A glutathione synthetase mutant and its application in glutathione synthesis

Abstract

The application discloses a mutant of glutamate-cysteine synthetase and application of the mutant in synthesis of glutathione. Single-site mutation is carried out on amino acid residues at positions 150, 188, 235, 236, 241, 300, 301, 302 and 330 of wild-type glutamate-cysteine synthetase (Glutamate-cysteine ligase [EC:6.3.2.2]) from Citrobacter koseri (strain ATCC BAA-895), and mutant sites at positions 150, 236, 302 and 330 are screened. Mutants M1-M6 are obtained by combination mutation of the mutant sites screened above, and the enzyme activity of the mutant M4 with the highest activity is increased by 477% compared with that of the wild-type glutamate-cysteine synthetase. The mutant M4 is used to catalyze synthesis of glutathione, and the highest concentration reaches 23 g / L, which is increased by 4.2 times compared with the wild type, and the production cost is greatly reduced, so the mutant has important application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a glutamylcysteine ​​synthase mutant and its application in glutathione synthesis. Background Technology

[0002] The enzymatic synthesis of glutathione involves two ATP-intensive steps. First, γ-glutamylcysteine ​​synthase (γ-GCS or gshA, EC 6.3.2.2) catalyzes the synthesis of γ-glutamylcysteine ​​(γ-GC) from L-glutamate and L-cysteine. Then, glutathione synthase (GS or gshB, EC 6.3.2.3) catalyzes the synthesis of glutathione from L-glycine and γ-glutamylcysteine ​​(γ-GC). In most cells, these two enzymes are encoded by separate genes, gshA and gshB, respectively. gshA has been confirmed as the key rate-limiting enzyme in the GSH biosynthesis pathway. However, the catalytic activity of gshA is subject to feedback inhibition by the product glutathione (GSH) to prevent excessive accumulation of GSH in the body. Therefore, it is necessary to reduce the feedback inhibition of gshA by GSH, improve its enzyme activity and stability, further increase the concentration of product inhibition, and thus improve the yield of glutathione. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a glutamylcysteine ​​synthase mutant in order to overcome the shortcomings of the prior art.

[0004] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned glutamylcysteine ​​synthase mutant in the preparation of glutathione by enzyme catalysis.

[0005] To address the aforementioned technical problems, this invention provides a glutamylcysteine ​​synthase mutant.

[0006] The amino acid sequence of the glutamylcysteine ​​synthase mutant is obtained by mutating any one or more of the following sites in the wild-type glutamylcysteine ​​synthase: histidine at position 150, tyrosine at position 188, arginine at position 235, leucine at position 236, tyrosine at position 241, tyrosine at position 300, alanine at position 301, proline at position 302, and arginine at position 330.

[0007] Specifically, histidine at position 150 becomes serine, tyrosine at position 188 becomes glutamine, arginine at position 235 becomes aspartic acid, leucine at position 236 becomes glutamic acid, tyrosine at position 241 becomes glutamine, tyrosine at position 300 becomes glutamine, alanine at position 301 becomes leucine, proline at position 302 becomes glycine, and arginine at position 330 becomes aspartic acid.

[0008] In some embodiments, after performing single-site mutations on the above-mentioned mutation sites, the relative enzyme activity was calculated by measuring the enzyme activity of the mutants with each single-site mutation. It was found that the enzyme activity was increased after performing single mutations on histidine at position 150, leucine at position 236, proline at position 302, and arginine at position 330 (H150S, L236E, P302G, and R330D).

[0009] Furthermore, in some embodiments, the mutation sites obtained by the above single-site mutation screening are combined with mutations to obtain glutamylcysteine ​​synthase mutants M1-M6.

[0010] The amino acid sequences of the glutamylcysteine ​​synthase mutants M1-M6 are shown in SEQ ID NO.2-7, and the nucleotide sequences of the corresponding encoding genes are shown in SEQ ID NO.9-14.

[0011] In some embodiments, the enzyme activity of the mutants of the above combined mutations is determined, and their relative enzyme activity is calculated to obtain preferred glutamylcysteine ​​synthase mutants M1, M2, M4 and M6, more preferably glutamylcysteine ​​synthase mutant M4.

[0012] In this invention, the wild-type glutamylcysteine ​​synthase WT is derived from Citrobacter koseri (strain ATCC BAA-895), whose amino acid sequence is shown in SEQ ID NO.1, and the corresponding nucleotide sequence encoding the wild-type glutamylcysteine ​​synthase gene is shown in SEQ ID NO.8.

[0013] A recombinant expression vector containing the nucleotide sequence of the above-mentioned glutamylcysteine ​​synthase mutant encoding gene is also within the scope of protection of this invention.

[0014] A recombinant strain containing the nucleotide sequence of the above-mentioned glutamylcysteine ​​synthase mutant encoding gene or containing the above-mentioned recombinant expression vector is also within the scope of protection of this invention.

[0015] The application of the above-mentioned glutamylcysteine ​​synthase mutant in the catalytic reaction to prepare glutathione is also within the scope of protection of this invention.

[0016] The catalytic reaction described herein uses L-cysteine, L-glutamic acid, L-glycine, and adenosine triphosphate as substrates, magnesium ions as cofactors, and glutathione synthase mutant and glutathione synthase as catalytic enzymes to catalyze the synthesis of glutathione.

[0017] The catalytic reaction is carried out under the following conditions: the reaction temperature is maintained at 35-40℃, the reaction time is 8-12 hours, and the pH range is maintained at 7.0-8.0.

[0018] Preferably, the reaction conditions are: reaction temperature maintained at 37°C, reaction time 10 hours, and pH 7.5.

[0019] Beneficial Effects: This invention, through single and combined mutations at specific sites in the wild-type glutathione synthase sequence, screened and obtained several glutathione synthase mutants. These mutants reduce the feedback inhibition of catalytic activity by the product glutathione (GSH), resulting in a significant increase in activity compared to the wild-type glutathione synthase, with the highest activity increasing by 477%. Using the most active mutant M4, reduced glutathione was finally obtained by adding substrates such as L-glutamate, L-glycine, L-cysteine, ATP, and magnesium ions. This invention utilizes this high-activity mutant production process to produce glutathione, increasing product concentration and reducing production costs, thus possessing significant application value. Attached Figure Description

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0021] Figure 1 It is a recombinant expression vector containing the gene encoding glutamylcysteine ​​synthase (gshA).

[0022] Figure 2 It is the protein structure of glutamylcysteine ​​synthase derived from Citrobacter koseri.

[0023] Figure 3 It is the non-covalent interaction between the active pocket residues of gshA and GSH.

[0024] Figure 4 It is the result of the synthesis of glutathione catalyzed by wild-type and mutant M4. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. The embodiments will help to understand the present invention, but the scope of protection of the present invention is not limited to the following embodiments.

[0026] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0027] Example 1: Construction and expression of wild-type glutamylcysteine ​​synthase

[0028] Based on the encoding gene (Gene ID: 45137681) of glutamylcysteine ​​synthase from Citrobacter koseri (strain ATCC BAA-895) in the NCBI database, a gene fragment was synthesized by General Biotechnology (Anhui) Co., Ltd. The amino acid sequence is shown in SEQ ID NO.1 and the corresponding nucleotide sequence is shown in SEQ ID NO.8.

[0029] Based on the nucleotide sequence corresponding to the wild-type glutamylcysteine ​​synthase, upstream primer F (5'-CCGGAATTCATGATCCCGGACGTATCACAGGC-3') and downstream primer R (5'-CCGCTCGAGTCAGGCGTGCTTTTCCAGCCAC-3') were designed and synthesized, and PCR amplification was performed using the synthesized wild-type glutamylcysteine ​​synthase gshA gene fragment as a template.

[0030] The PCR reaction system included: 25 μL 2×PCR Buffer, 10 μL 2 mM dNTPs, 1.5 μL each of 10 pmol / μL primer F and primer R, 1 μL template DNA, 1 μL high-fidelity polymerase, and finally, sterile double-distilled water to a final volume of 50 μL. The PCR reaction parameters were: 94℃ melting for 2 minutes, 98℃ denaturation for 10 seconds, 60℃ annealing for 30 seconds, 68℃ extension for 2 minutes, 30 cycles, with a final 5 minutes at 68℃, and then storage at 4℃.

[0031] The amplified gshA gene fragment and pET-28a expression vector (Novagen) were digested with EcoRI and XhoI restriction endonucleases (purchased from TaKaRa). The purified gshA DNA fragment and linearized pET28a expression vector were separated and recovered by 1% agarose gel electrophoresis using a DNA fragment purification kit (purchased from TaKaRa). The purified DNA fragment was ligated into the digested linearized pET28a expression vector using the following ligation system: 4 μL purified DNA fragment, 6 μL linearized pET-28a expression vector, and 10 μL Solution I. The mixture was incubated at 16°C for 1.5 h to obtain the recombinant expression vector pET28a-gshA (e.g., pET28a-gshA). Figure 1 As shown in the figure, the recombinant expression vector was then transformed into Escherichia coli BL21(DE3) competent cells using the calcium chloride method to obtain recombinant Escherichia coli. After culturing at 37°C for 12 hours on LB plates (containing a final concentration of 50 mg / L Kan), single colonies were picked and cultured in LB medium at 37°C and 200 rpm (containing a final concentration of 50 mg / L Kan) for 12 hours before being sent for sequencing to determine the sequence.

[0032] Recombinant *E. coli* containing wild-type glutamylcysteine ​​synthase was cultured in LB medium at 37°C and 200 rpm (containing a final concentration of 50 mg / L Kan) for 12 hours, and then transferred to 1 L TB medium (final concentration of 50 mg / L Kan) at a 1% v / v inoculum and cultured. When OD... 600 When the OD value is 0.6-0.8, 0.2 mM IPTG is added for induction, and the mixture is incubated at 30℃ and 150 rpm for 16 h. When the OD value is measured... 600 If the bacterial count is 5 or higher, collect the bacterial cells by centrifugation.

[0033] Example 2: Site-directed mutagenesis of glutamylcysteine ​​synthase

[0034] Firstly, the crystal structure of wild-type glutamylcysteine ​​synthase protein from *Citrobacter koseri* (strain ATCC BAA-895) could not be found in the PDB database. Therefore, its protein crystal structure was predicted using the AlphaFold ProteinStructure Database, and the results are as follows: Figure 2 As shown. Autodock Vina software was used to perform molecular docking of the product glutathione with glutamylcysteine ​​synthase to determine its active pocket location. Then, Protein-Ligand Interaction Profiler was used to analyze the non-covalent interactions between the ligand and receptor, as shown. Figure 3 As shown, it was determined that receptor amino acid residues HIS150, ARG235, and ARG330 form salt-bridge interactions with the ligand; receptor amino acid residues TYR188, TYR241, TYR300, and ALA301 form hydrogen-bonding interactions with the ligand; and receptor amino acid residues LEU236 and PRO302 form hydrophobic interactions with the ligand. To reduce the feedback inhibition of glutathione on the catalytic activity of glutamylcysteine ​​synthase, nine key residues in its active pocket were site-directedly mutated to reduce the feedback inhibition of glutathione on glutamylcysteine ​​synthase, thereby improving its enzyme activity and stability, further increasing the concentration of product inhibition, and thus increasing the yield of glutathione.

[0035] The amino acid sequence of the glutamylcysteine ​​synthase mutant is obtained by mutating any one or more of the following sites in the wild-type glutamylcysteine ​​synthase WT: histidine at position 150, tyrosine at position 188, arginine at position 235, leucine at position 236, tyrosine at position 241, tyrosine at position 300, alanine at position 301, proline at position 302, and arginine at position 330.

[0036] Specifically, histidine at position 150 mutates into serine, tyrosine at position 188 mutates into glutamine, arginine at position 235 mutates into aspartic acid, leucine at position 236 mutates into glutamic acid, tyrosine at position 241 mutates into glutamine, tyrosine at position 300 mutates into glutamine, alanine at position 301 mutates into leucine, proline at position 302 mutates into glycine, and arginine at position 330 mutates into aspartic acid.

[0037] All mutations were obtained using whole-plasmid PCR, with pET28a-gshA constructed in Example 1 as the plasmid template, and mutation primers as shown in Table 1 were designed. The PCR reaction system included: 25 μL 2×PCR Buffer, 10 μL 2 mM dNTPs, 1.5 μL each of 10 pmol / μL primer F and primer R, 1 μL plasmid template, 1 μL high-fidelity polymerase, and finally, brought to a final volume of 50 μL with sterile double-distilled water. The PCR reaction parameters were: 94℃ melting for 2 minutes, 98℃ denaturation for 10 seconds, 60℃ annealing for 30 seconds, 68℃ extension for 7 minutes, for 30 cycles, with a final 5 minutes at 68℃, followed by storage at 4℃.

[0038] Table 1 Primer sequences corresponding to each mutation site in whole plasmid PCR

[0039]

[0040] After confirming the correct sequencing of the mutants of each single-point mutation of glutamylcysteine ​​synthase, the mutant cells were induced to express the mutants, following the same process as in Example 1 for inducing the expression of wild-type glutamylcysteine ​​synthase WT. Example 3: Enzyme activity detection of wild-type glutamylcysteine ​​synthase WT and its single-point mutation mutants.

[0041] Wild-type glutamylcysteine ​​synthase (WT) and its single-point mutant, collected from Examples 1 and 2 after induced expression, were taken from a -80℃ freezer. 2g of each was weighed into 50mL centrifuge tubes, washed twice with 0.1mol / L PBS (pH 7.5), centrifuged to collect the cells, and resuspended in 20mL of 0.1mol / L PBS (pH 7.5) to obtain a 100g / L bacterial suspension. The cells were then disrupted using an ultrasonic disruptor in an ice-water bath. The instrument parameters were set to 200W power, with a 3s sonication pause followed by a 7s pause, for approximately 40 minutes until the suspension became clear. After the disruption, the cells were centrifuged at 8000rpm for 10 minutes at 4℃. The supernatant in the centrifuge tube was the crude enzyme solution of wild-type glutamylcysteine ​​synthase (WT) and its single-point mutant.

[0042] Preparation of a standard reaction mixture (10 mL): The reaction system consisted of 80 mM L-cysteine, 85 mM L-glycine, 85 mM M L-glutamate, 180 mM ATP, and 150 mM magnesium chloride. After adjusting the pH to 7.5, 1.5 mL of 100 g / L crude glutamylcysteine ​​synthase solution and 0.5 mL of 100 g / L crude glutathione synthase solution were added at 37 °C. The mixture was allowed to stand for 5 min, then shaken to react. Samples were taken every 15 min and quenched by boiling in a water bath. Enzyme activity was defined as the amount of enzyme required to produce 1 μmol of glutathione per minute. Relative enzyme activity = enzyme activity of the single-point mutation mutant / enzyme activity of wild-type glutamylcysteine ​​synthase.

[0043] Table 2. Relative enzyme activities of wild-type glutamylcysteine ​​synthase (WT) and its single-point mutant.

[0044] strains Wild-type WT H150S Y188Q R235D L236E Enzyme activity (IU) 56 86 25 30 101 Relative enzyme activity 100％ 154％ 45％ 54％ 180％ strains Y241Q Y300Q A301L P302G R330D Enzyme activity (IU) 6 22 51 125 152 Relative enzyme activity 11％ 39％ 91％ 223％ 271％

[0045] As shown in Table 2, the results obtained by comparing the relative enzyme activities of mutants and wild-type strains show that the relative enzyme activities of Y188Q, R235D, Y241Q, Y300Q, and A301L are reduced. Mutations may lead to a decrease in the binding capacity of glutamylcysteine ​​synthase to the substrate cysteine, resulting in reduced enzyme activity. Therefore, H150S, L236E, P302G, and R330D, which have increased enzyme activity, were selected for combined mutations. Considering that too many combined mutations would lead to significant changes in the catalytic pocket of glutamylcysteine ​​synthase, and also to reduce the number of screening samples, pairwise combined mutations were selected to obtain mutants M1-M6.

[0046] Example 4: Detection of the activity of combined mutant glutamylcysteine ​​synthase

[0047] The construction and induction of expression of the various mutant combinations of glutamylcysteine ​​synthase M1-M6 were carried out in the same manner as in Example 2. The preparation methods of the crude enzyme solution and standard reaction mixture of the various mutant combinations of glutamylcysteine ​​synthase were the same as in Example 3.

[0048] The amino acid sequences of the glutamylcysteine ​​synthase mutants M1-M6 are shown in SEQ ID NO.2-7, and the nucleotide sequences of the corresponding encoding genes are shown in SEQ ID NO.9-14.

[0049] The enzyme activity detection results of each combination mutant M1-M6 of glutamylcysteine ​​synthase are shown in Table 3.

[0050] Table 3. Relative Enzyme Activities of Different Mutant Combinations of Glutamine Cysteine ​​Synthase

[0051]

[0052]

[0053] As shown in Table 3, M1, M2, M4, and M6 are the best combination of mutants, among which mutant M4 shows the most outstanding performance.

[0054] Example 5: Glutamine cysteine ​​synthase mutant M4 used for enzymatic preparation of reduced glutathione

[0055] Reaction system: In a 2L reaction system, 80mM L-cysteine, 85mM L-glycine, 85mM L-glutamate, 180mM ATP, and 150mM magnesium chloride were added, and the pH was adjusted to 7.5. Then, at 37℃, 15g / L crude glutamylcysteine ​​synthase M4 enzyme solution (with wild-type glutamylcysteine ​​synthase WT as a control) and 5g / L crude glutathione synthase enzyme solution were added. The reaction was allowed to proceed for 10 hours, with 500µl samples taken every hour. The reaction was terminated by boiling in a water bath for 3 minutes, and the glutathione concentration was determined. See [link to results] for details. Figure 4 According to Table 4, after 10 hours of reaction, the highest concentration of 23 g / L was reached when the reaction time was 5 hours, compared to 5.5 g / L for the wild type, representing a 4.2-fold increase.

[0056] Table 4. Results of glutathione synthesis catalyzed by wild-type and mutant M4.

[0057]

[0058] This invention provides a mutant glutamylcysteine ​​synthase and its application in glutathione synthesis, along with its concept and method. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A glutamylcysteine ​​synthase mutant, characterized in that, The amino acid sequence of the glutamylcysteine ​​synthase mutant is that the histidine at position 150 of the wild-type glutamylcysteine ​​is replaced with serine. Alternatively, leucine at position 236 may be replaced with glutamic acid; Alternatively, proline 302 is replaced by glycine; Alternatively, it can be obtained by converting arginine (number 330) into aspartic acid. The amino acid sequence of the wild-type glutamylcysteine ​​is shown in SEQ ID NO.

1.

2. A glutamylcysteine ​​synthase mutant, characterized in that, The amino acid sequence of the glutamylcysteine ​​synthase mutant is obtained by changing histidine at position 150 of wild-type glutamylcysteine ​​to serine, and leucine at position 236 to glutamic acid. Alternatively, it can be obtained by changing histidine at position 150 to serine and simultaneously changing proline at position 302 to glycine. Alternatively, it can be obtained by changing leucine at position 236 to glutamic acid, and simultaneously changing proline at position 302 to glycine; Alternatively, it can be obtained by converting arginine (number 330) into aspartic acid and simultaneously converting proline (number 302) into glycine. The amino acid sequence of the wild-type glutamylcysteine ​​is shown in SEQ ID NO.

1.

3. The glutamylcysteine ​​synthase mutant according to claim 1 or 2, characterized in that, The wild-type glutamylcysteine ​​synthase described above is derived from Citrobacter. Citrobacter koseri The nucleotide sequence of the gene encoding wild-type glutamylcysteine ​​synthase corresponding to its amino acid sequence is shown in SEQ ID NO.

8.

4. The encoding gene for the glutamylcysteine ​​synthase mutant as described in claim 1 or 2.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the encoding gene as described in claim 4.

6. A recombinant bacterial strain, characterized in that, It contains the coding gene as described in claim 4 or the recombinant expression vector as described in claim 5.

7. The application of the glutamylcysteine ​​synthase mutant according to any one of claims 1-3 in the catalytic reaction to prepare glutathione.

8. The application according to claim 7, characterized in that, The catalytic reaction uses L-cysteine, L-glutamic acid, L-glycine, and adenosine triphosphate as substrates, magnesium ions as cofactors, and glutathione synthase mutant and glutathione synthase as catalytic enzymes to catalyze the synthesis of glutathione.

9. The application according to claim 8, characterized in that, The catalytic reaction is carried out under the following conditions: the reaction temperature is maintained at 35-40℃, the reaction time is 8-12 hours, and the pH range is maintained at 7.0-8.0.

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