Mutant with improved S-adenosylmethionine synthetase catalytic activity and application thereof

Abstract

The invention discloses a mutant with improved catalytic activity of S-adenosylmethionine synthetase and application of the mutant, and belongs to the technical field of biological enzyme engineering. According to the present invention, the site 189, the site 234, the site 266 and the site 371 of the S-adenosylmethionine synthetase MAT are subjected to site-specific combinatorial mutation to obtain a series of mutants with improved enzyme activity, the I189V / V266H improvement is the most obvious, and the specific enzyme activity is 3.5833 + / -0.1373 U.mg <-1 >, which is improved by 1353.08% compared to the parent WT; and for a whole-cell catalysis system, the yield of S-ademetionine synthesized by the mutant I189V / V266H / Q234N / N371R reaches 1751.01 mg / L, and the increase is most obvious. The enzyme mutant provided by the invention has a wide application prospect in the production of S-adenosylmethionine and the construction of genetic engineering microorganisms of S-adenosylmethionine.

Description

Technical Field

[0001] The present invention relates to a mutant of S-adenosylmethionine synthetase with improved catalytic activity and application thereof, belonging to the technical field of bioenzyme engineering. Background Art

[0002] S-adenosylmethionine synthase (EC 2.5.1.6) is an enzyme responsible for the biosynthesis of S-adenosylmethionine from L-methionine and ATP. The entire process is a two-step reaction. First, L-methionine reacts with ATP to form S-adenosylmethionine (SAM) and tripolyphosphate (PPPi). PPPi is then asymmetrically hydrolyzed into pyrophosphate (PPi) and orthophosphate (Pi) before SAM is released. SAM plays a vital role in cellular metabolism. It is not only a methyl donor for the synthesis of DNA, RNA, proteins, phospholipids and fatty acids, but also a precursor molecule for transsulfuration and polyamine synthesis. In clinical practice, SAM has been successfully used to treat diseases such as Alzheimer's disease, liver disease, osteoarthritis and depression.

[0003] Currently, the common industrial methods for producing SAM are fermentation and enzymatic methods. Fermentation primarily involves yeast cell fermentation, where SAM accumulates after culturing in a medium supplemented with L-methionine. To further accumulate SAM, most research focuses on fermentation processes using Saccharomyces cerevisiae or Pichia pastoris strains. However, fermentation-based SAM production suffers from disadvantages such as long fermentation cycles, low conversion rates, and complex extraction processes. Enzymatic synthesis, while renowned for its high efficiency and simple extraction process, also suffers from low enzyme activity and high fermentation costs. Compared to these two methods, whole-cell catalytic methods offer unique advantages and promising applications in biopharmaceuticals, biotransformation, and other fields, thanks to their low catalytic cost and the elimination of traditional enzymatic steps such as cell lysis and protein concentration. This advantage is particularly evident when complex transformations involving coenzymes are involved. However, naturally occurring S-adenosylmethionine synthase (MAT) suffers from low catalytic activity and strong substrate inhibition, resulting in low yields for whole-cell catalytic SAM production.

[0004] Currently, MAT derived from Escherichia coli possesses high specific activity, and the crystal structures of MAT extracted from E. coli and MAT complexed with AMPPNP and L-methionine have been reported. This has led to a significant increase in recent research on MAT derived from E. coli. However, a major drawback of this enzyme is that product inhibition severely limits the synthesis of SAM. Some studies have required the addition of high concentrations of sodium p-toluenesulfonate to the reaction system to overcome product inhibition, but p-toluenesulfonic acid has low bioavailability as a drug for SAM. MAT encoded by the sam2 gene from Saccharomyces cerevisiae has been widely cloned and used because it lacks product inhibition. There is an urgent need to increase the activity of MAT encoded by the sam2 gene to enable the industrialization of SAM synthesis using whole-cell catalysis. Summary of the Invention

[0005] In view of the problems of low catalytic efficiency and poor stability of the synthetase in the current industrial production of S-adenosylmethionine, in order to overcome the defects of the existing technology, the purpose of the present invention is to provide a mutant of S-adenosylmethionine synthetase with improved activity, and to increase the yield of S-adenosylmethionine synthesized by its whole-cell catalytic method, which has universal significance for promoting the synthesis of S-adenosylmethionine.

[0006] The technical solution adopted in the present invention is as follows:

[0007] The S-adenosylmethionine synthase MAT encoded by the sam2 gene from Saccharomyces cerevisiae was cloned, and a protein model was established using the Swiss-Model. The protein structure was analyzed, and the protein was molecularly docked with the substrate. Sites were selected for site-directed mutagenesis through computer-assisted semi-rational design. The activity of the mutated enzyme was measured to identify sites with significant effects on enzyme activity. Combinatorial mutagenesis was then performed to superimpose the mutation advantages and obtain mutants with improved activity and stability compared to the wild type.

[0008] The present invention provides an S-adenosylmethionine synthetase mutant, which is based on the amino acid sequence shown in SEQ ID NO.1 and undergoes at least one of the following mutations:

[0009] (1) mutating valine at position 266 to histidine;

[0010] (2) mutating asparagine at position 371 to arginine;

[0011] (3) mutating isoleucine at position 189 to valine;

[0012] (4) Mutate glutamine at position 234 to asparagine.

[0013] In one embodiment of the present invention, the mutation is to mutate the valine at position 266 to histidine to obtain the mutant V266H.

[0014] In one embodiment of the present application, the mutation is that the valine at position 266 is mutated to histidine and the asparagine at position 371 is mutated to arginine, obtaining mutant V266H / N371R.

[0015] In one embodiment of the present application, the mutation is that the valine at position 266 is mutated to histidine and the isoleucine at position 189 is mutated to valine, obtaining mutant I189V / V266H.

[0016] In one embodiment of the present application, the mutation is that the valine at position 266 is mutated to histidine, the asparagine at position 371 is mutated to arginine and the glutamine at position 234 is mutated to asparagine, obtaining mutant V266H / Q234N / N371R.

[0017] In one embodiment of the present application, the mutation is that the valine at position 266 is mutated to histidine, the asparagine at position 371 is mutated to arginine, the isoleucine at position 189 is mutated to valine and the glutamine at position 234 is mutated to asparagine, obtaining mutant I189V / V266H / Q234N / N371R.

[0018] The present application also provides a gene encoding the mutant.

[0019] The present application also provides an expression vector carrying the gene.

[0020] The present application also provides a microbial cell expressing the mutant or carrying the expression vector.

[0021] The present application also provides a method for producing S-adenosyl methionine, using the mutant or the microbial cell as catalyst for the reaction.

[0022] In one embodiment of the present 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 12-24h, and the bacterial cells are collected; the bacterial cells are added to a reaction system containing a substrate for reaction for 2h.

[0023] In one embodiment of the present application, the recombinant monoclonal is inoculated into 10mL LB liquid medium containing kanamycin, and cultured at 37°C at 220rpm for 10-12h, 2% of the culture is then inoculated into new 50mL LB liquid medium containing kanamycin for culture for about 2-3h to obtain OD=0.6-0.8, and then filter-sterilized inducer IPTG is added to a final concentration of 0.1mM, followed by culture at 25°C at 220rpm for 14-24h, and the bacterial cells are collected by centrifugation.

[0024] In an embodiment of the present application, the substrate comprises reduced glutathione, L-methionine, adenosine-5'-triphosphate disodium salt, potassium chloride, magnesium chloride, Tris-HCl. The concentration of the substrate in the reaction system is 20-100 mM, and the OD 600 = (3.2) ± 0.2, and the reaction is carried out at 37°C and pH 8 ± 1.

[0025] The present application also provides a method for improving the catalytic activity of S-adenosyl methionine synthetase, which comprises at least one mutation in the amino acid sequence shown in SEQ ID NO. 1:

[0026] (1) mutating the valine at position 266 to histidine;

[0027] (2) mutating the asparagine at position 371 to arginine;

[0028] (3) mutating the isoleucine at position 189 to valine;

[0029] (4) mutating the glutamine at position 234 to asparagine.

[0030] The present application also provides the use of the mutant or the microbial cell in the preparation of S-adenosyl methionine or a product containing S-adenosyl methionine.

[0031] In an embodiment of the present application, the product comprises food, medicine or daily chemical product.

[0032] Advantages:

[0033] (1) Mutating one or more of the positions 189, 234, 266 and 371 of S-adenosyl methionine synthetase MAT, a series of mutants with improved enzyme activity are obtained:

[0034] Mutating the valine at position 266 to histidine (V266H), the specific enzyme activity is improved by 272.63% compared with the parent;

[0035] Mutating the valine at position 266 to histidine and mutating the asparagine at position 371 to arginine (V266H / N371R), the specific enzyme activity is improved by 1060.38 compared with the parent;

[0036] Mutating the valine at position 266 to histidine and mutating the isoleucine at position 189 to valine (I189V / V266H), the specific enzyme activity is improved by 1353.08% compared with the parent;

[0037] The specific enzyme activity of the strain that mutated valine at position 266 to histidine, asparagine at position 371 to arginine, and glutamine at position 234 to asparagine (V266H / Q234N / N371R) was 340.84% ​​higher than that of the parent strain.

[0038] The valine at position 266 was mutated to histidine, the asparagine at position 371 was mutated to arginine, the isoleucine at position 189 was mutated to valine, and the glutamine at position 234 was mutated to asparagine to obtain a mutant (I189V / V266H / Q234N / N371R), whose specific enzyme activity was increased by 294.57% compared with the parent.

[0039] Moreover, the enzyme activities of the single point mutations at positions 189, 234, 266, and 371 were all improved to varying degrees compared with the parent, indicating that the enzyme activity of the mutant was greatly improved.

[0040] (2) The catalytic performance was compared by whole-cell catalysis. After 2 h of catalysis, the concentration of S-adenosylmethionine synthesized by the parent (WT) E. coli BL21-pET28a-sam2 was 276.32 mg / L, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-V266H / N371R was 1087.26 mg / L, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / V266H was 550.57 mg / L, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-V266H / Q234N / N371R was 1572.81 mg / L, and the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / V266H was 550.57 mg / L. The concentration of S-adenosylmethionine synthesized by BL21-pET28a-sam2-I189V / V266H / Q234N / N371R was 1751.01 mg / L, which was significantly higher than that of the parent. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The relative enzyme activities of the mutants obtained by site-directed mutagenesis were set to 100% with the wild-type enzyme as the control;

[0042] Figure 2 Comparison of whole-cell catalysis between the wild-type enzyme and the mutant. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0044] The enzyme activities involved in the following examples are:

[0045] Definition of enzyme activity: The amount of enzyme required to catalyze the production of 1 μmol S-adenosylmethionine (SAM) per minute under these conditions is one enzyme activity unit (1U).

[0046] Definition of specific enzyme activity: the number of units of enzyme activity per unit weight (mg) of protein. The calculation formulas for enzyme activity and specific enzyme activity are as follows:

[0047]

[0048]

[0049] Where A SAM is the molar concentration of the product S-adenosylmethionine in the reaction solution, V is the volume of the reaction solution, t is the conversion time (min), and the unit of enzyme activity is U = μmol·min -1 In the following examples, crude enzyme activity refers to the activity per unit volume of crude enzyme solution, and the unit is U·mL -1 .

[0050] Example 1 Construction of mutants

[0051] 1. Construction of E. coli BL21 / pET-28a(+)-sam2 wild-type strain

[0052] The S-adenosylmethionine synthetase gene sam2, which was artificially synthesized by GENEWIZ (Suzhou Jinweizhi Biotechnology Co., Ltd.) from Saccharomyces cerevisiae and codon-optimized in Escherichia coli, has a nucleotide sequence as shown in SEQ ID NO.2 and an amino acid sequence encoded by the gene as shown in SEQ ID NO.1.

[0053] The target fragment sam2 was amplified using the primers in Table 1, and then ligated with the plasmid pET-28a double-digested with restriction endonucleases (EcoR I and Hind III) using the homologous recombinase ClonExpress II One Step Cloning Kit in a 37°C metal bath for 30 minutes to obtain the recombinant plasmid pET28a-sam2. The recombinant plasmid pET28a-sam2 was transformed into Escherichia coli BL21 (DE3) and spread on an LB plate containing Kan. After single colonies grew, transformants were picked for colony PCR verification. If the bands were consistent, they were sent to Tianlin Biotechnology (Shanghai) Co., Ltd. for sequencing to obtain recombinant E. coli BL21-pET28a-sam2 expressing wild-type S-adenosylmethionine synthetase.

[0054] Table 1 Primers used for construction of recombinant strains

[0055]

[0056] Note: The lowercase sequence is the recombinant sequence added to the 5' end

[0057] 2. Construction of mutant strains

[0058] (1) Screening of mutation sites

[0059] AlphaFold 2 was used to predict the three-dimensional model of the MAT protein and the model was evaluated. The substrate and protein were docked using AutoDockVina, and the optimal docking model was selected based on the docking analysis. The protein was visualized and analyzed using the software Pymol.

[0060] Single-site mutant construction:

[0061] Site-directed mutagenesis sites were introduced by inverse PCR, using the recombinant plasmid pET28a-sam2 obtained in Step 1 as a template for single-point mutagenesis to obtain a mutant plasmid. Primers are listed in Table 2. The methylated sites were then recognized and digested with Dpn I. The template was then transformed into E. coli BL21(DE3) and plated onto LB plates containing Kan. Single colonies were grown and confirmed by picking and sequencing to obtain recombinant E. coli expressing the mutant. Taking the construction of recombinant Escherichia coli expressing mutant I189V as an example, the recombinant plasmid pET28a-sam2 was used as a template and I189V-F and I189V-R were used as primers for reverse PCR to obtain the recombinant plasmid pET28a-sam2-I189V. The methylation site was then recognized by Dpn I enzyme and the template was digested with enzyme. The template was transformed into Escherichia coli BL21 (DE3) and spread on an LB plate containing Kan. After single colonies grew, the cells were picked and sequenced to obtain recombinant E. coli BL21-pET28a-sam2-I189V expressing mutant I189V.

[0062] Table 2 Primers used for site-directed mutagenesis

[0063]

[0064]

[0065] The PROSS method mainly combines conservation analysis and protein conformational free energy calculation to generate multiple mutations of the target protein. Therefore, highly expressed and stable mutants can be obtained without changing the function of the protein. Studies have shown that the use of PROSS can improve the thermal stability and catalytic activity of proteases. Based on this, this study used the PROSS strategy to calculate the conservation and conformational free energy of MAT. In addition, the European protein database PDBePISA can be used to analyze the interface residues of polymerases. In this way, key amino acid sites on the polymer enzyme interface can be identified, and the interactions between the polymer enzyme interfaces can be improved through mutations, thereby improving the performance of the enzyme. We selected mutation sites based on the semi-rational design prediction results, successfully constructed mutant strains, and conducted SAM whole-cell catalytic fermentation experiments to verify them.

[0066] Construction of multi-site mutants:

[0067] Taking the strain E. coli BL21-pET28a-sam2-I189V / V266H / Q234N / N371R as an example, firstly, using the plasmid pET28a-sam2-I189V as a template, primers V266H-F and V266H-R were selected for inverse PCR to construct the recombinant plasmid pET28a-sam2-I189V / V266H; then, using the plasmid pET28a-sam2-I189V / V266H as a template, primers Q234N-F and Q234N-R were selected for inverse PCR to construct the recombinant plasmid pET28a-sam2-I189V / V266H. 4N-R were used as primers to introduce site-directed mutation sites by inverse PCR to construct the recombinant plasmid pET28a-sam2-I189V / V266H / Q234N; then, using pET28a-sam2-I189V / V266H / Q234N as template, primers N371R-F and N371R-R were selected to perform inverse PCR to construct the recombinant plasmid pET28a-sam2-I189V / V266H / Q234N / N371R. The methylation site was recognized by enzyme I and the template was digested by enzyme I. The template was then transformed into Escherichia coli BL21 (DE3) and plated onto an LB plate containing Kan. Single colonies were grown and finally verified by picking and sequencing to obtain recombinant E. coli BL21-pET28a-sam2-I189V / V266H / Q234N / N371R expressing the mutant I189V / V266H / Q234N / N371R.

[0068] Mutant screening:

[0069] In order to screen mutants with enhanced enzyme activity, the enzyme activity of the unmutated strain E. coli BL21 / pET-28a-sam2 (WT) was used as a blank control, defined as 100%. The constructed mutants and WT were induced at 25°C for 24 hours, and their enzyme activities were measured. The culture conditions were as follows: the seed solution cultured at 37°C and 220 rpm for 12 hours was transferred to the fermentation medium at a 2% inoculum size, and cultured for about 1-2 hours until the OD 600 =0.6-0.8, add IPTG with a final concentration of 0.1mM, and culture at 25°C and 220rpm for 24h. At the end of the culture, the bacterial concentration in the fermentation liquid is controlled to be OD 600 About 3.3±0.3.

[0070] Take 2mL of fermentation liquid and heat it at 4℃ and 8000r·min -1 Centrifuge for 5 minutes, resuspend and wash the cells with 50mM Tris-HCl (pH 8.0) buffer, centrifuge for 5 minutes, and repeat the washing twice. Take 1mL of buffer to suspend the cells, perform ultrasonic disruption, and then resuspend the cells at 4℃ and 12000r·min. -1 After centrifugation for 10 minutes, the supernatant was the crude enzyme solution, and its crude enzyme activity was determined. The mutant crude enzyme was purified by nickel column affinity chromatography and its specific enzyme activity was determined under optimal conditions.

[0071] The enzyme activity unit (U) is defined as the amount of MAT enzyme required to produce 1 μmol SAM per minute, and the specific enzyme activity (U·mg -1 ) to measure. SAM enzyme activity assay system (based on final concentration): 100mM Tris-HCl (pH 8.0), 20mM L-Met, 8mM reduced glutathione, 50mM KCl, 20mM MgCl2, 20mM ATP. Add 400μL of crude enzyme solution to each 1mL reaction system. Incubate in a water bath at 37°C for 30min. Remove 500μL and mix with an equal volume of 20% perchloric acid solution to terminate the reaction. After an ice bath for 30min, rotate at 12000r / min. -1 After centrifugation for 10 min, the supernatant was collected and the SAM concentration was determined by HPLC.

[0072] S-adenosylmethionine content was determined by high-performance liquid chromatography (HPLC). The fermentation supernatant was filtered through a 0.22 μm aqueous membrane filter. The HPLC column used was a Diamonsil C18 (250 x 4.6 mm, 5 μm) column with a mobile phase of 82:18 (V (2 mM sodium heptanesulfonate and 40 mM ammonium dihydrogen phosphate solution): V (methanol). The pH was adjusted to 3.0 with phosphoric acid. The flow rate was 0.8 mL / min, the column temperature was 30°C, the UV detection wavelength was 254 nm, and the injection volume was 10 μL.

[0073] Under the same conditions, the relative enzyme activity of the mutant and the wild type was calculated with the wild type relative enzyme activity as 100%. Figure 1 and Table 3.

[0074] Table 3 Enzyme activities of mutants

[0075]

[0076] The results are as follows Figure 1 As shown in Table 3, the specific enzyme activity was 0.2466±0.0022 U·mg compared to the original -1 The specific enzyme activities of WT, mutants V266H / N371R, I189V / V266H, V266H / Q234N / N371R, and I189V / V266H / Q234N / N371R were all improved to varying degrees, among which I189V / V266H showed the most significant improvement, with a crude enzyme activity of 0.3247 U·mL -1 , which was increased by 1193.62% compared with the parent WT, and the specific enzyme activity was 3.5833±0.1373U·mg -1 , an increase of 1353.08% compared with the parent WT.

[0077] Example 2: Whole-cell catalytic synthesis of SAM

[0078] The wild-type E. coli BL21-pET28a-sam2 and mutant strains E. coli BL21-pET28a-sam2-V266H / N371R, E. coli BL21-pET28a-sam2-I189V / V266H, E. coli BL21-pET28a-sam2-V266H / Q234N / N371R, and E. coli BL21-pET28a-sam2-I189V / V266H / Q234N / N371R were streaked on plates for activation. Single colonies were picked and transferred into 10 mL LB liquid medium containing Kan, and cultured at 37°C and 220 rpm for 12 h. The inoculum was then transferred into 50 mL TB medium containing Kan with a 2% inoculum size and cultured at 37°C and 220 rpm for about 1.5-2.5 h. The biomass OD 600 =0.6-0.8, add IPTG to a final concentration of 0.1 mM, and culture at 25°C, 220 rpm for 24 h. Collect the cells, wash them twice with 50 mM Tris-HCl (pH 8.0) buffer, and perform whole-cell catalysis.

[0079] Whole cell catalytic system (based on final concentration): biomass OD 600=5±0.4, 100mM Tris-HCl Buffer (pH 8.0), 20mM L-Met, 50mM KCl, 20mM MgCl2, 20mM ATP, 220rpm, 37℃ for 2h, then add an equal volume of 20% perchloric acid to terminate the reaction, centrifuge and collect the supernatant, and use HPLC to detect the production of SAM. Specific results are shown in Figure 2 . Figure 2 The results showed that the catalytic performance was compared by whole-cell catalysis. After 2 h of catalysis, the concentration of S-adenosylmethionine synthesized by the parent (WT) was 276.32 mg / L, the concentration of S-adenosylmethionine synthesized by V266H / N371R was 1087.26 mg / L, the concentration of S-adenosylmethionine synthesized by I189V / V266H was 550.57 mg / L, the concentration of S-adenosylmethionine synthesized by V266H / Q234N / N371R was 1572.81 mg / L, and the concentration of S-adenosylmethionine synthesized by I189V / V266H / Q234N / N371R was 1751.01 mg / L, which was significantly improved compared with the parent. It is speculated that this is due to the increase in enzyme expression caused by the mutation.

[0080] In summary, the present invention heterologously expresses S-adenosylmethionine synthetase from Saccharomyces cerevisiae, constructs recombinant Escherichia coli, modifies the S-adenosylmethionine synthetase protein, and performs site-directed mutagenesis to improve enzyme activity. Through whole-cell catalysis, the mutant I189V / V266H / Q234N / N371R synthesizes S-adenosylmethionine at a yield of 1751.01 mg / L. This invention successfully constructs an exogenous expression pathway for S-adenosylmethionine, further improves enzyme activity, saves raw materials and costs, and improves economic benefits, laying the foundation for industrial production.

[0081] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A mutant of S-adenosylmethionine synthetase, characterized in that Based on the amino acid sequence shown in SEQ ID NO.1, at least one of the following mutations is performed: (1) mutating valine at position 266 to histidine; (2) mutating asparagine at position 371 to arginine; (3) mutating isoleucine at position 189 to valine; (4) Mutate glutamine at position 234 to asparagine.

2. The mutant according to claim 1, characterized in that The mutation includes any of the following: (1) mutating valine at position 266 to histidine; (2) mutating valine at position 266 to histidine and asparagine at position 371 to arginine; (3) mutating valine at position 266 to histidine and isoleucine at position 189 to valine; (4) mutating valine at position 266 to histidine, asparagine at position 371 to arginine, and glutamine at position 234 to asparagine; (5) The valine at position 266 was mutated to histidine, the asparagine at position 371 was mutated to arginine, the isoleucine at position 189 was mutated to valine, and the glutamine at position 234 was mutated to asparagine.

3. A gene encoding the mutant according to claim 1 or 2.

4. An expression vector carrying the gene according to claim 3.

5. A microbial cell expressing the mutant according to claim 1 or 2, or carrying the expression vector according to claim 4.

6. A method for producing S-adenosylmethionine, characterized in that: The reaction is carried out using the mutant according to claim 1 or 2, or the microbial cells according to claim 5 as a catalyst, and L-methionine, adenosine-5'-triphosphate disodium salt, sodium chloride, and magnesium chloride as substrates.

7. The method according to claim 6, wherein React at pH 7-9 and 37-60°C for at least 2 hours.

8. A method for improving the catalytic activity of S-adenosylmethionine synthetase, characterized in that: Based on the amino acid sequence shown in SEQ ID NO.1, any of the following mutations is performed: (1) mutating valine at position 266 to histidine; (2) mutating valine at position 266 to histidine and asparagine at position 371 to arginine; (3) mutating valine at position 266 to histidine and isoleucine at position 189 to valine; (4) mutating valine at position 266 to histidine, asparagine at position 371 to arginine, and glutamine at position 234 to asparagine; (5) The valine at position 266 was mutated to histidine, the asparagine at position 371 was mutated to arginine, the isoleucine at position 189 was mutated to valine, and the glutamine at position 234 was mutated to asparagine.

9. Use of the mutant according to claim 1 or 2, or the microbial cell according to claim 5, in the preparation of S-adenosylmethionine or a product containing S-adenosylmethionine.

10. The use according to claim 9, characterized in that The products include food, medicine or daily chemical products.

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