1361 mutant of dihydroxyacetone transaminase is formed by mutating propionic acid at position 223 of the amino acid sequence shown in SEQ ID NO.2 to glycine (A223G), aspartic acid at position 156 to lysine (D156K), and aspartic acid at position 270 to tryptophan (D270W); preferably, the CJ1361 mutant of dihydroxyacetone transaminase is CJ1361-D156K / A223G / D270W, with the amino acid sequence shown in SEQ ID NO.3 and the nucleotide sequence shown in SEQ ID NO.4; Alternatively, the CJ1361 mutant of dihydroxyacetone transaminase is formed by mutating propionic acid at position 223 of the amino acid sequence shown in SEQ ID NO.2 to glycine (A223G) and serine at position 279 to alanine (S279A); preferably, the CJ1361 mutant of dihydroxyacetone transaminase is CJ1361-A223G / S279A, with the amino acid sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.

6.

6. A biological material relating to the CJ1361 mutant of dihydroxyacetone phosphate transaminase according to any one of claims 1-5, wherein the biological material is any one of E1) to E5) below: E1) encodes the nucleic acid molecule of the CJ1361 mutant of dihydroxyacetone phosphate transaminase mentioned above; E2) contains an expression cassette containing the nucleic acid molecules described in E1; E3) A recombinant vector containing the nucleic acid molecule described in E1), or a recombinant vector containing the expression cassette described in E2; E4) Recombinant microorganisms containing the nucleic acid molecules described in E1), or recombinant microorganisms containing the expression cassette described in E2), or recombinant microorganisms containing the recombinant vector described in E3); E5) A transgenic cell line containing the nucleic acid molecule described in E1) or a transgenic cell line containing the expression cassette described in E2).

7. The biomaterial according to claim 6, characterized in that, The biomaterial optionally further comprises a second group of polynucleotides, the second group of polynucleotides comprising at least one polynucleotide encoding a phosphatase; Preferably, the biomaterial optionally further comprises a third group of polynucleotides, the third group of polynucleotides comprising at least one polynucleotide encoding an amino donor; Preferably, the biomaterial optionally further comprises a fourth group of polynucleotides, the fourth group of polynucleotides comprising at least one polynucleotide encoding the following proteins: serine transporter, O-acetylserine transporter, cysteine / O-acetylserine efflux protein, threonine transporter, homoserine and homoserine lactone efflux transporter, threonine efflux protein, and threonine intima transporter. Preferably, the expression of any one, two or more of the following genes is weakened or eliminated in the biological material: pyruvaldehyde synthase encoding gene, glycerol-3-phosphate dehydrogenase encoding gene, triose phosphate isomerase encoding gene, and glyceraldehyde-3-phosphate dehydrogenase encoding gene. Preferably, the biological material optionally comprises polynucleotides encoding the following enzymes: hexokinase, phosphohexokinase, 6-phosphofructokinase, and aldolase.

8. The method for preparing the CJ1361 mutant of dihydroxyacetone transaminase according to any one of claims 1-5, wherein the CJ1361 mutant of dihydroxyacetone transaminase is prepared by site-directed saturation mutagenesis.

9. The use of the CJ1361 mutant of dihydroxyacetone phosphate transaminase according to any one of claims 1-5, or the biomaterial according to any one of claims 6-7, in the production of serine.

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