CN116814586A - Pyridoxal kinase mutant and application thereof in pyridoxine phosphate synthesis - Google Patents

Pyridoxal kinase mutant and application thereof in pyridoxine phosphate synthesis Download PDF

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CN116814586A
CN116814586A CN202311049480.6A CN202311049480A CN116814586A CN 116814586 A CN116814586 A CN 116814586A CN 202311049480 A CN202311049480 A CN 202311049480A CN 116814586 A CN116814586 A CN 116814586A
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梁海龙
任丽梅
韩中博
穆玉敏
张彦青
裴红硕
王亮
刘�东
高文杲
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Meibangmeihe Biotechnology Co ltd
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Abstract

The invention provides a pyridoxal kinase mutant and application thereof in pyridoxine phosphate synthesis, and belongs to the field of enzyme engineering. The mutant is obtained by one or more mutations of an amino acid sequence shown in SEQ ID NO.1, wherein the 51 st (N51K) and 175 th (V175E) are contained as mutant 1, the 51 st (N51K) and 152 th (D152E) are contained as mutant 2, and the 51 st (N51K) is contained as mutant 3. The invention also provides recombinant plasmids containing any pyridoxal kinase mutant gene, recombinant strains, and methods for expressing enzymes and applications thereof in pyridoxine phosphate preparation. Compared with the wild type pyridoxal kinase, the pyridoxal kinase mutant has higher catalytic efficiency, wherein the enzyme activity of the mutant 3 is improved by 23.3 times compared with that of the wild type, and the mutant can realize the conversion rate of more than 99.8 percent in 2-5h when the mutant is used for synthesizing pyridoxine phosphate. Compared with the prior chemical synthesis method, the method has the advantages of simple process, high conversion rate, relatively low production cost and good industrial application prospect.

Description

Pyridoxal kinase mutant and application thereof in pyridoxine phosphate synthesis
Technical Field
The invention belongs to the field of enzyme engineering, and particularly relates to a pyridoxal kinase mutant and application thereof in pyridoxine phosphate synthesis.
Background
Vitamin B 6 (Vitamin B6, VB 6) is a generic name for a class of pyridines that can be converted to each other, including Pyridoxine (PN), pyridoxamine (PM), pyridoxal (Pydaxal, PL). The corresponding phosphate forms are Pyridoxine-5' -phosphate (PNP), pyridoxamine-5' -phosphate (PMP) and pyridoxal-5 ' -phos-phate (PLP).
PLP is a coenzyme for more than 140 enzymes in a cell. At present, the PLP is produced by a chemical method, and Chinese patent CN 110016049A discloses a method for preparing pyridoxal phosphate by the chemical method, which is time-consuming, energy-consuming, serious in pollution, high in cost and low in yield. In organisms PLP cannot be synthesized directly by the organism, often by various forms of VB6 through metabolic pathways. PNP, a precursor of PLP, is an important source of pyridoxal phosphate in organisms. Therefore, the one-step oxidation of PNP to PLP is an important route for the biological preparation of PLP. Chinese patent CN202110644237 discloses a two-step method for producing PLP, in which, in the first step, PNP is produced by catalyzing vitamin B6 with phosphotransferase, but the final PNP production rate is not high.
Song Shina and the like describe pyridoxal kinase (pyridoxal kinasePLK) as vitamin B 6 (VB 6) key metabolizing enzyme, PLK phosphorylates PN to PNP (Song Shina, wang Tinghua. Pyridoxal kinase research progress [ J)]J.Sichuan anatomy, 2010,18 (04): 38-42.). Therefore, to increase the PNP production rate and further increase the PLP yield, it is necessary to develop a pyridoxal kinase with high catalytic efficiency and a process route for producing PNP with high efficiency.
Disclosure of Invention
The invention aims to provide a pyridoxal kinase mutant and application thereof in pyridoxine phosphate synthesis, so as to solve the problem of low catalytic efficiency of the existing pyridoxal kinase.
The invention aims at realizing the following technical scheme:
a pyridoxal kinase mutant which is any one of the following mutants:
mutant 1, wherein the mutant 1 is obtained by mutating Asn at 51 st position into Lys and Val at 175 th position into Glu on the basis of wild pyridoxal kinase with an amino acid sequence shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 3;
mutant 2, wherein the mutant 2 is obtained by mutating Asn at 51 st position into Lys and Asp at 152 th position into Glu on the basis of wild pyridoxal kinase with an amino acid sequence shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 5;
mutant 3, wherein the mutant 3 is obtained by mutating Asn at 51 st position into Lys on the basis of wild pyridoxal kinase with an amino acid sequence shown as SEQ ID NO.1, and the amino acid sequence of the mutant 3 is shown as SEQ ID NO. 7;
wherein, the nucleotide sequence of the optimized PDXK codon of the encoding gene of the wild pyridoxal kinase is shown as SEQ ID NO. 2.
The invention also provides a nucleotide sequence for encoding the pyridoxal kinase mutant:
the nucleotide sequence of the coding mutant 1 is shown as SEQ ID NO. 4; the nucleotide sequence of the coding mutant 2 is shown as SEQ ID NO. 6; the nucleotide sequence of the coding mutant 3 is shown as SEQ ID NO.8.
The methods for obtaining pyridoxal kinase mutants and the mutation positions are shown in Table 1.
TABLE 1 wild type and mutant mutation positions and methods of obtaining
Further, the present invention provides a recombinant expression vector comprising a gene encoding any one of the pyridoxal kinase mutants described above.
Preferably, the recombinant expression vector takes pET-29a (+) as a primary expression vector.
Still further, the present invention also provides a recombinant bacterium comprising the recombinant vector.
Preferably, the host bacterium isEscherichia coliBL21(DE3)。
The construction method of the recombinant bacteria comprises the following steps:
1) Inserting the coding gene fragment of any pyridoxal kinase mutant into the original expression vector pET-29a (+)NdeI andEcorecombinant vectors are obtained between RI enzyme cutting sites;
2) Transformation of recombinant vectors by hot-shockEscherichia coliBL21 (DE 3) to obtain recombinant bacteria.
The application of the pyridoxal kinase mutant in the synthesis of pyridoxine phosphate.
Specifically, under the condition of stirring, adding a substrate VB6 into a conversion tank containing a certain amount of pure water, respectively adding magnesium salt, ATP sodium salt and sodium hexametaphosphate, heating to 37 ℃ and stirring for dissolution, and then regulating the reaction by using sodium hydroxidepH to 5-6, adding pyridoxal kinase mutant enzyme solution and polyphosphorokinase (PpK) enzyme solution, fixing volume, and carrying out catalytic reaction at 36-40 ℃ to synthesize pyridoxine phosphate.
Preferably, the mass ratio of VB6, magnesium salt, ATP sodium salt and sodium hexametaphosphate is 5:1:5:5.
preferably, the magnesium salt is magnesium chloride hexahydrate.
Preferably, the reactionpH was 5 and the catalytic reaction temperature was 37 ℃.
Preferably, 1400U-5600U pyridoxal kinase mutant enzyme solution and 800U-1340U polyphosphate kinase enzyme solution are added into each 1 liter of the system; more preferably, 1400U pyridoxal kinase mutant enzyme solution and 800U polyphosphate kinase enzyme solution are added per 1 liter of the system.
In the synthesis system, the enzyme solution of the pyridoxal kinase mutant can generate pyridoxine phosphate by catalyzing VB6, and the conversion rate of more than 99.8% can be achieved after 2-5h of reaction.
The invention also provides a preparation for catalyzing VB6 to synthesize pyridoxine phosphate, wherein the preparation contains the pyridoxal kinase mutant.
The invention has the beneficial effects that:
on the basis of the traditional chemical synthesis method and biological enzyme method, the invention designs a pyridoxal kinase mutant, and compared with the wild pyridoxal kinase, the enzyme has higher catalytic efficiency. VB6 is used as a substrate, and can be used for synthesizing pyridoxine phosphate through a specific catalytic process, and the conversion rate of more than 99.8% is realized in 2-5 h. Compared with the prior chemical synthesis method and biological enzyme method, the method has shorter reaction time, lower cost and good industrial application prospect.
Drawings
FIG. 1 is an agarose gel electrophoresis of the successful construction of pyridoxal kinase mutant engineering bacteria. Wherein M is DNA Marker; no.1 is recombinant plasmid pET29a-PDXK-M3.
FIG. 2 is an SDS-PAGE electrophoresis of pyridoxal kinase mutant engineering bacteria after fermentation induced expression. Wherein M is a protein Marker; no.1 is a negative control bacterium; sample number 2 is post induction.
FIG. 3 is a liquid phase detection chromatogram of pyridoxal kinase mutant catalyzed synthesis of pyridoxine phosphate as a precursor and product.
FIG. 4 is a liquid phase detection chromatogram of pyridoxine phosphate end substrates and products catalyzed by pyridoxal kinase mutants.
Detailed Description
The following examples serve to further illustrate the invention in detail, but do not limit it in any way. The procedures and methods not described in detail in the examples below are conventional methods well known in the art, and the reagents used in the examples are either commercially available or prepared by methods well known to those of ordinary skill in the art. The following examples all achieve the object of the invention.
DESCRIPTION OF THE SEQUENCES
SEQ ID NO.1 is the amino acid sequence of the wild-type pyridoxal kinase PDXK-WT: full length 271 amino acids.
SEQ ID NO.2 is the nucleotide sequence of the wild-type pyridoxal kinase PDXK-WT: full length 816 bases.
SEQ ID NO.3 is the amino acid sequence of pyridoxal kinase mutation 1 PDXK-M1: the full length 271 amino acids, the mutation sites relative to the wild-type pyridoxal kinase are N51K and V175E.
SEQ ID NO.4 is the nucleotide sequence of pyridoxal kinase mutant 1 PDXK-M1: full length 816 bases.
SEQ ID NO.5 is the amino acid sequence of pyridoxal kinase mutant 2 PDXK-M2: the full length 271 amino acids, the mutation sites relative to the wild-type pyridoxal kinase are N51K and D152E.
SEQ ID NO.6 is the nucleotide sequence of pyridoxal kinase mutant 2 PDXK-M2: full length 816 bases.
SEQ ID NO.7 is the amino acid sequence of pyridoxal kinase mutant 3 PDXK-M3: the full length 271 amino acids, the mutation site relative to the wild-type pyridoxal kinase is N51K.
SEQ ID NO.8 is the nucleotide sequence of pyridoxal kinase mutant 3 PDXK-M3: full length 816 bases.
Definition of enzyme Activity
The amount of enzyme required to convert 1. Mu. Mole of pyridoxine hydrochloride or to produce 1. Mu. Mole of pyridoxine phosphate in 1 minute is one viability unit (U) at a specific temperature, pH and stirring speed.
Enzyme activity determination method
The reaction solution is added into a triangular flask, wherein the reaction solution comprises VB6, ATP sodium salt, magnesium chloride hexahydrate, sodium hexametaphosphate and pyridoxal kinase mutant enzyme solution. Put into a shaking table for reaction, immediately after sampling, the metal bath is used for inactivation, dilution and HPLC analysis, and the enzyme activity is calculated through PNP peak area change.
EXAMPLE 1 construction of wild-type pyridoxal kinase recombinant vector and acquisition of corresponding Strain
The nucleotide sequence shown in SEQ ID No.2 (protein sequence number P39610.1 from NCBI, bacillus subtilis) was artificially synthesized by an artificial synthesis method and ligated to pET-29a (+) expression vectorNdeI andEcoand (3) obtaining a recombinant vector pET29a-PDXK-WT between RI enzyme cutting sites. Transformation of recombinant vector pET29a-PDXK-WT into E.coliE.coliBL21 (DE 3), sent to sequence, and the successful sequence is the wild pyridoxal kinase strain PDXK-WT.
EXAMPLE 2 construction of mutant 1, mutant 2 recombinant vector and obtaining of corresponding Strain
Random mutation was introduced by a random mutation PCR kit (cat# BTN 1010055), and pyridoxal kinase mutant and wild type pyridoxine were screened for differences in their ability to synthesize by a method of screening for viability under the same conditions using pET29a-PDXK-WT as a template as described above. Selecting high-activity high-stability strain, extracting plasmid and sequencing. According to the result of the enzyme activity measurement of the near hundred strains, 2 mutants with increased activity and increased stability are obtained, wherein two amino acid mutations are introduced into wild amino acid sequences, and the obvious change sequences are respectively named as a mutant 1 and a mutant 2. Wherein, the 51 st (N51K) and 175 th (V175E) are mutant 1, and the obtained recombinant plasmid is named pET29a-PDXK-M1; contains mutant 2 at 51 st (N51K) and mutant 2 at 152 th (D152E), and the obtained recombinant plasmid is named pET29a-PDXK-M2. Transferring the two recombinant vectors into a host cellE.coliBL21 (DE 3), the strain which was sequenced and successfully sequenced was named PDXK-M1 and PDXK-M2, respectively.
The enzyme activities of the wild pyridoxal kinase strain and the 2 mutant strains are respectively detected, the detection results are shown in table 2, and the enzyme activities of the mutant strains are obviously improved.
TABLE 2 influence of amino acid sequence mutation positions on enzyme activity
EXAMPLE 3 construction of mutant 3 recombinant vector and obtaining of corresponding Strain
The inventors found that mutant 1 and mutant 2 each contain a 51 st (N51K) mutation site, and therefore, site-directed mutagenesis primers Fl primer and Rlprimer were designed for mutation site N51K, wherein Fl primer specifically is: 5'-CCGAACAACAGCTGGAAACATCAGGTTTTTCCG-3'; the Rlprimer is specifically: 5'-CGGAAAAACCTGATGTTTCCAGCTGTTGTTCGG-3' by PCR reaction with vector pET29a-PDXK-WT as template, the gene N51K is obtained, and the nucleotide sequence of the gene is SEQ ID NO.8. The resulting recombinant expression vector was designated pET29a-PDXK-M3. The PCR reaction system is shown in Table 3, and the PCR reaction conditions are shown in Table 4.
TABLE 3 PCR reaction System
TABLE 4 PCR reaction conditions
The result of gel electrophoresis detection of the PCR product is shown in figure 1, and the target band is consistent with theory. mu.L of the PCR product was taken and 1. Mu.L was addedDpnI restriction enzyme digestion of the template plasmid, incubation at 37℃for 3h. And (3) absorbing 10 mu L of enzyme digestion products, converting into escherichia coli BL21 (DE 3) to obtain corresponding recombinant escherichia coli, coating the corresponding recombinant escherichia coli on a calicheamicin (100 mg/L) -containing LB plate, culturing overnight at 37 ℃, randomly picking clone products for colony PCR identification and sequencing verification, and successfully converting a recombinant expression vector containing a pyridoxal kinase mutant gene into an expression host escherichia coli BL21 (DE 3). The strain with successful mutation by sequencing verification is named as PDXK-M3, and the bacterial liquid is taken, added with glycerol and preserved in a refrigerator at the temperature of minus 70 ℃.
The enzyme activity of the mutant 3 obtained by enzyme activity measurement is 28U/mL, which is 23.3 times higher than that of the wild pyridoxal kinase, 21.7% higher than that of the mutant 1, and 64.7% higher than that of the mutant 2. Thus, mutant 3 was selected as a test strain for later test development.
Example 4 catalytic synthesis of pyridoxine phosphate by pyridoxal kinase mutant 3
Catalytic reaction of 50mL system under different temperature conditions
In a 50mL system, 0.5g VB was added 6 0.1g ATP sodium salt, 0.5g magnesium chloride hexahydrate and 0.5. 0.5g sodium hexametaphosphate, adjusted by 5M NaOHpH was 5.0, pyridoxal kinase mutant enzyme solution 280U and PpK kinase enzyme solution 67. 67U were added to a volume of 50mL, and the reaction was started at different temperatures with stirring at 200 rpm. After 4 hours of reaction, the conversion and the yield under different reaction conditions were measured as shown in Table 5 below:
TABLE 5 optimization of reaction system temperature
As can be seen from the results of the above table, the catalytic efficiency of the enzyme was highest at 37℃with the best results, the conversion was 100%, and the production was 99.7%. At 37℃the conversion rates of mutant 1 and mutant 2 were 83.65% and 80.24%, respectively, which were less effective than mutant 3.
Catalytic reaction of 50mL systems under different pH conditions
In a 50mL system, 0.5g VB6,0.1g ATP sodium salt, 0.5g magnesium chloride hexahydrate and 0.5g sodium hexametaphosphate were added, adjusted by 5M NaOHpH was varied, pyridoxal kinase mutant enzyme solution 280U and PpK kinase enzyme solution 67U were added to a volume of 50mL and the reaction was started with stirring at 200 rpm at 37 ℃. After 4 hours of reaction, the conversion and the yield under different reaction conditions were measured as shown in Table 6 below:
TABLE 6 pH optimization Table of reaction systems
As can be seen from the results of the above table, the reactionpThe enzyme has a catalytic efficiency of 5 or 6, a conversion of 100% and a production rate of 99% or more, but is selected in consideration of costpH is 5 as the final reactionpH. And is the same aspUnder H5 conditions, the conversion rates of mutant 1 and mutant 2 were 81.12% and 80.08%, respectively, which were less effective than mutant 3.
Catalytic reaction of 50mL systems with different pyridoxal kinase enzyme throwing amounts
In a 50mL system, 0.5g VB6,0.1g ATP sodium salt, 0.5g magnesium chloride hexahydrate and 0.5g sodium hexametaphosphate were added, adjusted by 5M NaOHpH was 5.0, pyridoxal kinase mutant enzyme solution 70U-280U and PpK kinase enzyme solution 67U were added to a volume of 50mL, and the reaction was started under stirring at 200 rpm at 37 ℃. After 4 hours of reaction, the conversion and the yield of the different reactions were measured as shown in Table 7 below:
TABLE 7 optimization of enzyme solution dosage of pyridoxal kinase mutants
As can be seen from the results of the table, the enzyme solution dosage of the pyridoxal kinase mutant is 70U-280U, which has higher conversion rate and production rate, and the enzyme dosage is 70U in consideration of cost. Mutant 1 and mutant 2 were dosed in the same amount as 70U, and the amount of the added mutant was slightly greater than that of mutant 3, so that the mixed protein was more brought in, the conversion rates were 93.12% and 87.61%, respectively, and the effect was slightly poor.
Catalytic reaction of 50mL systems with different PpK enzyme throwing amounts
In a 50mL system, 0.5g VB6,0.1g ATP sodium salt, 0.5g magnesium chloride hexahydrate and 0.5g sodium hexametaphosphate were added, adjusted by 5M NaOHpH was 5.0, pyridoxal kinase mutant enzyme solution 70U and PpK kinase enzyme solution 40U-67U were added to a volume of 50mL, and the reaction was started under stirring at 200 rpm at 37 ℃. After 4 hours of reaction, the conversion and the yield of the different reactions were measured as shown in Table 8 below:
TABLE 8 PpK enzyme solution dosage optimization Table
As can be seen from the results of the table, the addition amount of the PpK kinase enzyme solution is 40U-67U, the conversion rate and the generation rate are high, and the addition amount of the PpK kinase enzyme solution is 40U in consideration of cost. According to the PpK enzyme solution dosage of 40U, mutant 1 has more foreign proteins introduced because of the pyridoxal kinase dosage problem similar to mutant 2, the conversion rates are 92.36% and 88.57%, respectively, and the effect is poor.
Example 5 pyridoxal kinase mutant 1 catalytic synthesis of pyridoxine phosphate (catalytic reaction of 1L System)
In a 1L system, 10g of VB6, 2g of ATP sodium salt, 10g of magnesium chloride hexahydrate and 10g of sodium hexametaphosphate were added and adjusted by 5M NaOHpH was 5.0, pyridoxal kinase mutant 1 enzyme solution 1400U was added, ppK kinase enzyme solution 800U was set to a volume of 1L, and the reaction was started under stirring at 200 rpm at 37 ℃. After reaction 5h, pyridoxine phosphate concentration was measured to be 10.2g/L and conversion was 93.5%.
Example 6 pyridoxal kinase mutant 2 catalytic synthesis of pyridoxine phosphate (catalytic reaction of 1L System)
In a 1L system, 10g of VB6, 2g of ATP sodium salt, 10g of magnesium chloride hexahydrate and 10g of sodium hexametaphosphate were added and adjusted by 5M NaOHpH was 5.0, pyridoxal kinase mutant 2 enzyme solution 1400U was added, ppK kinase enzyme solution 800U was set to a volume of 1L, and the reaction was started under stirring at 200 rpm at 37 ℃. After reaction 5h, pyridoxine phosphate concentration was measured at 9.29g/L and conversion was 88.1%.
Example 7 pyridoxal kinase mutant 3 catalytic synthesis of pyridoxine phosphate (catalytic reaction of 1L System)
In a 1L system, 10g of VB6, 2g of ATP sodium salt, 10g of magnesium chloride hexahydrate and 10g of sodium hexametaphosphate were added and adjusted by 5M NaOHpH was 5.0, pyridoxal kinase mutant 3 enzyme solution 1400U was added, ppK kinase enzyme solution 800U was set to a volume of 1L, and the reaction was started under stirring at 200 rpm at 37 ℃. After reaction 5h, pyridoxine phosphate concentration was measured at 12.1g/L and conversion was 99.8%.
Further, the initial and end VB6 and pyridoxine phosphate were subjected to liquid chromatography, and the liquid chromatography is shown in FIG. 3 and FIG. 4, respectively.
Example 8 pyridoxal kinase mutant 3 catalytic synthesis of pyridoxine phosphate (catalytic reaction of 10L System)
In a 10L system, 100g of VB6, 20 g of ATP sodium salt, 100g of magnesium chloride hexahydrate and 100g of sodium hexametaphosphate were added and adjusted by 5M NaOHpH was 5.0, and pyridoxal kinase mutant 3 enzyme solution 14000U and PpK kinase enzyme solution 8000U were added to a volume of 10L, and the reaction was started under stirring at 200 rpm at 37 ℃. After reaction 5h, pyridoxine phosphate concentration was measured to be 120.6 g/L and conversion was 99.9%. The reaction amplification is not problematic, and the industrial production can be guided.

Claims (9)

1. Pyridoxal kinase mutant, characterized in that it is any of the following mutants:
mutant 1, wherein the mutant 1 is obtained by mutating Asn at 51 st position into Lys and Val at 175 th position into Glu on the basis of wild pyridoxal kinase with an amino acid sequence shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 3;
mutant 2, wherein the mutant 2 is obtained by mutating Asn at 51 st position into Lys and Asp at 152 th position into Glu on the basis of wild pyridoxal kinase with an amino acid sequence shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 5;
mutant 3, wherein the mutant 3 is obtained by mutating Asn at 51 st position into Lys on the basis of wild pyridoxal kinase with an amino acid sequence shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 7.
2. The pyridoxal kinase mutant according to claim 1, wherein the nucleotide sequence encoding mutant 1 is shown in SEQ ID No.4, the nucleotide sequence encoding mutant 2 is shown in SEQ ID No.6, and the nucleotide sequence encoding mutant 3 is shown in SEQ ID No.8.
3. Recombinant bacterium expressing a pyridoxal kinase mutant according to claim 1, characterized in that the original expression vector of said recombinant bacterium is pET-29a (+).
4. A recombinant bacterium according to claim 3 expressing any one of the pyridoxal kinase mutants according to claim 1, characterized in that the host bacterium of the recombinant bacterium isEscherichia coli BL21（DE3）。
5. Use of a pyridoxal kinase mutant according to claim 1 for the synthesis of pyridoxine phosphate.
6. The method according to claim 5, wherein VB6 is used as substrate, magnesium salt, ATP sodium salt and sodium hexametaphosphate are added in sequence, and stirred for dissolution and adjustmentpH to 5-6, adding pyridoxal kinase mutant enzyme solution and polyphosphorokinase enzyme solution, fixing the volume, and carrying out catalytic reaction at 36-40 ℃ to generate pyridoxine phosphate.
7. The use according to claim 6, wherein VB 6 The mass ratio of magnesium salt, ATP sodium salt and sodium hexametaphosphate is 5:1:5:5.
8. the use according to claim 6, characterized in that 1400U-5600U pyridoxal kinase mutant enzyme solution and 800U-1340U polyphosphate kinase enzyme solution are added per 1 liter of the synthesis system.
9. A preparation for catalyzing synthesis of pyridoxine phosphate from VB6, wherein the preparation comprises the pyridoxal kinase mutant of claim 1.
CN202311049480.6A 2023-08-21 2023-08-21 Pyridoxal kinase mutant and application thereof in pyridoxine phosphate synthesis Pending CN116814586A (en)

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