Prevotella meso - diaminopimelate dehydrogenase mutant and its application

Abstract

The present invention discloses a meso-diaminopimelate dehydrogenase mutant of Prevotella and its application. The modification method of the present invention designs the optimal mutant through combinatorial mutation, modifies the protein of PtDAPDH, overcomes the limitation of the low catalytic activity of the previous wild-type enzyme, and finally obtains the optimal mutant T5 (PtDAPDH<supgt;N13E / S90R / P254A< / supgt;), whose catalytic half-life and D-p-hydroxyphenylglycine production are 1.10 times and 2.67 times that of the wild-type respectively. Using L-p-hydroxyphenylglycine as the substrate, the production in a 0.1 L shake flask after conversion is 3.2 g / L, and the conversion rate is 32%. With high catalytic efficiency and production intensity, it greatly reduces the previous high cell mass and expensive substrate cost, laying a foundation for the industrial production of D-p-hydroxyphenylglycine.

Description

Technical Field

[0001] The present invention relates to a mutant of meso-diaminopimelate dehydrogenase of Prevotella and its application, belonging to the technical field of bioengineering. Background Art

[0002] D-p-hydroxyphenylglycine (D-HPG), also known as 4-hydroxyphenylglycine, is an unnatural amino acid with a chiral center. D-HPG is an important intermediate in amino acid biosynthesis and is widely used in the synthesis of peptide hormones and pesticides. Especially as a pharmaceutical intermediate, it is widely used in the preparation of β-lactam antibiotics such as amoxicillin, ampicillin, and cefoperazone. These antibiotics have good bactericidal effects on Gram-positive bacteria, Gram-negative bacteria, pathogenic spirochetes, etc., and have the advantages of small side effects and good oral effects. Clinically, β-lactam antibiotics can be used to treat organ and soft tissue infections such as pneumonia, bacteremia, and urethritis caused by sensitive bacteria.

[0003] Currently, the main production methods of D-HPG are chemical synthesis method and hydantoinase conversion method. Among them, the chemical synthesis method mainly includes asymmetric conversion method and chemical resolution method. However, these methods all have problems to varying degrees, such as harsh reaction conditions, large environmental pollution, low production efficiency, and high production cost; and they will have a toxic effect on the environment, which does not meet the requirements of green production, safe production, and sustainable development. Currently, the enzyme conversion method is mainly the hydantoinase method. Due to the characteristics of high specificity, diverse types, and environmental friendliness of enzyme catalysts, the enzyme conversion method has become an important technical means for synthesizing D-HPG; therefore, more and more researchers are seeking to use the enzyme conversion method to synthesize D-HPG. Among them, the preparation of chiral D-p-hydroxyphenylglycine by one-step reductive amination of keto acid has the characteristics of mild production process conditions, high efficiency, and environmental protection, which can reduce the environmental and resource pressure. Therefore, there is an urgent need for a biological method for efficiently preparing D-p-hydroxyphenylglycine.

[0004] The production of D-p-hydroxyphenylglycine by one-step reductive amination of keto acid involves a key enzyme, meso-diaminopimelate dehydrogenase (meso-DAPDH, EC 1.4.1.16), which has a relatively broad substrate spectrum and can catalyze the reductive amination of aliphatic keto acids and some aromatic keto acids, enabling the reductive amination of the α-carbonyl group of keto acids to form the corresponding D-amino acids.

[0005] In addition, multi-enzyme cascade reaction is also an important means in enzyme biotransformation and has many advantages. For example, it can avoid the accumulation of reaction intermediates, use cheap and easily available raw materials as the starting substrates of the reaction, and the synergy between multi-enzyme reactions can be regulated by controlling the expression ratio between enzymes, thus solving the problems of high cost or difficulty in obtaining the substrate keto acid. The commonly used cascade pathway couples meso-diaminopimelate dehydrogenase (DAPDH), L-amino acid oxidase and glucose dehydrogenase. L-amino acid oxidase can use L-p-hydroxyphenylglycine as the substrate, catalyze the deamination of L-p-hydroxyphenylglycine to generate keto acid, and then finally generate D-p-hydroxyphenylglycine through the reductive amination of DAPDH. At the same time, the regeneration of reduced coenzyme NADPH is achieved by introducing a coenzyme regeneration system.

[0006] In recent decades, protein engineering has become an effective strategy to improve the properties of enzymes at the molecular level, such as the most effective method to expand the substrate range, improve the enzyme activity and improve the enzyme stability. Therefore, it is possible to solve the problem of its low catalytic activity by designing PtDAPDH through protein engineering. Protein engineering transformation can be mainly divided into four categories: traditional directed evolution (i.e., irrational design), semi-rational design, rational design (based on structure and computer technology) and the combined application of multiple strategies. At present, certain research progress has been made on the transformation of PtDAPDH by protein engineering. However, the improvement effect of catalytic activity is still limited and far from meeting the actual industrial demand. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a PtDAPDH mutant capable of efficiently preparing D-p-hydroxyphenylglycine and its modification method, uses the mutant protein to catalyze p-hydroxyphenylglyoxylate to prepare D-p-hydroxyphenylglycine, and further couples the obtained mutant with L-amino acid oxidase and glucose dehydrogenase to prepare D-p-hydroxyphenylglycine with the cheap substrate L-p-hydroxyphenylglycine. The strain constructed by the present invention has high production intensity and high catalytic stability in the preparation of D-p-hydroxyphenylglycine, reduces the amount of inoculated bacteria in the transformation, and greatly saves the production cost of industrialization.

[0008] The first object of the present invention is to provide a mutant of Prevotella meso-diaminopimelate dehydrogenase (PtDAPDH), wherein one or more of the amino acids at positions 13, 90, and 254 of the meso-diaminopimelate dehydrogenase PtDAPDH parent having the amino acid sequence shown in SEQ ID NO.1 are mutated.

[0009] Furthermore, the nucleotide sequence of the meso-diaminopimelate dehydrogenase PtDAPDH parent is shown in SEQ ID NO.2.

[0010] Further, the mutant is obtained by mutating asparagine at position 13 of the meso-diaminopimelate dehydrogenase PtDAPDH parent with the amino acid sequence shown in SEQ ID NO.1 to glutamic acid, and the mutant is named N13E.

[0011] Further, the mutant is obtained by mutating serine at position 90 of the meso-diaminopimelate dehydrogenase PtDAPDH parent with the amino acid sequence shown in SEQ ID NO.1 to arginine, and the mutant is named S90R.

[0012] Further, the mutant is obtained by mutating proline at position 254 of the meso-diaminopimelate dehydrogenase PtDAPDH parent with the amino acid sequence shown in SEQ ID NO.1 to alanine, and the mutant is named P254A.

[0013] Further, the mutant is obtained by mutating asparagine at position 13 of the meso-diaminopimelate dehydrogenase PtDAPDH parent with the amino acid sequence shown in SEQ ID NO.1 to glutamic acid and mutating serine at position 90 to arginine, and the mutant is named N13E / S90R.

[0014] Further, the mutant is obtained by mutating asparagine at position 13 of the meso-diaminopimelate dehydrogenase PtDAPDH parent with the amino acid sequence shown in SEQ ID NO.1 to glutamic acid, mutating serine at position 90 to arginine, and mutating proline at position 254 to alanine, and the mutant is named N13E / S90R / P254A.

[0015] The second object of the present invention is to provide a gene encoding the mutant of Prevotella meso-diaminopimelate dehydrogenase (PtDAPDH).

[0016] The third object of the present invention is to provide an expression vector containing the gene.

[0017] Further, the expression vector is based on pRSFDuet.

[0018] Further, the expression vector further contains the PmLAAD coding gene and the BmGDH coding gene.

[0019] Further, the nucleotide sequence of the PmLAAD coding gene is as shown in SEQ ID NO.3.

[0020] Further, the nucleotide sequence of the BmGDH coding gene is as shown in SEQ ID NO.4.

[0021] The fourth object of the present invention is to provide a recombinant bacterium expressing the said vector.

[0022] Furthermore, the said recombinant bacterium uses Escherichia coli as the host.

[0023] Furthermore, the said recombinant bacterium uses Escherichia coli BL21(DE3) as the host.

[0024] The fifth object of the present invention is to provide the application of the said recombinant bacterium in the preparation of D-p-hydroxyphenylglycine.

[0025] Furthermore, the said application uses L-p-hydroxyphenylglycine as the reaction substrate and the said recombinant bacterium cells as the catalyst to catalyze the production of D-p-hydroxyphenylglycine.

[0026] Furthermore, in the catalytic reaction, the reaction conditions are to react for 12 - 24 h under the conditions of pH 8.0 - 8.5 and 30 - 37 °C.

[0027] Furthermore, in the catalytic reaction, the addition amount of the recombinant bacterium cells is 10 - 60 g / L of the final concentration. Further preferably, it is 10 - 30 g / L.

[0028] The beneficial effects of the present invention are as follows:

[0029] The present invention constructs a mutant of Prevotella meso-diaminopimelate dehydrogenase PtDAPDH for catalytic production of D-p-hydroxyphenylglycine. The catalytic half-life and the yield of D-p-hydroxyphenylglycine of the mutant of the present invention are 1.10 times and 2.67 times that of the wild type respectively, which improves the production capacity of the unit catalyst, effectively reduces the production cost, and only uses water as the catalytic medium in the reaction, having the advantages of mild reaction conditions, simple operation, high yield, etc. When the mutant obtained by the present invention uses L-p-hydroxyphenylglycine as the substrate in a 0.5 L shake flask, the yield of D-p-hydroxyphenylglycine can reach 3.2 g / L and the conversion rate is 32%, which accelerates the industrialization process of producing D-p-hydroxyphenylglycine by the enzyme conversion method. Description of the Drawings

[0030] Figure 1 It is the connection mode of the three-enzyme recombinant vector.

[0031] Figure 2 It is the relationship between the whole-cell catalytic concentration and the production amount of D-p-hydroxyphenylglycine. Detailed Embodiments

[0032] The following further illustrates the present invention with specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments do not limit the present invention.

[0033] Gene source: The PtDAPDH gene of the biological enzyme involved in this patent is derived from Prevotellatimonensis. The pRSFDuet plasmid was purchased from Novagen (Madison, WI, U.S.A.). Restriction endonucleases, T4 DNA ligase, primeSTAR, etc. were purchased from TaKaRa (Dalian, China). Benzeneacetic acid and the standard sample were purchased from SIGMA. All PtDAPDH mutants were obtained by molecular modification, and the rest of the reagents were purchased from the market.

[0034] Preparation of LB medium: Peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, sterilized at 121 °C for 20 min.

[0035] Preparation of fermentation medium: Tryptone 12 g / L, yeast extract (Angel yeast powder 802) 24 g / L, glycerol 4 mL / L, KH2PO4 2.31 g / L, and K2HPO4 12.31 g / L.

[0036] Determination of D-p-hydroxyphenylglycine by HPLC: For the specific steps, please refer to the literature: Song Huimin, Tu Chunyan, Ouyang Pingkai. Determination of D-p-hydroxyphenylglycine by High Performance Liquid Chromatography [J]. Journal of Nanjing University of Technology (Natural Science Edition), 2002(04): 97-99.

[0037] Method for determining specific enzyme activity: The absorbance method was used to measure the enzyme activity of DAPDH. One unit of DAPDH enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of NADPH (U). The enzyme activity could be calculated by measuring the consumption of NADPH.

[0038] The specific enzyme activity was defined as the number of enzyme activity units per milligram of protein (U / mg protein).

[0039]

[0040] The present invention will be further described below in conjunction with the embodiments:

[0041] Example 1: Construction and screening of single mutant

[0042] (1) Construction of single mutant: Primers for the mutation sites of PtDAPDH N13E 、PtDAPDH S90R and PtDAPDH P254A were designed as shown in Table 1, and the mutants were constructed by whole plasmid PCR.

[0043] Table 1 Primer sequences for single mutants

[0044]

[0045] Construct a reaction PCR amplification system: 0.5 μL of PrimSTAR enzyme, 10 μL of 5×PrimeSTAR Buffer, 4 μL of dNTP, 1 μL of each of the two primers for each mutation site, 4 μL of template (PtDAPDH WT ) and 32.5 μL of water; The reaction conditions are as follows: ① 94°C for 3 min; ② 98°C for 10 s; ③ 55°C for 30 s; ④ 72°C for 3 min; ⑤ Cycle steps ② - ④ for 29 times; ⑥ 72°C for 5 min; ⑦ Incubate at 12°C.

[0046] Incubate the above reaction system at 37°C for 3 h to digest the plasmid template (the digestion system is: 0.5 μL of DpnI, 45 μL of the above reaction PCR product, 5 μL of 10×T Buffer). After digestion, the digested product is introduced into Escherichia coli BL21 competent cells by chemical transformation method. The specific steps of the chemical transformation method are as follows:

[0047] (1) Introduce 10 μl of the homologous recombination product into 100 μl of BL21 competent cells;

[0048] (2) Incubate on ice for 15 - 30 min;

[0049] (3) Heat shock in a 42°C water bath for 90 s, then quickly take it out and place it on ice for 3 - 5 min;

[0050] (4) Add 800 μl of antibiotic - free LB medium and mix well, culture at 37°C and 200 rpm for 1 h;

[0051] (5) Centrifuge at 5000 rpm for 2 min to collect bacteria;

[0052] (6) Remove the supernatant, and resuspend the remaining 100 - 200 μl by pipetting and spreading it onto a kanamycin - resistant plate containing 0.05 mg / mL, and culture at 37°C for about 12 h.

[0053] (7) Pick a single colony into kanamycin - resistant LB containing 0.05 mg / mL, culture at 200 rpm and 37°C for 12 h, and then send it to the company for sequencing. The correctly sequenced ones are positive transformants.

[0054] Example 2: Construction and screening of double - mutant and triple - mutant mutants

[0055] (1) Construction of double - mutant mutants: On the basis of the mutant PtDAPDH N13 , use the mutant primers S90R - S and S90R - A (Table 1) to construct double - mutant mutants by whole - plasmid PCR. The specific implementation method refers to step (1) in Example 2 to prepare the double - mutant mutant PtDAPDH N13E / S90R .

[0056] (2) Construction of triple mutants: In the mutant PtDAPDH N13E / S90R Based on the above, the triple mutant PtDAPDH was constructed by whole plasmid PCR using mutation primers P254A-S and P254A-A (Table 1). The specific implementation method is shown in Example 1. N13E / S90R / P254A .

[0057] (3) Screening of multiple mutants: The mutant strains with correct sequencing were inoculated into LB seed medium and cultured at 200 rpm and 37°C for about 10 h. 5% of the inoculum was inoculated into shake flask fermentation medium and cultured at 200 rpm and 37°C until OD 600 = 0.8, add lactose with a final concentration of 5g / L for induction, and the induction conditions are 200rpm, 25℃ for 16h.

[0058] The conversion conditions were: conversion temperature 37°C, reaction pH 8.5, conversion time 24h, and rotation speed 550rpm.

[0059] The conversion solution after the conversion was determined by HPLC method. The results are shown in Table 2. The final triple mutant PtDAPDH N13E / S90R / 254A The effect is best.

[0060] Table 2 Results of shake flask screening of different mutants

[0061]

[0062] Example 3: Expression and purification method of mutant enzyme

[0063] The positive transformants of the mutant recombinant strain prepared in Example 2 were inoculated into LB medium and cultured at 37°C until OD 600 When the pH is 0.6-1.0, add 5g / L lactose at a final concentration to induce the expression of the enzyme, the induction temperature is 25°C, the induction time is 16h, and the fermentation broth is obtained. The fermentation broth is centrifuged at 4°C and 6000rpm for 10min to obtain the bacteria. Add 10mL of binding solution A (20mM sodium phosphate, 0.5mMNaCl, 20mM imidazole, 1% glycerol, adjust the pH to 8.5 with HCl) to fully resuspend the bacteria, then place the centrifuge tube in an ice bath and put it into an ultrasonic cell disruptor. The conditions for ultrasonic disruption are: working time 4s, interval time 4s, a total of 10min. The obtained disrupted liquid is subjected to low-temperature high-speed centrifugation, centrifuged at 4°C and 8000rpm for 30min to obtain a crude enzyme solution. Filter with a 0.22μm microporous filter membrane and set aside.

[0064] Prepare a nickel ion affinity chromatography column. First, use a constant flow pump to pump ultrapure water into the column to wash the column (about 6 - 12 times the column volume) at 4°C, and then balance the column environment with 10 mL of binding solution A. When the pH value of the effluent at the lower end of the column is the same as that of the low-salt concentration buffer solution pumped into the column (about 5 times the column volume of buffer solution is required), add the obtained crude enzyme solution after passing through the membrane to the column. First, wash the miscellaneous proteins with binding solution A until baseline balance, and then elute with elution solution B (20 mM sodium phosphate, 0.5 mM NaCl, 250 mM imidazole). Collect the eluate of the absorption peak, measure the enzyme activity, and obtain the target protein that reaches electrophoretic purity.

[0065] Example 4: Determination of kinetic parameters of parental enzyme and mutants

[0066] To evaluate the mutants, the present invention determined the kinetic parameters of mutant parent T0 and mutants T1 to T5 at 37°C.

[0067] k cat / K m It was calculated by measuring the initial rate of D - p - hydroxyphenylglycine produced from different concentrations of L - p - hydroxyphenylglycine at 37°C. The catalytic half - lives of the parental enzyme and mutants were determined by the residual enzyme activity experiment during the conversion process (performed in a whole - cell system). The wet cells of the PtDAPDH parental enzyme strain and mutant strains were each added to the reaction solution at a final concentration of 20 g / L. Using L - p - hydroxyphenylglycine as the substrate, their residual enzyme activities were measured every 1 h during the whole conversion process (the initial enzyme activity at 0 h was set as 100%), and a total of 12 h was measured.

[0068] As shown in Table 3, compared with Q0, the catalytic half - lives of all mutants became longer. Among them, T5 was 10.1 h, which was 110% longer than 4.8 h of Q0. Consistent with this, the total turnover number (TTN) of each mutant was increased compared with T0. The TTN of T5 was 29200, which was 2.59 times that of 11200 of T0.

[0069] Table 3 Kinetic parameters of PtDAPDH parental enzyme and its mutants

[0070]

[0071] Example 5: Construction of a co - expression strain of PtDAPDH, EcLAAD, and BsGDH

[0072] The mutant gene of PtDAPDH N13E / S90R / P254A and the PmLAAD gene with the nucleotide sequence shown in SEQ ID NO.3 and the BmGDH gene with the nucleotide sequence shown in SEQ ID NO.4 were tandemly connected in sequence to the pRSFDuet vector ( Figure 1Thus, a co-expression vector was obtained. By using the method of double digestion and ligation of the fragment and the vector, the PtDAPDH N13E / S90R / P254A mutant gene was ligated to the pRSFDuet vector, and finally the PmLAAD gene and the BmGDH gene were ligated to the pRSFDuet vector. The specific primers are shown in Table 4, and the underlined parts are the restriction enzyme sites.

[0073] Table 4 Primer sequences

[0074]

[0075] The recombinant plasmid was introduced into Escherichia coli BL21(DE3) competent cells. For the specific transformation steps, refer to Example 1 for introducing into Escherichia coli BL21 competent cells by the chemical transformation method. Finally, the BL21-LAAD-DAPDH-GDH strain (mutant T5 strain) was constructed.

[0076] Example 6: Optimization of the production of D-p-hydroxyphenylglycine from L-p-hydroxyphenylglycine at the 0.5L shake flask level

[0077] The mutant T5 strain with correct sequencing on the plate was inoculated into LB containing 0.05 mg / mL kanamycin resistance and cultured at 200 rpm and 37 °C for 10 - 12 h. Then, it was inoculated into the TB medium at an inoculation amount of 5% by volume and cultured at 200 rpm and 37 °C until the OD 600 reached 3, and then lactose was added at a final concentration of 5 g / L for induction. The induction temperature was 25 °C. After 14 h of induction, the cells were collected by centrifugation at 6,000×g for 8 min and placed at 37 °C for 16 h for use in the transformation.

[0078] (1) Effect of different whole-cell catalyst concentrations on the concentration of D-p-hydroxyphenylglycine

[0079] In the shake flask, a transformation reaction system was prepared: 20 g / L of L-p-hydroxyphenylglycine was adjusted to pH 8.5 with NaOH, the liquid loading volume was 0.1 L, and 1 g, 2 g, 3 g, 4 g, and 5 g of wet mutant cells (i.e., the whole-cell catalyst) were added to the reaction system respectively, so that the concentrations of the whole-cell catalyst were 10 g / L, 20 g / L, 30 g / L, 40 g / L, and 50 g / L respectively.

[0080] The transformation reaction was carried out at 37 °C and 550 rpm. During the reaction, solid powder of L-p-hydroxyphenylglycine was added to maintain the pH of the transformation solution at about 8.5. The powder addition time continued for 11 h after the start of the transformation, and then the transformation was carried out for 1 h. During this period, the addition of the powder was stopped, and the pH was adjusted to 8.5 with sodium hydroxide. The total volume after the transformation reaction was 0.1 L.

[0081] After the conversion reaction was completed, a part of the conversion solution was centrifuged at 12,000×g for 15 min, and the supernatant was filtered through a 0.22-μm microfiltration membrane and then analyzed by HPLC. The results are as Figure 2 shown. When the concentration of the catalyst increased from 10 g / L to 50 g / L, the concentration of D-p-hydroxyphenylglycine increased from 1.3 g / L to 3.2 g / L. And under the condition of a whole-cell catalyst concentration of 20 g / L, increasing the whole-cell catalyst concentration did not improve the conversion rate. Considering the industrial requirements for high yield and low catalyst (cell mass) usage, a whole-cell catalyst concentration of 20 g / L, which simultaneously has a relatively high concentration of D-p-hydroxyphenylglycine and a high product / catalyst ratio (i.e., the ability of unit cell mass to produce D-p-hydroxyphenylglycine), was used for the conversion experiment.

[0082] (2) Effect of different conversion reaction pH on the concentration of D-p-hydroxyphenylglycine

[0083] The specific steps were the same as in (1). The whole-cell catalyst concentration was controlled at 20 g / L, and the pH during the conversion reaction was controlled at 7.5, 8.0, 8.5, and 9.0, respectively. The results are shown in Table 5. The slightly alkaline condition was more suitable for the synthesis of D-p-hydroxyphenylglycine. Therefore, the conversion pH was selected to be controlled at about 8.5, and at this time, the concentration of D-p-hydroxyphenylglycine was 3.2 g / L.

[0084] Table 5 Results of pH optimization for conversion

[0085]

[0086] (3) Effect of different conversion durations on the concentration of D-p-hydroxyphenylglycine

[0087] The specific steps were the same as in (1). The conversion pH was controlled at 8.5, and the whole-cell catalyst concentration was controlled at 20 g / L. Samples were taken at different conversion times to measure the yield of D-p-hydroxyphenylglycine (12 - 24 h) (the results are shown in Table 6). As the conversion time extended, the yield continuously increased, but after 24 h, the increase was not obvious. Considering the industrial operation cost, the conversion time was controlled at 24 h. Finally, the yield of D-p-hydroxyphenylglycine reached 3.2 g / L, and the conversion rate was 32%.

[0088] Table 6 Results of fermentation time optimization

[0089]

[0090] Comparative Example 1:

[0091] For the specific implementation manner, refer to Example 6. The difference is that the mutant T5 strain is replaced with the wild-type T0 strain for fermentation and transformation experiments. After the transformation is completed, a part of the transformation broth is centrifuged at 12,000×g for 15 min, and the supernatant is filtered through a 0.22-μm microfiltration membrane and then subjected to HPLC analysis. The results of the HPLC chromatogram show that the yield of D-p-hydroxyphenylglycine is 1.2 g / L and the conversion rate is 12%.

[0092] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A Prevotella meso-diaminopimelate dehydrogenase Pt DAPDH mutant, characterized in that The mutant is meso-diaminopimelate dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1 Pt The amino acids at positions 13, 90, and 254 of the DAPDH parent are mutated as follows: (1) The asparagine at position 13 is mutated to glutamic acid; or, (2) The serine at position 90 is mutated to arginine; or, (3) The proline at position 254 is mutated to alanine; or, (4) The asparagine at position 13 is mutated to glutamic acid and the serine at position 90 is mutated to arginine; or, (5) The asparagine at position 13 is mutated to glutamic acid, the serine at position 90 is mutated to arginine, and the proline at position 254 is mutated to alanine.

2. A gene encoding the Prevotella meso-diaminopimelate dehydrogenase Pt Pt DAPDH mutant as claimed in claim 1.

3. An expression vector comprising the gene according to claim 2.

4. The expression vector according to claim 3, wherein The expression vector further comprises Pm the LAAD coding gene and Bm the GDH coding gene.

5. The expression vector according to claim 4, wherein The Pm nucleotide sequence of the LAAD-encoding gene is as shown in SEQ ID NO.3, and the Bm nucleotide sequence of the GDH-encoding gene is as shown in SEQ ID NO.

4.

6. A recombinant bacterium containing the expression vector according to any one of claims 3 - 5.

7. Use of the recombinant bacterium according to claim 6 in the preparation of D - p - hydroxyphenylglycine.

8. The application according to claim 7, characterized in that, The said use uses L - p - hydroxyphenylglycine as a reaction substrate and the said recombinant bacterium cells as a catalyst to catalytically produce D - p - hydroxyphenylglycine.

9. The application according to claim 8, characterized in that, In the catalytic reaction, the reaction conditions are to react for 12 - 24 h at pH 8.0 - 8.5 and 30 - 37 °C, and the addition amount of the recombinant bacterium cells is a final concentration of 10 - 60 g / L.

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