Glutathione bifunctional synthetase mutant with improved catalytic activity and application

Abstract

The invention discloses a glutathione bifunctional synthetase mutant with improved catalytic activity and application, and belongs to the field of gene engineering and fermentation engineering. A series of mutants with improved enzyme activity are obtained by carrying out site-specific combinatorial mutation on a site 61, a site 136, a site 485, a site 498 and a site 722 of the glutathione bifunctional synthetase GshFst, the specific enzyme activity reaches 13.58 U.mg <-1 >-27.34 U.mg <-1 >, the half-life period at 37 DEG C reaches 195-320 min, and the enzyme activity and the thermal stability are remarkably improved compared with those of a wild type. The enzyme mutant provided by the invention has a wide application prospect in the production of glutathione and the construction of genetically engineered microorganisms for producing glutathione.

Description

TECHNICAL FIELD

[0001] The present application relates to a glutathione bifunctional synthetase mutant with improved catalytic activity and application, and belongs to the field of genetic engineering and fermentation engineering. BACKGROUND

[0002] Glutathione (GSH) is a tripeptide substance containing γ-amide bond and sulfhydryl group, which widely exists in organisms and maintains the redox homeostasis and detoxification of cells. Glutathione is condensed by L-cysteine (L-Cys), L-glutamic acid (L-Glu) and glycine (Gly). Glutathione has two forms in organisms, reduced form and oxidized form, and the reduced form is the main active state and has wide application value. The physiological functions of glutathione mainly include antioxidant, detoxification, amino acid transport and free radical clearance in organisms, and it is widely used in medicine, food, cosmetics and breeding fields.

[0003] The biosynthesis of glutathione is a two-step enzyme-catalyzed reaction involving ATP, in which L-Glu and L-Cys are catalyzed by γ-glutamyl cysteine synthetase (γ-GCS) to generate γ-glutamyl cysteine (γ-Glu-Cys), and then γ-Glu-Cys is catalyzed by glutathione synthetase (GS) to generate glutathione, wherein the γ-GCS enzyme activity is inhibited by glutathione feedback. In recent years, a glutathione bifunctional synthetase (GshF) has been found in Streptococcus agalactiae, which can catalyze the two-step reaction of γ-GCS and GS, and there is no feedback inhibition. However, GshF has the problems of low catalytic efficiency and poor stability. Liu Xiaodong expressed GshF from Streptococcus agalactiae in Escherichia coli, established a high-throughput screening method based on anti-arsenic ion pressure, screened a mutant with 2.28-fold improved enzyme activity through directed evolution, coupled the acetic acid kinase gene ack from E. coli K-12 to construct an ATP regeneration system, and used GshFSA and ACK to produce glutathione. In a 1L reaction system, the yield of glutathione was 12.64g·L -1 .

[0004] The main production methods of glutathione are extraction, chemical synthesis, enzyme conversion and microbial fermentation. Due to the limited raw materials and low purity, the extraction method, the complex reaction process, the need for optical resolution of the product and the pollution of the environment, the enzyme method and the fermentation method are more studied. The enzyme method for synthesizing glutathione has the advantages of high specificity, rapid reaction and high purity, but needs cell crushing, purification and immobilization steps; the commonly used strains for microbial fermentation are Escherichia coli and yeast, and the production cost is low and the environment is green, but since glutathione is an intracellular product, the downstream glutathione purification cost is high. The whole cell catalysis method combines the advantages of enzyme method and fermentation method, and has high catalytic efficiency, mild reaction conditions, simple reaction liquid composition, easy downstream purification and reusable cells.

[0005] At present, GshF has been successfully expressed in Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Corynebacterium glutamicum. Li et al. expressed GshFst in E. coli JM109, and synthesized 11.05 g·L -1 Glutathione. Liu et al. expressed GshFsa in Corynebacterium glutamicum as a host, and strengthened the supply of precursor L-cysteine by modifying the metabolic pathway including increasing the L-cysteine biosynthesis flux, knocking out the L-cysteine degradation gene and deleting the transcription factor factor, etc. Under the condition of not adding L-cysteine and L-glutamic acid exogenously, 756 mg·L -1 Glutathione. Cui et al. [ By alkali mutation or deletion of plasmid pUC18 to reduce the combination of repressor and operator, a constitutive promoter is constructed, and GshFss is expressed, and 7.37 g·L -1 GSH.

[0006] The bifunctional enzyme GshF has been studied more in expression, and great progress has been made in the synthesis of glutathione, but the enzyme activity and stability are still a factor limiting its efficient production, and it is still of important economic value to screen glutathione synthetase enzyme with high performance and obtain mutant with good catalytic activity and stability. SUMMARY

[0007] In view of the problems of low catalytic efficiency and poor stability of the synthesis enzyme in the current enzyme method for producing glutathione, in order to overcome the defects existing in the prior art, the purpose of the present application is to provide a mutant of glutathione bifunctional synthesis enzyme with improved activity, and to improve the yield of glutathione synthesized by enzyme method, which has universal significance for promoting the synthesis of glutathione.

[0008] The technical scheme adopted by the present application is as follows:

[0009] The application clones a bifunctional enzyme GshF from Streptococcus thermophilus, establishes a protein model by AlaphFold 2, analyzes the protein structure, carries out molecular docking of the protein and the substrate, carries out site-directed mutagenesis on the selected site, determines the site which has an important influence on the enzyme activity, then carries out combined mutation, superimposes the mutation advantages, and obtains the mutant with improved activity and stability compared with the wild type.

[0010] The application provides a glutathione bifunctional synthetic enzyme mutant, on the basis of an amino acid sequence shown in SEQ ID NO. 1, any one of the following mutations is carried out:

[0011] (1) the threonine at the 485th position is mutated into valine, to obtain a mutant T485V;

[0012] (2) the phenylalanine at the 61st position is mutated into asparagine, and the threonine at the 485th position is mutated into valine, to obtain a mutant F61N / T485V;

[0013] (3) the phenylalanine at the 61st position is mutated into asparagine, the threonine at the 485th position is mutated into valine, and the leucine at the 136th position is mutated into lysine, to obtain a mutant F61N / T485V / L136K;

[0014] (4) the phenylalanine at the 61st position is mutated into asparagine, the leucine at the 136th position is mutated into lysine, the threonine at the 485th position is mutated into isoleucine, and the serine at the 722nd position is mutated into alanine, to obtain a mutant F61N / L136K / T485V / S722A;

[0015] (5) the phenylalanine at the 61st position is mutated into asparagine, the leucine at the 136th position is mutated into lysine, the threonine at the 485th position is mutated into isoleucine, the serine at the 722nd position is mutated into alanine, and the valine at the 498th position is mutated into cysteine, to obtain a mutant F61N / L136K / T485V / S722A / V498C.

[0016] In an embodiment, the wild-type glutathione bifunctional synthetic enzyme GshF is derived from Streptococcus thermophilus, and the nucleotide sequence encoding the glutathione bifunctional synthetic enzyme is shown in SEQ NO. 2.

[0017] The application also provides a gene encoding the mutant.

[0018] The application also provides an expression vector carrying the gene.

[0019] The application also provides a microbial cell expressing the mutant or carrying the expression vector.

[0020] The application also provides a genetically engineered bacterium expressing the mutant, with Escherichia coli BL21 (DE3) as a host and pET-28a (+) as a vector.

[0021] In one embodiment, the genetically engineered bacterium further knocks out genes ggt and pepT and overexpresses polyphosphate kinase PPK2; the nucleotide sequence of the gene ggt is shown in SEQ ID NO. 3, the nucleotide sequence of the gene pepT is shown in SEQ ID NO. 4, and the amino acid sequence of the polyphosphate kinase PPK2 is shown in SEQ ID NO. 5.

[0022] The application also provides a method for fermentatively producing glutathione by using the genetically engineered bacterium expressing the mutant.

[0023] In one embodiment of the application, a microbial cell expressing the mutant glutathione bifunctional synthetase is used to convert a substrate to produce glutathione, and the substrate is a combination of L-glutamic acid, L-cysteine and glycine.

[0024] In one embodiment of the application, the microbial cell is cultured in a culture system to OD 600 0.6-0.8, an inducer is added at 25°C for induction for 10-12 hours, and the bacterial cells are collected; the bacterial cells are added to a reaction system containing the substrate and reacted for 1-10 hours.

[0025] In one embodiment of the application, the recombinant monoclonal is inoculated into 10 mL of LB liquid medium containing kanamycin, cultured at 37°C at 220 rpm for 10-12 hours, 2% of the culture is transferred to a new 50 mL of LB liquid medium containing kanamycin, and cultured for about 2-3 hours to OD = 0.6-0.8, then a sterilized inducer IPTG is added to a final concentration of 0.2 mM, and then cultured at 25°C at 220 rpm for 10-12 hours, and the bacterial cells are collected by centrifugation.

[0026] In one embodiment of the application, the substrate concentration in the reaction system is 20-60 mM, and the strain OD 600 =(4-60)±0.5, and the reaction is carried out at pH 8±1 and 30-45°C.

[0027] The application also provides glutathione obtained by the method.

[0028] The application also provides application of the mutant, the expression vector, the microbial cell or the genetically engineered bacterium in the production of glutathione and products containing glutathione in the fields of food, cosmetics, and drug preparation.

[0029] Beneficial effects:

[0030] (1) The glutathione bifunctional synthetase GshFst was subjected to site-directed combined mutation at positions 61, 136, 485 and 722, and a series of mutants with improved enzyme activity were obtained, wherein the enzyme activities of T485V, F61N / T485V, F61N / T485V / L136K, F61N / L136K / T485V / S722A, F61N / L136K / T485V / S722A / V498C were 1.22 U·mL -1 , 1.45 U·mL -1 , 2.05 U·mL -1 , 3.03 U·mL -1 and 1.91 U·mL -1 , which were 75.22%, 109.63%, 196.66%, 339.14% and 177.09% higher than that of the wild type, respectively; the specific enzyme activities were 13.62 U·mg -1 , 13.58 U·mg -1 , 20.34 U·mg -1 , 27.34 U·mg -1 and 19.57 U·mg -1 , which were 1.11, 1.11, 2.16, 3.24 and 2.04 times higher than that of the wild type (6.44 U·mg -1 ), respectively; and the half-lives at 37°C were 285 min, 210 min, 195 min, 245 min and 320 min, which were 2.00, 1.21, 1.05, 1.58 and 2.37 times higher than that of the wild type, respectively, indicating that the enzyme activity of the mutant was greatly improved.

[0031] (2) The mutant F61N / L136K / T485V / S722A had the highest glutathione concentration of 5.33 g·L -1 after 4 h of cell catalysis, while the wild type had the highest glutathione concentration of 5.08 g·L -1 after 6 h of catalysis. The production intensity of the mutant was 2.01 g·(L·h) -1 after 2 h of catalysis, which was 28.84% higher than that of the wild type (1.56 g·(L·h) -1 ). By knocking out the glutathione degradation genes ggt and pepT and coupling with the polyphosphate kinase PPK2 from Corynebacterium glutamicum, the mutant synthesized glutathione at a concentration of 12.58 g·L -1 after 6 h of catalysis in a 5 L fermenter. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1Whole-cell catalytic synthesis of glutathione was achieved for wild-type and mutant F61N / L136K / T485V / S722A.

[0033] Figure 2 Construction of whole-cell catalytic system for mutant F61N / L136K / T485V / S722A and scale-up catalysis in a 5L fermenter: A shows the effect of knocking out the GSH degradation gene on whole-cell catalysis, with ECG00 being the strain without knockout, ECG01 being the strain with the ggt gene knocked out, ECG02 being the strain with the pepT gene knocked out, and ECG03 being the strain with both ggt and pepT genes knocked out; B shows the effect of introducing an ATP regeneration system on whole-cell catalysis, introducing polyphosphate kinase from Corynebacterium glutamicum into strains with double deletion of ggt and pepT, and adding different concentrations of ATP to promote the synthesis of glutathione in whole-cell catalysis; C shows the scale-up of the catalytic system for glutathione synthesis in a 5L fermenter. Detailed Implementation

[0034] The present invention aims to provide several mutants of bifunctional glutathione synthase and their applications in glutathione synthesis. For further detailed explanation, the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0035] Example 1: Determination and Construction of Site-Directed Mutation Sites

[0036] 1. Construction of wild-type GshF recombinant Escherichia coli

[0037] The glutathione bifunctional synthase gene gshFst, derived from Streptococcus thermophilus and with codon optimization for Escherichia coli, was artificially synthesized by GENEWIZ (Suzhou Genewiz Biotechnology Co., Ltd.). The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.1.

[0038] The target fragment gshFst was amplified using the primers in Table 1, and then ligated with the restriction endonuclease (Hind III) digested plasmid pET-28a using the ClonExpress II One Step Cloning Kit at 37°C for 30 min to obtain the recombinant plasmid pET28a-gshFst. The recombinant plasmid pET28a-gshFst was then transformed into *E. coli* BL21(DE3) and plated on LB agar plates containing Kans. After single colonies grew, transformants were picked for colony PCR verification. If the bands matched, the transformed bacteria were sent to Tianlin Biotechnology (Shanghai) Co., Ltd. for sequencing to obtain recombinant *E. coli* BL21-pET28a-gshFst expressing wild-type glutathione bifunctional synthase.

[0039] Table 1 primers used for construction of recombinant strains

[0040]

[0041] 2. Construction of mutant recombinant E. coli

[0042] The protein structure of GshFst was predicted using AlphaFold 2 and SWISS MODEL (https: / / swissmodel.expasy.org / ), and the model was evaluated. GshFst was subjected to molecular docking with substrates L-cysteine, L-glutamic acid and glycine using Autodock Vina, and potential hot spot residues were screened out by performing alanine scanning on points within the range HotSpot Wizard can predict mutation hot spots based on evolutionary data, so the hot spot residues of GshFst were predicted in combination with HotSpot Wizard, and after excluding highly conserved sites through multiple sequence alignment, GshFst was subjected to site-directed mutagenesis.

[0043] The site-directed mutagenesis site was introduced by reverse PCR, and the recombinant plasmid pET28a-gshFst constructed above was used as a template for single-point mutation. The primers are shown in Table 2. The reverse PCR amplification product was used to recognize the methylation site and digest the template with Dpn I enzyme, and the chemical transformation method was used to transform into E. coli BL21(DE3), which was coated on LB plates containing Kan, and incubated at 37°C, 220r·min -1 for 12h, and single colonies were picked for colony PCR verification. The agarose gel electrophoresis results were consistent with the theoretical values, and the transformants were sent to Tianlin Biotechnology Co., Ltd. for sequencing to obtain recombinant E. coli expressing mutants.

[0044] Table 2 primers used for site-directed mutagenesis

[0045]

[0046]

[0047] Note: lowercase bases are mutation sites

[0048] The transformants with correct sequencing results were picked and inoculated into 10mL LB small bottles containing antibiotics, and incubated at 37°C, 220r·min -1 for 12h, and inoculated into 50mL LB liquid medium containing antibiotics at an inoculation amount of 2%, and incubated at 37°C. When the OD 600= 0.6-0.8, then add IPTG to a final concentration of 0.2 mM, then 25°C, 220 r / min -1 Induce expression, induce for 12 h. Take 2 mL of fermentation broth, centrifuge at 8000 r / min at 4°C -1 Centrifuge for 5 min, resuspend the bacterial cells with 20 mM Tris-HCl (pH 8.0) buffer, centrifuge for 5 min, and repeat the washing twice. Take 1 mL of buffer to suspend the bacterial cells, and then perform ultrasonic disruption, followed by centrifugation at 12000 r / min at 4°C -1 Centrifuge for 10 min, and the supernatant is the crude enzyme solution, and its crude enzyme activity is determined.

[0049] The enzyme activity (U) of GshFst is defined as the amount of GshFst required to produce 1 μmol of GSH per minute, and is measured by volume-specific enzyme activity (U·mL -1 ). The enzyme activity reaction system (in terms of final concentration) is 40 mM L-glutamic acid, 20 mM L-cysteine, 40 mM glycine, 20 mM ATP, 20 mM magnesium chloride, 100 mM Tris-HCl buffer (pH 8.0), 100 μL of crude enzyme solution, 37°C water bath for 20 min, and then add an equal volume of 10% (w / v) trichloroacetic acid solution to terminate the reaction, ice bath for 30 min, and centrifuge at 12000 r / min at 4°C -1 Centrifuge for 10 min, and the supernatant is the crude enzyme solution, and its crude enzyme activity is determined.

[0050] The detection method of GSH is high-performance liquid chromatography, and the supernatant of the reaction solution (diluted by the corresponding multiple) is filtered with a 0.22 μm water filter. HPLC analysis: the mobile phase is V(0.01 M sodium heptanesulfonate and 0.05 M potassium dihydrogen phosphate solution):V(methanol)=95:5, and the pH is adjusted to 3.0 with phosphoric acid; the chromatographic column is Diamonsil C18 (250x 4.6 mm, 5 μm), the flow rate is 0.6 mL / min, the column temperature is 30°C, the ultraviolet detection wavelength is 210 nm, and the injection volume is 10 μL.

[0051] Under the same conditions, the activity of the wild-type crude enzyme solution is taken as 100%, and the relative enzyme activity of the mutants and the wild type is calculated, as shown in Table 3. The enzyme activity of the wild type is 0.69 U·mL -1 , and the enzyme activities of the mutants F61N, Y220A, T485V, T485Q, T485Q and T485A are 1.21, 0.88, 1.22, 0.81, 0.75 and 0.84 U·mL -1 , respectively, wherein compared with the wild type, the enzyme activities of the mutants F61N and T485V are increased by 75.15 and 75.22%, respectively.

[0052] Table 3 Enzyme activity of mutants

[0053]

[0054]

[0055] The T485V, F61N, L136K, V498C and S722A are combined and mutated in the order of the best to the worst effect. Taking the construction of the combined mutation F61N / L136K / T485V / S722A as an example, first, the mutant plasmid pET28a-gshFst F61N is constructed by taking the recombinant plasmid pET28a-gshFst as a template and taking F61N-F and F61N-R in Table 1 as primers, then the mutant plasmid pET28a-gshFst F61N is constructed by taking the plasmid pET28a-gshFst F61N / L136K as a template and taking L136K-F and L136K-R in Table 1 as primers, then the mutant plasmid pET28a-gshFst F61N / L136K is constructed by taking the plasmid pET28a-gshFst F61N / L136K / T485V as a template and taking T485V-F and T485V-R in Table 1 as primers, and the mutant plasmid pET28a-gshFst F61N / L136K / T485V / S722A is constructed by taking the plasmid pET28a-gshFst-F61N / L136K / T485V as a template and taking S722A-F and S722A-R in Table 1 as primers. The obtained mutant plasmid is transformed into E. coli BL21 (DE3) and verified, and the recombinant E. coli BL21-pET28a-gshFst F61N / L136K / T485V / S722A of the expression mutant L136K / V498C is obtained.

[0056] The recombinant E. coli BL21-pET28a-gshFst F61N / L136K / T485V / S722A is cultured according to the above method, the crude enzyme solution is collected and the enzyme activity is determined, as shown in Table 3, the enzyme activity of the mutant F61N / L136K / T485V / S722A is 3.03 U·mL -1 , which is 4.39 times that of the wild type.

[0057] Definition of specific enzyme activity: the number of enzyme activity units possessed by unit weight (mg) of GshF.

[0058] The calculation formula of specific enzyme activity is as follows:

[0059]

[0060] The wild type and mutant F61N, L136K, T485V, V498C, S722A, F61N / T485V, F61N / T485V / L136K, F61N / L136K / T485V / S722A and F61N / L136K / T485V / S722A / V498C were purified by nickel column affinity chromatography respectively, and their specific enzyme activities and half-lives were determined under optimal conditions. The specific enzyme activity of the combined mutant F61N / L136K / T485V / S722A / V498C was 27.34 U·mg -1 , which was 3.24 times higher than that of the wild type (6.44 U·mg -1 ). The half-life at 37°C was 245 min, which was 1.58 times higher than that of the wild type (95 min). See Table 4 for details.

[0061] Table 4 Specific enzyme activity and half-life of mutants

[0062]

[0063] Example 2: Whole-cell catalysis of glutathione bifunctional synthetase mutants for synthesis of glutathione

[0064] Whole-cell catalysis combines the advantages of enzymatic method and microbial fermentation method, such as no need for separation and purification of enzymes, reuse of cells and relatively short catalytic cycle, and thus has attracted widespread attention. The catalytic ability of wild type and mutant F61N / L136K / T485V / S722A was compared by whole-cell catalysis.

[0065] The wild type E. coli BL21-pET28a-gshFst and mutant strain E. coli BL21-pET28a-gshFst F61N / L136K / T485V / S722A were activated by plate streaking, and a single colony was picked and transferred to 10 mL LB liquid medium containing Kan, which was cultured at 37°C and 220 rpm for 11 h. Then, 2% of the culture was transferred to 50 mL SB medium (yeast extract 20 g·L -1 , peptone 32 g·L -1 , sodium chloride 5 g·L -1 ) containing Kan, which was cultured at 37°C and 220 rpm for about 1.5-2.5 h. When the biomass OD 600 was 0.8, sterile IPTG was added to a final concentration of 0.2 mM, and the culture was incubated at 25°C and 220 rpm for 11 h. The bacterial cells were collected and washed twice with 20 mM Tris-HCl (pH 8.0) buffer, and then subjected to whole-cell catalysis.

[0066] Whole-cell catalysis reaction system: the amount of bacterial cells was controlled at OD 600= 30, 40 mM L-glutamic acid, 20 mM L-cysteine, 40 mM glycine, 20 mM ATP, 20 mM magnesium chloride, 100 mM Tris-HCl buffer (pH 8.0), 220 r·min -1 , 37℃ shaking bed reaction, timed sampling, 4℃ 12000 r·min -1 centrifugation for 10 min, and the supernatant was diluted to the corresponding multiple to determine the yield of GSH.

[0067] The results are shown in Figure 1 , the mutant catalyzed 4h, the concentration of glutathione was 5.33 g·L -1 , and the wild type catalyzed 4h, the concentration of glutathione was 4.53 g·L -1 , the glutathione yield of the mutant was 1.18 times that of the wild type; catalyzed 6h, the highest concentration of glutathione of the wild type was 5.08 g·L -1 , the production intensity of the mutant was 2.01 g·(L·h) -1 , which was 28.84% higher than that of the wild type (1.56 g·(L·h) -1 ).

[0068] Example 3: Full-cell catalytic synthesis of glutathione in a 5L fermenter

[0069] According to the literature, with the extension of the catalytic or fermentation time, the degradation of glutathione is shown, which often leads to the decrease of glutathione yield, and the key enzymes related to the degradation of glutathione include γ-glutamyl transpeptidase encoded by ggt and tripeptidase encoded by pepT. The synthesis of glutathione needs ATP to provide energy, and the energy can be provided by glycolysis pathway in Saccharomyces cerevisiae as a chassis cell, while the synthesis of glutathione in Escherichia coli usually needs exogenous addition of ATP to provide energy for catalysis. However, direct addition of ATP is not conducive to industrial production, which greatly increases the production cost. In order to improve the synthesis yield of glutathione and reduce the addition amount of exogenous ATP, the GSH degradation genes ggt and pepT were knocked out by CRISPR / Cas9 gene editing technology, and the polyphosphate kinase PPK2 from Corynebacterium glutamicum was expressed to construct ATP regeneration pathway.

[0070] The knockout process is as follows:

[0071] 1) Construct the knockout frame: search the E. coli BL21 genome sequence on the NCBI database to select the fragments of about 500 bp around the upstream and downstream of the genes ggt and pepT to be knocked out for fusion PCR to construct the knockout frame.

[0072] 2) Construct the target plasmid: predict http: / / crispr.dfci.harvard.edu / SSC / Suitable target points are mainly the first 20 bp bases of NGG in the gene sequence to be knocked out as the recognition site of sgRNA, and primers are designed for reverse PCR to replace and construct a targeted plasmid.

[0073] 3) Knockout gene: pCas plasmid with cutting function is introduced into E. coli BL21, and after ensuring successful introduction, the method of 2.4.1(1) is used to prepare electrotransformation competent cells. The knockout frame and the targeted plasmid are electrotransformed into the competent cells, which are cultured at 30°C for 24h, and single colonies are picked for colony PCR verification. Positive strains are screened according to the band size of electrophoresis.

[0074] 4) Loss of plasmid: the strain with successful knockout verification is picked into 10mL LB liquid medium, IPTG is added, and it is cultured at 30°C for 10h to eliminate the targeted plasmid. After successful loss of the targeted plasmid in the double-antibiotic medium, the pCas plasmid is lost again by culturing in 10mL LB liquid at 42°C for 10h, and then it is verified in 10mL LB liquid medium containing Kan.

[0075] 5) Knockout verification: single colonies after loss of pCas and pTarget plasmids are picked, and colony PCR verification is performed using verification primers to determine whether ggt and pepT are successfully knocked out. The recombinant strain with successful knockout is named BL21-Δggt&ΔpepT.

[0076] Table 5 primers for knockout

[0077]

[0078] The expression of PPK is as follows:

[0079] The genome of Corynebacterium glutamicum is extracted according to the instructions of the Novagen gene extraction kit, and the extracted genome is used as a template to amplify the target gene polyphosphate kinase PPK2. The plasmid pET-28a-gshFst is digested with restriction endonuclease Xho I F61N / L136K / T485V / S722A , and the ppk2 fragment and the plasmid are connected by homologous recombination enzyme, and the chemical transformation method is used to transform into competent BL21-Δggt&ΔpepT. Single colonies are picked for colony PCR verification, and sent for testing to construct the recombinant strain BL21-Δggt&ΔpepT-pET-28a-ppk2-gshFst F61N / L136K / T485V / S722A .

[0080] Table 6 primers for expressing PPK

[0081]

[0082] The mutant F61N / L136K / T485V / S722A was scaled up in a 5L fermentor to catalyze the synthesis of GSH. The suitable catalytic conditions were as follows: the cell was treated by freezing and thawing once, the reaction temperature was 37°C, the Tris-HCl buffer had a pH of 8.5 and a concentration of 100mM, the cell amount was OD 600 =40, the initial concentration of L-cysteine and ATP was 25mM, and the ATP and L-cysteine were supplemented twice (the final concentration of ATP and L-cysteine was 15mM at 1h and 3h, respectively), and the concentration of GSH synthesized was 12.58g·L -1 after 6h of catalysis, and the conversion rate of L-cysteine was 74.51%. The results are shown in Table 1. Figure 2 .

[0083] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and the protection scope of the present application should be defined by the claims.

Claims

1. A mutant of glutathione difunctional synthetase characterized in that, Based on the amino acid sequence shown in SEQ ID NO. 1, any one of the following mutations is made: (1) the threonine at position 485 is mutated into valine; (2) the phenylalanine at position 61 is mutated into asparagine, and the threonine at position 485 is mutated into valine; (3) the phenylalanine at position 61 is mutated into asparagine, the threonine at position 485 is mutated into valine, and the leucine at position 136 is mutated into lysine; (4) the phenylalanine at position 61 is mutated into asparagine, the leucine at position 136 is mutated into lysine, the threonine at position 485 is mutated into isoleucine, and the serine at position 722 is mutated into alanine; (5) the phenylalanine at position 61 is mutated into asparagine, the leucine at position 136 is mutated into lysine, the threonine at position 485 is mutated into isoleucine, the serine at position 722 is mutated into alanine, and the valine at position 498 is mutated into cysteine.

2. A gene encoding the mutant of claim 1.

3. An expression vector carrying the gene of claim 2.

4. A microbial cell expressing the mutant of claim 1, or carrying the expression vector of claim 3.

5. A genetically engineered bacterium expressing the mutant of claim 1, characterized in that, Escherichia coli BL21 (DE3) is used as the host, and pET-28a (+) is used as the vector.

6. The genetically engineered bacteria as described in claim 5, characterized in that, The genetically engineered bacteria also knock out genes ggt and pepT, and overexpress polyphosphate kinase PPK2; the nucleotide sequence of the gene ggt is shown in SEQ ID NO. 3, the nucleotide sequence of the gene pepT is shown in SEQ ID NO. 4, and the amino acid sequence of the polyphosphate kinase PPK2 is shown in SEQ ID NO.

5.

7. A method for producing glutathione, characterized by, The mutant of claim 1, the microbial cell of claim 4, or the genetically engineered bacteria of claim 5 or 6 is used as the catalyst in a reaction system containing L-glutamic acid, L-cysteine and glycine.

8. The method of claim 7, wherein, The addition amount of the microbial cells or genetically engineered bacteria in the reaction system is OD 600 =(4-60).

9. The method of claim 8, wherein, The reaction is carried out at pH 7-9 and 30-45℃.

10. Use of the mutant of claim 1, the expression vector of claim 3, the microbial cell of claim 4, or the genetically engineered bacteria of claim 5 or 6 in the production of glutathione and products containing glutathione in the fields of food, medicine and cosmetics.

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