A polyphosphoryl kinase mutant with high catalytic activity and its application in synthesis of glutathione

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a polyphosphate kinase mutant with high catalytic activity and application thereof in synthesis of glutathione. The present application provides a polyphosphate kinase mutant with high catalytic activity, which is characterized in that the polyphosphate kinase mutant is mutated from an amino acid sequence shown in SEQ ID NO: 2 into one of the following: (1) single mutation is made to the 234th amino acid in the amino acid sequence shown in SEQ ID NO: 2; (2) double mutations are made to the 234th and 166th amino acids in the amino acid sequence shown in SEQ ID NO: 2; wherein a nucleotide sequence of a coding gene coding the amino acid sequence shown in SEQ ID NO: 2 is as shown in SEQ ID NO: 1. ch PPK-N234A and double-site mutation polyphosphate kinase mutant ch PPK-N234A / K166D has high activity, and the enzyme activity is increased by 4.19 times and 6.12 times, respectively, compared with wild-type polyphosphate kinase.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to a polyphosphate kinase mutant with high catalytic activity and application thereof in synthesizing glutathione. Background Art

[0002] Glutathione (GSH) is a biologically active, non-protein, short thiol peptide formed by the condensation of glutamic acid, glycine, and cysteine. It possesses antioxidant, detoxifying, and immune-boosting properties and is widely used in the medical, food, and cosmetic industries. Enzymatic synthesis of GSH, with its advantages of simple components, high product concentration, short cycle times, and high yields, has become a hot topic of research. However, the synthesis of each GSH molecule consumes two molecules of adenosine triphosphate (ATP). Therefore, the recycling of ATP in the synthesis system is a key factor in the efficiency of GSH biosynthesis.

[0003] The cleavage of ATP high-energy phosphate bonds provides the energy required for the enzymatic synthesis of GSH. However, ATP is expensive, and direct addition significantly increases the cost of industrial production. It also produces by-products such as the accumulation of adenosine diphosphate or adenosine monophosphate, making downstream separation and purification difficult. n ) provides a phosphate source, using ADP or AMP as substrates to synthesize ATP. Glutathione bifunctional enzyme (GshF) catalyzes the synthesis of GSH in conjunction with PPK, achieving efficient GSH synthesis. Simultaneously, ADP / AMP can be utilized by polyphosphate, achieving ATP regeneration and GSH synthesis.

[0004] The ATP regeneration system based on polyphosphate kinase is efficient, stable, and easily scaled up for industrial use. However, the low catalytic efficiency of polyphosphate kinase in this ATP cycle limits its industrial application. Therefore, molecular engineering of polyphosphate kinase to generate highly active polyphosphate kinase and construct an efficient ATP regeneration system has important application value and research significance for large-scale enzymatic GSH synthesis. Summary of the Invention

[0005] The present invention is made to solve the above-mentioned problems, and its object is to provide a polyphosphate kinase mutant with high catalytic activity.

[0006] In a first aspect of the present invention, a polyphosphate kinase mutant with high catalytic activity is provided, characterized in that: the polyphosphate kinase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO: 2 to one of the following: (1) a single mutation at position 234 of the amino acid sequence shown in SEQ ID NO: 2; or (2) a double mutation at positions 234 and 166 of the amino acid sequence shown in SEQ ID NO: 2.

[0007] Wherein, the nucleotide sequence encoding the gene encoding the amino acid sequence shown in SEQ ID NO: 2 is as SEQ ID NO: 1.

[0008] In some embodiments, the single mutation comprises mutating asparagine at position 234 to alanine, the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.3.

[0009] In some embodiments, the double mutation comprises mutating asparagine at position 234 to alanine and mutating lysine at position 166 to aspartic acid, the amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.5.

[0010] The second aspect of the present invention provides a gene encoding the polyphosphate kinase mutant as described in the first aspect.

[0011] The third aspect of the present invention provides a recombinant vector comprising a gene encoding the polyphosphate kinase mutant as described in the second aspect.

[0012] The fourth aspect of the present invention provides a recombinant strain comprising a gene encoding the polyphosphate kinase mutant as described in the second aspect.

[0013] A fifth aspect of the present invention provides a method for preparing a polyphosphate kinase mutant, which has the following characteristics, comprising the following steps:

[0014] (1) The coding gene shown in SEQ ID NO.1 was connected to the original expression vector pET-28a (+) to obtain the recombinant vector pET28a- Ch PPK, the recombinant vector pET28a- Ch PPK transformed host cells to obtain ch PPK recombinant strains;

[0015] (2) Design the primers for site-directed mutagenesis of N234A with the recombinant vector pET28a- Ch PPK was used as a template for overlap extension PCR to obtain the amino acid sequence shown in SEQ ID NO.4 in which the asparagine at position 234 was mutated to alanine. ChPPK -N234A mutant vector;

[0016] (3) Ch The PPK-N234A mutant vector was transformed into the host bacteria E. coli BL21 (DE3) was screened and obtained Ch PPK-N234A polyphosphate kinase mutant expression strain, induced expression, obtained Ch PPK-N234A polyphosphate kinase mutant.

[0017] A sixth aspect of the present invention provides a method for preparing a polyphosphate kinase mutant, which has the following characteristics, comprising the following steps:

[0018] (1) The coding gene shown in SEQ ID NO.1 was connected to the original expression vector pET-28a (+) to obtain the recombinant vector pET28a- Ch PPK, the recombinant vector pET28a- Ch PPK transformed host cells to obtain ch PPK recombinant strains;

[0019] (2) Design the primers for site-directed mutagenesis of N234A with the recombinant vector pET28a- Ch PPK was used as a template for overlap extension PCR to obtain the amino acid sequence shown in SEQ ID NO.4 in which the asparagine at position 234 was mutated to alanine. Ch PPK -N234A mutant vector;

[0020] (3) Design the K166D site-directed mutagenesis primers, using the primers in step (2) Ch The PPK-N234A mutant vector was used as a template for overlap extension PCR to obtain the amino acid sequence shown in SEQ ID NO.6, in which the 166th lysine was mutated to aspartic acid. Ch PPK-N234A / K166D mutant vector;

[0021] (4) Ch The PPK-N234A / K166D mutant vector was transformed into the host bacteria E. coli BL21 (DE3) was screened and obtained Ch PPK-N234A / K166D polyphosphate kinase mutant expression strain, induced expression, obtained Ch PPK-N234A / K166D polyphosphate kinase mutant.

[0022] The seventh aspect of the present invention provides a use of the polyphosphate kinase mutant as described in the first aspect, characterized in that the polyphosphate kinase mutant is used to construct an ATP regeneration system.

[0023] In an eighth aspect, the present invention provides an application of a polyphosphate kinase mutant, characterized in that the polyphosphate kinase mutant, a polyphosphate kinase mutant-expressing microorganism, or a cell disrupted liquid thereof is used as an ATP regenerating agent for synthesizing glutathione.

[0024] By implementing the above technical solution, the present invention has the following beneficial effects:

[0025] Single-site mutation polyphosphate kinase mutant provided by the present invention ch PPK-N234A and double-site mutation polyphosphate kinase mutants ch PPK-N234A / K166D has high activity, with the enzyme activity increased by 4.19 times and 6.12 times compared with the wild-type polyphosphate kinase, respectively.

[0026] The polyphosphate kinase mutant provided by the present invention is used to construct an ATP regeneration system. The polyphosphate kinase mutant has high catalytic activity and can catalyze the byproduct ADP in the reaction process to synthesize ATP, thereby achieving cyclic regeneration of ATP. The ADP conversion rate of the polyphosphate kinase mutant is higher than that of the wild-type polyphosphate kinase mutant.

[0027] The present invention provides the use of polyphosphate kinase mutants in glutathione synthesis. Two polyphosphate kinase mutants are used as ATP regenerators to assist glutathione synthesis reaction, significantly accelerating the reaction rate. ch PPK-N234A can produce 17.86 g / L of glutathione within 4 h. ch PPK-N234A / K166D could produce 18.31 g / L of glutathione within 4 h. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the relative enzyme activity of the wild type polyphosphate kinase and its mutants in Example 4;

[0029] Figure 2 is a curve showing the change in ADP conversion rate of polyphosphate kinase over time in Example 5;

[0030] Figure 3 This is a curve showing the change of glutathione production over time when polyphosphate kinase is used for glutathione synthesis in Example 6. DETAILED DESCRIPTION

[0031] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is described in detail below with reference to embodiments and drawings.

[0032] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., were mainly performed with reference to Molecular Cloning Laboratory Manual (3rd edition, edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002).

[0033] LB medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 2 g / L agar powder (solid medium), solvent is water, pH natural.

[0034] Example 1: Site-directed mutagenesis of positions 234 and 166 of polyphosphate kinase

[0035] 1. Site-directed mutagenesis

[0036] In this study, we first used Alphafold 3 to ch PPK homology modeling was performed to predict its 3D structure. ch PPK and ADP molecular docking generation ch PPK_ADP.pdb file, and then visualized the docking results in Pymol and found amino acid residues within 20 Å of ADP, initially constructing a large mutation library. In order to reduce the size of the library, the active site that directly interacts with ADP and amino acid residues located in key structures (lid structure, loop, Walker A and Walker B) were manually excluded. Although the mutation library was relatively small, it was still too large. Therefore, FoldX was further used to repair the ch The PPK_ADP.pdb file was used to perform virtual saturation mutations on the amino acid residues in the reduced mutation library. Some of the results are shown in Table 1. Theoretically, higher predicted free energies represent greater enzyme structural flexibility and are more likely to improve catalytic activity. Based on this, high-energy sites with significantly higher free energy were selected for further experimental verification. This strategy ultimately resulted in a smaller, experimentally feasible mutant library and identified beneficial mutations at positions N234 and K166.

[0037] To mutate positions 234 and 166 of wild-type polyphosphate kinase to predicted amino acids, chThe coding gene sequence of PPK was used as a template, and its amino acid sequence was shown in SEQ ID NO.2, and its nucleotide sequence was shown in SEQ ID NO.1. The corresponding primers were designed and the pET28a- Ch PPK was used for full plasmid amplification, and the primer sequences are shown in Table 2.

[0038] The PCR amplification system (50 µL) was as follows: 1 µL template DNA, 25 µL 2× Phanta Max Buffer, 1 µL dNTPs Mix, 1 µL each of upstream and downstream primers, 1 µL Phanta DNA Polymerase, and ddH2O was added to a total volume of 50 µL.

[0039] PCR reaction parameters: (1) pre-denaturation at 95°C for 30 s; (2) denaturation at 95°C for 30 s; (3) annealing at 55°C for 30 s; (4) extension at 72°C for 6 min, and steps (2)-(4) were cycled 30 times; (5) complete extension at 72°C for 7 min, and storage at 16°C. After the PCR product was positive after 0.9% agarose gel electrophoresis analysis, 1 μL of endonuclease Dpn I was added to the PCR reaction solution and digested at 37°C for 2 h to remove the template plasmid DNA.

[0040] Table 1. Virtual saturation mutation energy table based on FoldX (partial)

[0041] Amino acid N234 K166 E152 S101 A 1.287 0.307 1.246 -0.403 C 1.863 0.836 0.793 -0.816 D 1.917 -0.032 0.531 -0.598 E 1.563 -0.391 0.000 -1.167 F 1.118 0.305 -1.372 -0.178 G 2.114 0.913 1.783 0.046 H 1.925 0.761 0.202 -1.277 I 1.203 0.534 -0.758 -2.309 K 1.106 0.000 0.718 -1.111 L 1.183 0.211 -0.534 -1.969 M 0.658 -0.060 -0.095 -1.475 N 0.000 0.218 1.139 -0.709 P 5.804 -0.892 1.406 -0.419 Q 1.014 -0.048 -0.003 -0.818 R 1.032 0.053 0.774 -0.460 S 1.282 0.538 2.103 0.000 T 1.062 0.731 1.545 -1.103 V 1.548 0.761 -0.290 -1.645 W 1.424 0.391 -0.878 -0.915 Y 1.382 0.447 -1.302 -0.280

[0042] Table 2 Primers for site-directed mutagenesis of polyphosphokinase at sites 234 and 166

[0043]

[0044] 2. Transformation and culture of polyphosphate kinase mutants

[0045] Take 10 μL of the PCR product from step 1 and add E. coliBL21 (DE3) competent cells were placed on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, and then added with 600 μL of antibiotic-free LB medium. The cells were incubated at 37°C at 180 rpm for 1 hour. After recovery, they were plated onto LB plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Single colonies from the plates were randomly picked and transferred to 10 mL of LB medium containing 50 μg / mL kanamycin. After incubation at 37°C for 8 hours, 500 μL of the bacterial solution was mixed with 30% glycerol (v / v = 1:1) in a glycerol tube and stored at -80°C. The remaining bacterial solution was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, resulting in the generation of 10 recombinant engineered polyphosphate kinase mutants.

[0046] Example 2: Construction of a polyphosphate kinase iterative mutation library

[0047] The best single mutant in Example 1 ch PPK-N234A was iteratively mutated to ch The coding gene sequence of PPK-N234A was used as a template, and the primers designed in Example 1 were used to clone the pET28a- Ch The whole plasmid amplification and mutant transformation and culture of PPK-N234A were performed as shown in Example 1, and the polyphosphate kinase double-site mutant was finally obtained. ch PPK-N234A / K166H and ch PPK-N234A / K166D.

[0048] Example 3: Preparation of cell lysate of recombinant Escherichia coli containing polyphosphate kinase mutants

[0049] The recombinant engineered bacteria involved in Example 1 were inoculated into 10 mL LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 8 h as seed liquid. The seed liquid was then inoculated into fresh 50 mL LB liquid medium containing 50 μg / mL kanamycin at a 2% (v / v) inoculum and cultured at 37°C and 180 rpm for 2-3 h to a bacterial cell concentration of OD 600 When the pH reaches 0.5–0.7, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM and induce the culture at 28°C and 180 rpm for 12 h. Harvest the cells by centrifugation at 4°C and 8000 rpm for 10 min to collect the wet cells for later use.

[0050] The collected cells were resuspended at 1 g / L in 50 mM Tris-HCl buffer (pH = 7.5) and disrupted by ultrasound, with each working cycle lasting 1 s and a rest period of 2 s for a total working time of 10 min. The resulting cell disruption solution was diluted 10-fold to a final concentration of 0.1 g / L.

[0051] Example 4: Determination of polyphosphate kinase activity

[0052] The enzyme activity of the cell lysate containing the wild-type polyphosphate kinase and the polyphosphate kinase mutant obtained in Example 3 was determined.

[0053] To 100 µL of 0.1 g / L cell lysate, 50 µL of 25 mM ADP, 50 mM sodium hexametaphosphate, and 100 mM anhydrous magnesium sulfate, prepared in 50 mM Tris-HCl buffer (pH 7.5), were added. The mixture was incubated at 35°C and 600 rpm for 5 minutes. The reaction was terminated by the addition of 50 µL of 2 M HCl, and the pH was neutralized by the addition of 50 µL of 2 M NaOH. The reaction mixture was centrifuged at 1000 rpm for 1 minute. The supernatant was diluted two-fold with ultrapure water and filtered through a 0.22 µm filter. ATP content was determined by high-performance liquid chromatography (HPLC).

[0054] Enzyme activity definition: One enzyme activity unit (U) is the amount of enzyme required to generate 1 µmol ATP per minute at 35°C and pH 7.5.

[0055] HPLC detection conditions for ATP content: C18 column (4.6×mmol / L, 5 µm), 50 mM dipotassium hydrogen phosphate as mobile phase (pH = 7.0 adjusted with potassium dihydrogen phosphate, freshly prepared for use), 10 µL injection volume, 0.8 mL / min flow rate, 40°C, 8 min retention time, detection at 254 nm, ATP, ADP, and AMP eluted in that order.

[0056] The results of enzyme activity assay are shown in Figure 1 , single-site mutation polyphosphate kinase mutant ch PPK-N234A and double-site mutation polyphosphate kinase mutants ch The enzyme activity of PPK-N234A / K166D was significantly improved, which was 4.19 times and 6.12 times higher than that of wild-type polyphosphate kinase, respectively.

[0057] Example 5: Wild-type polyphosphate kinase ch Application of PPK and its mutants in catalyzing ATP synthesis

[0058] Prepared according to Example 3ch PPK, ch PPK-N234A, ch To 10 mL of 0.1 g / L PPK-N234A / K166D cell lysate, 5 mL of 25 mM ADP, 50 mM sodium hexametaphosphate, and 100 mM anhydrous magnesium sulfate, prepared in 50 mM Tris-HCl buffer (pH 7.5), were added. The mixture was shaken in a water bath at 35°C and 600 rpm. 250 µL of the sample was collected at different time points, and 50 µL of 2 M HCl and NaOH were added sequentially. Sample preparation and HPLC detection methods are as described in Example 4.

[0059] The reaction process of polyphosphate kinase changes with time Figure 2 As shown, wild-type polyphosphate kinase ch The conversion rate of PPK tended to be stable at 125 min, and the final conversion rate was about 63%. ch PPK-N234A and ch The conversion rates of PPK-N234A / K166D were approximately 39% and 57% at 2 minutes, respectively, compared to only about 3% for the wild type. The conversion rates of both mutants plateaued at 100 minutes, ultimately reaching approximately 68%.

[0060] Example 6: Application of polyphosphate kinase in glutathione synthesis

[0061] The reaction system was composed and operated as follows: 60 mM cysteine, 90 mM sodium glutamate, 100 mM glycine, 60 mM anhydrous magnesium sulfate, 35 mM sodium hexametaphosphate, and 12.5 g / L and 5 g / L wet bacterial cell disruption solutions of glutathione bifunctional enzyme (GshF) and polyphosphate kinase, respectively, were added to 100 mM Tris-HCl buffer (pH = 7.5), and then 2 mM ATP was added. The reaction was shaken at 35°C and 600 rpm for 4 h, during which nitrogen was continuously introduced and the pH was adjusted to 7.0.

[0062] Intermittently remove 1 mL of the reaction mixture and add 100 µL of 2 M HCl to terminate the reaction. Add NaOH at the same volume concentration. Centrifuge at 12,000 rpm for 1 min. Dilute the supernatant 10-fold with pure water, filter through a 0.22 µm water filter, and analyze the glutathione (GSH) content by HPLC.

[0063] HPLC detection conditions: C18 column (4.6×mmoL / L, 5 µm), a solution of potassium hydrogen phosphate and sodium heptanesulfonate was prepared with final concentrations of 6.8 g / L and 2.02 g / L, respectively. The pH was adjusted to 2.8 with phosphoric acid. 950 mL of the mixed solution was added with 50 mL of pure methanol to prepare the mobile phase. The injection volume was 10 µL, the flow rate was 1.0 mL / min, the column temperature was 30°C, the retention time was 20 min, and detection was at 210 nm.

[0064] Wild-type polyphosphate kinase ch PPK and its mutants ch PPK-N234A, ch The reaction process of PPK-N234A / K166D for glutathione synthesis changes with time Figure 3 Wild type ch After 4 hours of glutathione synthesis reaction, the glutathione content was 16.31 g / L. ch After 4 h of reaction, the glutathione production of PPK-N234A increased to 17.86 g / L. ch After 4 h of reaction, the glutathione production of PPK-N234A / K166D was 18.31 g / L.

[0065] Example 7: ch Comparison examples between different PPK mutants

[0066] In our previous work (application number: CN202310871912.5), we ch Single-site saturation mutations were performed at positions 56 and 125 of the PPK amino acid sequence, successfully constructing two polyphosphate kinase mutants with significantly improved activity: ch PPK-E56K and ch PPK-T125S. ch The enzyme activity of PPK-E56K was increased by 4.8 times compared with the wild type. ch The enzyme activity of PPK-T125S reached 5.3 times that of the wild type.

[0067] In this study, we further constructed ch The PPK-N234A / K166D double mutant further enhanced enzyme activity. Under the same reaction conditions, the double mutant's enzyme activity increased 6.12-fold compared to the wild type, demonstrating excellent catalytic efficiency.

[0068] also, ch PPK-E56K and chThe application of PPK-T125S as an ATP regenerator in glutathione synthesis shows that when the enzyme dosage is 25 g / L, the constructed ATP regeneration system can support the effective glutathione synthesis reaction. ch PPK-N234A / K166D can reduce the amount of PPK enzyme to 5 g / L, which is 20% of the previous dosage.

[0069] In summary, ch The PPK-N234A / K166D double mutant not only exhibits the highest relative enzyme activity compared to the wild type, significantly accelerating ATP production and improving reaction efficiency, but also, in practical applications, further reduces the amount of enzyme required for the reaction when coupled with the glutathione bifunctional enzyme (GshF) to catalyze glutathione synthesis, effectively lowering production costs.

[0070] The above embodiments are preferred examples of the present invention and are not intended to limit the scope of protection of the present invention.

Claims

1. A polyphosphate kinase mutant with high catalytic activity, characterized in that The amino acid sequence of the mutant is shown in SEQ ID NO.4 or SEQ ID NO.

6.

2. A gene encoding the polyphosphate kinase mutant according to claim 1.

3. A recombinant vector comprising the coding gene according to claim 2.

4. A recombinant strain comprising the coding gene according to claim 2.

5. A method for preparing a polyphosphate kinase mutant according to claim 1, characterized in that: The following steps are involved: (1) The coding gene shown in SEQ ID NO.1 was connected to the original expression vector pET-28a (+) to obtain the recombinant vector pET28a- Ch PPK, the recombinant vector pET28a- Ch PPK transformed host cells to obtain ch PPK recombinant strains; (2) Design the primers for site-directed mutagenesis of N234A with the recombinant vector pET28a- Ch PPK was used as a template for overlap extension PCR to obtain the amino acid sequence shown in SEQ ID NO.4 in which the asparagine at position 234 was mutated to alanine. Ch PPK-N234A mutation vector; (3) Ch The PPK-N234A mutant vector was transformed into the host bacteria E. coli BL21 (DE3) was screened and obtained Ch PPK-N234A polyphosphate kinase mutant expression strain, induced expression, obtained Ch PPK-N234A polyphosphate kinase mutant.

6. A method for preparing a polyphosphate kinase mutant according to claim 1, characterized in that: The following steps are involved: (1) The coding gene shown in SEQ ID NO.1 was connected to the original expression vector pET-28a (+) to obtain the recombinant vector pET28a- Ch PPK, the recombinant vector pET28a- Ch PPK transformed host cells to obtain ch PPK recombinant strains; (2) Design the primers for site-directed mutagenesis of N234A with the recombinant vector pET28a- Ch PPK was used as a template for overlap extension PCR to obtain the amino acid sequence shown in SEQ ID NO.4 in which the asparagine at position 234 was mutated to alanine. Ch PPK-N234A mutation vector; (3) Design the K166D site-directed mutagenesis primers, using the primers in step (2) Ch The PPK-N234A mutant vector was used as a template for overlap extension PCR to obtain the amino acid sequence shown in SEQ ID NO.6, in which the 166th lysine was mutated to aspartic acid. Ch PPK -N234A / K166D mutant vector; (4) Ch The PPK-N234A / K166D mutant vector was transformed into the host bacteria E. coli BL21 (DE3) was screened and obtained Ch PPK-N234A / K166D polyphosphate kinase mutant expression strain, induced expression, obtained Ch PPK-N234A / K166D polyphosphate kinase mutant.

7. A use of the polyphosphate kinase mutant according to claim 1, characterized in that: The polyphosphate kinase mutant is used to construct an ATP regeneration system.

8. A use of the polyphosphate kinase mutant according to claim 1, characterized in that: The polyphosphate kinase mutant, the polyphosphate kinase mutant-expressing microorganism or the cell disrupted liquid thereof is used as an ATP regenerating agent to synthesize glutathione.

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