A method for producing S-adenosylmethionine by microbial transformation of inexpensive substrates

Abstract

The present invention relates to a method for producing S-adenosylmethionine by microbial transformation of cheap substrates, belonging to the field of industrial microorganisms. In the present invention, Escherichia coli BL21(DE3) is used as the chassis cell, and the S-adenosylmethionine synthase (MAT) is modified through rational design to obtain the double mutant MAT<supgt;I195V / S150A< / supgt;. Its specific enzyme activity is increased by 176% relative to the wild strain. At the same time, by introducing an ATP regeneration pathway, efficient intracellular ATP supply is achieved, a recombinant Escherichia coli strain producing SAM is constructed, and its whole-cell transformation is carried out, so that the final SAM yield reaches 13.6 g / L, achieving the purpose of efficiently producing SAM from cheap substrates, and having important industrial application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of industrial microorganisms, and particularly to a method for producing S-adenosylmethionine by microbial conversion of inexpensive substrates. Background Art

[0002] S-adenosylmethionine (SAM) is an essential metabolite and methyl donor in all organisms, participating in a variety of important biochemical reactions, including transmethylation, transsulfuration, and transaminopropyl action. SAM not only has important biological functions in organisms but also has wide application values in the fields of medicine and bioengineering, and can be used for the treatment of various diseases, including cardiovascular diseases, liver diseases, nervous system diseases, depression, etc.

[0003] Methionine adenosyltransferase (MAT, EC 2.5.1.6) is the key enzyme for catalyzing the synthesis of SAM. Under the catalysis of SAM synthetase (MAT), the substrate L-methionine and ATP react to generate the product SAM. Usually, metK is the gene that expresses MAT in most organisms, and its expression is regulated together with the genes in the methionine biosynthesis pathway. MATs derived from Escherichia coli and yeast have advantages such as high substrate affinity, short reaction time, high substrate conversion rate, and high final product concentration in the enzymatic synthesis of SAM.

[0004] Currently, the production methods of SAM mainly include chemical synthesis, enzymatic conversion, and microbial conversion. Among them, the chemical method has basically been no longer used due to problems such as complex reactions, harsh conditions, low yields, difficult separation, and environmental pollution. The main challenges faced by the enzymatic catalysis method include the complex purification process of SAM synthetase, it is difficult to obtain a large amount of pure enzyme in batches, and the high cost of the precursor ATP. Therefore, the enzymatic synthesis of SAM is currently mainly used for the research on enzymatic properties and catalytic mechanisms, and there are still some limitations in large-scale industrial production. The microbial method for producing SAM is the most commonly used method at present. Generally, yeast cells are used as hosts for fermentation production. Although using yeast as a fermentation strain has a relatively high yield in SAM production, since SAM mainly exists inside the cells, the cell wall needs to be broken first during the extraction and purification process. In addition, the complexity of the components inside yeast cells also increases the difficulty of separating and purifying SAM, resulting in a low purification rate of SAM.

[0005] S-adenosylmethionine (SAM), as an important bioactive molecule, plays an important role in life activities. However, due to the limitations of the high cost and low-efficiency synthesis methods of SAM, its widespread use in industrial applications has been restricted. The currently widely used MAT has problems of low catalytic activity and low substrate conversion rate, resulting in difficulty in achieving high yields in SAM production. Using ATP as a substrate in the enzymatic synthesis of SAM also leads to the problem of excessively high production costs. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a method for producing S-adenosylmethionine by microbial transformation of cheap substrates. The recombinant Escherichia coli engineering bacteria of the present invention overexpress S-adenosylmethionine synthase derived from Enterococcus faecalis in Escherichia coli, and its specific enzyme activity can reach 4.8 U / mg, and a double mutant MAT with improved specific enzyme activity is obtained through rational design. I195V / S150A Its specific enzyme activity can reach 12.9 U / mg, which is 1.7 times higher than that of the wild type. At the same time, to reduce production costs, an ATP regeneration system is constructed, and adenosine kinase (Adenosine kinase, AK, EC2.7.1.20) and polyphosphate kinase (ATP-polyphosphate phosphotransferase, PPK, EC2.7.4.1) are overexpressed in recombinant Escherichia coli for ATP synthesis.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide a mutant of S-adenosylmethionine synthase, wherein the mutant of S-adenosylmethionine synthase mutates serine at position 150 and / or isoleucine at position 195 of the S-adenosylmethionine synthase with the amino acid sequence shown in SEQ ID NO.1.

[0009] In one embodiment of the present invention, the S-adenosylmethionine synthase is one of the following mutants:

[0010] Serine at position 150 of the S-adenosylmethionine synthase with the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine, and the mutated amino acid sequence is shown in SEQ ID NO.2;

[0011] Isoleucine at position 195 of the S-adenosylmethionine synthase with the amino acid sequence shown in SEQ ID NO.1 is mutated to valine, and the mutated amino acid sequence is shown in SEQ ID NO.3;

[0012] The serine at position 150 of S-adenosylmethionine synthase with the amino acid sequence as shown in SEQ ID NO.1 was mutated to alanine; and the isoleucine at position 195 was mutated to valine; the mutated amino acid sequence is as shown in SEQ ID NO.4.

[0013] The second object of the present invention is to provide a gene encoding the S-adenosylmethionine synthase mutant.

[0014] The third object of the present invention is to provide an expression vector carrying the gene.

[0015] The fourth object of the present invention is to provide a recombinant bacterium expressing the S-adenosylmethionine synthase mutant.

[0016] In one embodiment of the present invention, the recombinant bacterium also expresses adenosine kinase and polyphosphate kinase.

[0017] In one embodiment of the present invention, the recombinant bacterium uses Escherichia coli E.coli BL21(DE3) as the host and pET28a plasmid and pACYC plasmid as expression vectors.

[0018] In one embodiment of the present invention, BL21(DE3) is used as the host.

[0019] In one embodiment of the present invention, the MAT is derived from Enterococcus faecalis.

[0020] In one embodiment of the present invention, the AK is derived from Arabidopsis thaliana; the nucleotide sequence is as shown in SEQ ID NO.6, and the amino acid sequence is as shown in SEQ ID NO.7.

[0021] In one embodiment of the present invention, the PPK is derived from Cytophaga hutchinsonii; the nucleotide sequence is as shown in SEQ ID NO.6, and the amino acid sequence is as shown in SEQ ID NO.7.

[0022] In one embodiment of the present invention, the pET28a plasmid is used as the expression vector to express MAT.

[0023] In one embodiment of the present invention, the pET28a plasmid is used as the expression vector to express MAT I195V / S150A 。

[0024] In one embodiment of the present invention, the pACYC plasmid is used as the expression vector to express AK and PPK.

[0025] The fifth object of the present invention is to provide the use of the S-adenosylmethionine synthase mutant or the recombinant bacterium in the production of S-adenosylmethionine.

[0026] In one embodiment of the present invention, in the said use, the S-adenosylmethionine synthase mutant, adenosine kinase and polyphosphate kinase are used as catalysts, and a buffer solution containing 5 - 10 g / L methionine, 5 - 10 g / L adenosine, 10 - 30 g / L sodium hexametaphosphate, 10 - 30 mM magnesium chloride, and 100 - 200 mM potassium chloride is used as the reaction system to catalytically produce S-adenosylmethionine;

[0027] Or, in the said use, the cell lysate of the recombinant bacterium is used as the catalyst, and a buffer solution containing 5 - 10 g / L methionine, 5 - 10 g / L adenosine, 10 - 30 g / L sodium hexametaphosphate, 10 - 30 mM magnesium chloride, and 100 - 200 mM potassium chloride is used as the reaction system to catalytically produce S-adenosylmethionine.

[0028] In one embodiment of the present invention, in the said cell lysate, the cell density OD 600 is 5 - 20; and the buffer solution is 50 - 200 mM Tris-HCl buffer solution.

[0029] The present invention uses the above-mentioned recombinant bacterium for whole-cell catalytic production of SAM. To achieve this purpose, in a basic implementation scheme, the present invention provides a method for preparing SAM using the aforementioned recombinant Escherichia coli engineering bacterium, and the said method includes the following steps:

[0030] (1) Activate and culture and induce the said recombinant Escherichia coli;

[0031] (2) After induction, centrifuge to collect the bacterial cells and resuspend the bacterial cells with a buffer solution;

[0032] (3) Add the substrate to the bacterial cell resuspension for reaction to prepare SAM.

[0033] In one embodiment of the present invention, in step (1), the recombinant bacterium is inoculated into an LB medium and cultured at 37 °C and 220 r / min for 12 h.

[0034] In one embodiment of the present invention, in step (1), 1 mL of the above-mentioned medium is transferred to 50 mL of an LB liquid medium. After culturing at 37 °C and 220 r / min for about 2 h, IPTG with a final concentration of 0.2 - 0.8 mM is added, and then cultured at 16 °C and 220 r / min for 12 h.

[0035] In one embodiment of the present invention, in step (2), the above-mentioned medium is centrifuged to remove the supernatant, and the obtained cell precipitate is suspended with a PBS buffer solution.

[0036] In one embodiment of the present invention, in step (3), 5 g / L adenosine, 7.5 g / L methionine, 20 g / L sodium hexametaphosphate, 20 mM magnesium chloride, 150 mM potassium chloride, and 0.1% Triton are added to the Tris-HCl buffer, and the bacterial suspension with an OD 600 of 10.

[0037] Advantages of the present invention:

[0038] The present invention provides a method for producing S-adenosylmethionine by microbial conversion of inexpensive substrates. Using Escherichia coli BL21(DE3) as the chassis cell, the S-adenosylmethionine synthase (MAT) is modified through rational design to obtain mutants. Its specific enzyme activity is increased by up to 176% compared to the wild strain, laying a foundation for improving the SAM yield.

[0039] Meanwhile, in the present invention, the S-adenosylmethionine synthase MAT is overexpressed in Escherichia coli, and the mutant MAT obtained through rational design I195V / S150A , lays a foundation for improving the SAM yield. An ATP regeneration system is constructed to achieve efficient intracellular ATP supply, promote SAM production, and produce SAM through whole-cell catalytic conversion. Finally, the SAM yield reaches 13.6 g / L, having important industrial application prospects. Description of the Drawings

[0040] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings, where

[0041] Figure 1 is the verification result of colony PCR of the recombinant plasmid pET28a-MAT construction in the present invention;

[0042] Figure 2 is the verification result of colony PCR of the recombinant plasmid pACYC-AK-PPK construction in the present invention;

[0043] Figure 3 is the schematic diagram of SAM production by the recombinant strain BL21 / pET28a-MAT-I195V / S150A

[0044] -pACYC-AK-PPK in the present invention;

[0045] Figure 4 is the SAM production yield and molar conversion rate of the recombinant bacterium BL21 / pET28a-MAT-I195V / S150A

[0046] -pACYC-AK-PPK in the present invention. Detailed implementation manners

[0047] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0048] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0049] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0050] In the quantitative experiments in the following embodiments, three repeated experiments are set, and the results are averaged.

[0051] The host used for constructing the recombinant plasmid in the following embodiments is Escherichia coli E.coli BL21(DE3), which is purchased from Benna Biology, and the pET28 plasmid and pACYC plasmid are both purchased from the BioVector Plasmid Vector Strain Cell Gene Preservation Center.

[0052] The preparation of Escherichia coli competent cells and the chemical transformation method involved in the following embodiments are as follows:

[0053] Prepare Escherichia coli chemically competent cells using the Competent Cell Preparation Kit from TaKaRa. The detailed operation refers to the instruction manual. Transform into E.coli BL21 by heat shock at 42°C, and obtain positive transformants through screening on antibiotic-resistant plates. Extract the plasmid for PCR verification and send it to Genewiz for sequencing verification.

[0054] The method for extracting the relevant plasmids involved in the following embodiments:

[0055] When extracting the plasmid from the recombinant strain of Escherichia coli, centrifuge the bacterial solution with an appropriate concentration and remove the supernatant, and then extract it using the Jierui Mini Plasmid Extraction Kit. The detailed operation refers to the instruction manual.

[0056] The amplification system of PCR in the following embodiments is: Primer F 1.0 μL, Primer R 1.0 μL, Template 1.0 μL, PhantaR Max(p515) DNA polymerases 25 μL, Nuclease-free water 22 μL.

[0057] The PCR amplification program in the following examples is as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; the annealing temperature is generally set at 58 - 60°C for 30 - 60 s; the extension at 72°C is set according to the time for amplifying 1500 bp gene per minute; the denaturation to extension program is carried out for 30 cycles; then extension at 72°C for 5 min; and preservation at 4°C.

[0058] The method for inducing expression of the recombinant strain in the following examples is as follows:

[0059] Pre-culture the recombinant Escherichia coli strain containing the target gene on a medium containing the corresponding resistance to make the strain reach a suitable growth state. During the culture process, take about 1 mL of the bacterial suspension and measure the wavelength using an ultraviolet spectrophotometer. When the OD of the bacterial solution 600 reaches 0.6 - 0.8, immediately add isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mmol / L and induce overnight at 16°C to express the protein. After induction, centrifuge at 4°C and 8000 r / min for 10 min to collect the cells. Suspend the collected cell precipitate with PBS buffer (0.1 mmol / L, pH 7.4), and centrifuge again to collect the cell precipitate. Repeat the operation three times to completely remove the medium to avoid the medium components in the enzyme solution affecting the subsequent experiments. Resuspend the cell precipitate in PBS buffer again, and use an ultrasonic cell disruptor to break the cells. The program is set to break for 3 s and stop for 2 s each time, and the total program time is set to 15 min until the bacterial solution becomes clear. Centrifuge the cell lysate at 4°C and 12000 r / min for 20 min, and the supernatant is the crude enzyme solution. Collect the crude enzyme solution for SDS-PAGE analysis of the target protein expression and subsequent enzyme activity determination or protein purification experiments.

[0060] The method for measuring the enzyme activity of MAT in the following examples is as follows:

[0061] Enzyme activity assay system for MAT: The premixed solution with a total volume of 0.4 mL contains 100 mM Tris-HCI buffer (pH 8.0), 10 mM ATP, 10 mM L-Met, 20 mM MgCl2, and 150 mM KCl. Add 0.1 mL of the crude enzyme solution, react at 40°C with a rotation speed of 150 rpm, and react in a constant-temperature metal bath for 5 min. Terminate the enzyme reaction with 0.5 mL of 20% perchloric acid solution. After centrifuging at 4°C and 12000 rpm for 10 min, measure the SAM concentration by high-performance liquid chromatography (HPLC). One unit of enzyme activity (1 U) is defined as the amount of enzyme that converts 1 μmol of SAM per minute.

[0062] The media involved in the following examples:

[0063] LB liquid medium (g / L): 5 yeast extract, 10 tryptone, 10 sodium chloride.

[0064] LB solid medium: On the basis of LB liquid medium, add 1.5 - 2.0% agar powder.

[0065] The detection methods involved in the following examples are as follows:

[0066] The high performance liquid chromatography detection conditions are: UV detector, C18 column (Agilent 5μm, 4.6mm×250mm), using an aqueous solution of 40mM ammonium dihydrogen phosphate and methanol with a volume fraction of 18% as the mobile phase, the flow rate is 0.8mL / min, the detection wavelength is 254nm, the column temperature is 30°C, and the injection volume is 10μL.

[0067] Example 1: Construction of recombinant bacterium BL21 / pET28a-MAT

[0068] Using Enterococcus faecalis genome as a template, the metK gene (nucleotide sequence as shown in SEQ ID NO.5) was amplified by PCR with P1 and P2 primers to obtain an amplification product of the metK gene fragment. The gene fragment was ligated to the linearized plasmid pET28a using homologous recombinase, and then the ligation product was chemically transformed into BL21 competent cells to obtain transformants. The transformants were spread on LB solid medium containing kanamycin and cultured at 37°C for 12h. Positive colonies were picked and verified by colony PCR to determine whether the plasmid was successfully constructed. Colony PCR verification of single colonies was performed using P3 and P4 as primers through Taq DNA polymerase (see Figure 1 ); After inoculating the positive single colonies with the target band size into a vial containing LB liquid medium and culturing for 12h, the plasmid was extracted and sent to Genewiz for correct sequencing, and the recombinant plasmid pET28a-MAT was successfully constructed.

[0069] P1:

[0070] 5’-GCGGATCCATGACAGAAAGACATTTATTTACATCAGAATCCGT-3’;

[0071] P2:

[0072] 5’-GCTCGAATTCTTATTCAGCTAAACTAGCTTTTAACGCTTCAACT-3’.

[0073] P3: 5’-CATGACTGGTGGACAGCAAAT-3’;

[0074] P4: 5’-GCTTTGTTAGCAGCCGGAT-3’.

[0075] Example 2: Construction of Recombinant Bacterial Mutant BL21 / pET28a-MAT-I195V / S150A

[0076] In this invention, MAT (amino acid sequence shown in SEQ ID NO.1) derived from Enterococcus faecalis was used as the parent for mutagenesis and transformation, and computer-aided design was adopted to design mutants. First, the AlphaFold2.0 was used to perform structural modeling on the EfMAT enzyme sequence, and after modeling, it was uploaded to the PROSS program for calculation. Nine mutants with higher rankings were selected from the calculation results of PROSS, primers were designed, and the recombinant expression plasmid pET28a-MAT prepared in Example 1 was used as the template to construct mutants through whole plasmid PCR, obtaining mutants E202D, L53N, K362G, Q67A, V304C, D200A, S150A, E214N, I195V. The recombinant Escherichia coli wild strain and each mutant were induced for expression and protein purification, and the specific enzyme activity determination results of each EfMAT mutant are shown in Table 1. The specific enzyme activities of mutants I195V and S150A increased most significantly compared with the wild type, reaching 6.50 U / mg and 5.19 U / mg respectively, and the specific enzyme activities increased by 35.4% and 8.1% compared with the wild type.

[0077] Since the specific enzyme activities of mutants I195V and S150A increased most significantly compared with the wild type, and these mutation sites are both near the substrate binding site, the mutation may have a synergistic promoting effect on the improvement of catalysis. To further improve the performance of the mutants, a double mutant MAT was constructed I195V / S150A , and its specific enzyme activity reached 13.35 U / mg, and the specific enzyme activity increased by 176% compared with the wild type.

[0078] Table 1 Determination of Specific Enzyme Activity of EfMAT Mutants

[0079]

[0080] Example 3: Construction of Recombinant Bacterium BL21 / pET28a-MAT-I195V / S150A-pACYC-AK-PPK

[0081] The AK gene derived from Arabidopsis thaliana was synthesized by the company (the nucleotide sequence is shown in SEQ ID NO.6, and the amino acid sequence is shown in SEQ ID NO.7), amplified by P5 and P6, and the plasmid pACYC-AK was constructed. Then, using the pACYC-AK plasmid as a template, the pACYC-AK plasmid was linearized. The ppk gene fragment (the nucleotide sequence is shown in SEQ ID NO.8, and the amino acid sequence is shown in SEQ ID NO.9) was amplified by PCR using the P7 and P8 primers. The ppk gene fragment and the linearized pACYC-AK plasmid were ligated using a homologous recombinase, and the ligation product was transformed into BL21 competent cells. The success of plasmid construction was verified by colony PCR (see Figure 2 ). After culturing the positive single colony with the size of the target band in a vial containing LB liquid medium for 12 h, the plasmid was extracted. Then, the plasmids pET28a-MAT-I195V / S150A and pACYC-AK-PPK were simultaneously chemically transformed into BL21 competent cells to obtain transformants. The transformants were spread on LB solid medium containing kanamycin and chloramphenicol and cultured at 37 °C for 12 h to obtain the recombinant strain BL21 / pET28a-MAT-I195V / S150A-pACYC-AK-PPK.

[0082] P5: 5’-TCGCGGATCCATGACCGCACCATTGGTAGTATTGGG-3’;

[0083] P6: 5’-CTATTTAGAGTAAGATATTTTTTCGGAAGGGTAAGAGGGAC-3’;

[0084] P7: 5’-GTGCGGTCATGGATCCGCGACCCATTTGCT-3’;

[0085] P8: 5’-AAATATCTTACTCTAAATAGGAATTCGAGCTCCGTCGACAAGC-3’;

[0086] Example 4: Production of SAM by the recombinant bacterium BL21 / pET28a-MAT-I195V / S150A-pACYC-AK-PPK

[0087] The process of generating SAM by the recombinant bacterium BL21 / pET28a-MAT-I195V / S150A-pACYC-AK-PPK in cells is as Figure 3As shown, one molecule of adenosine reacts with one molecule of ATP under the catalysis of AK to generate one molecule of AMP and ADP. AMP and ADP are respectively converted into ADP and ATP under the catalysis of PPK, and sodium hexametaphosphate provides phosphate groups in this process. ATP and methionine serve as substrates and generate SAM under the catalysis of MAT. Therefore, only a small amount of ATP and adenosine are needed to initiate the reaction in the reaction system. The ATP produced by the endogenous growth of cells in the whole-cell reaction is sufficient to meet the demand. One molecule of adenosine and sodium hexametaphosphate generate one molecule of ATP net under the catalysis of AK and PPK for SAM synthesis.

[0088] The prices of adenosine and sodium hexametaphosphate of the same mass are only about one-third and one-fiftieth of that of ATP respectively. In the reaction of adding adenosine and sodium hexametaphosphate to generate ATP, the cost can be greatly reduced, achieving the purpose of efficiently producing SAM with cheap substrates.

[0089] Recombinant Escherichia coli strains containing the target gene were pre-cultured on a medium containing the corresponding resistance to make the strains reach a suitable growth state. During the culture process, about 1 mL of the bacterial suspension was taken, and the wavelength was measured using an ultraviolet spectrophotometer. When the OD of the bacterial solution 600 reached 0.6 - 0.8, IPTG with a final concentration of 0.5 mmol / L was immediately added, and induction was carried out overnight at 16 °C to express the protein. After induction, centrifugation was carried out at 4 °C and 8000 r / min for 10 min to collect the cells. The collected cell precipitate was suspended using PBS buffer (0.1 mmol / L, pH 7.4), and then centrifuged again to collect the cell precipitate. The operation was repeated three times to completely remove the culture medium to prevent the culture medium components from being mixed in the enzyme solution and affecting the subsequent experiments.

[0090] Whole-cell SAM production system: Tris-HCl buffer (pH 8.0, 100 mM) containing 7.5 g / L of methionine, 0.5 g / L of adenosine, 20 g / L of sodium hexametaphosphate, 20 mM of magnesium chloride, 150 mM of potassium chloride, and a cell suspension with an OD 600 of 10.

[0091] After each sampling, the reaction sample was terminated with an equal volume of 20% perchloric acid solution. After centrifugation at 4 °C and 12000 rpm for 10 minutes, the SAM concentration was measured by HPLC. As Figure 4 shown, the recombinant strain BL21 / pET28a-MAT-I195V / S150A-pACYC-AK-PPK can accumulate up to 13.6 g / L of SAM during the production process, and the molar conversion rate reaches 68.1%, achieving the purpose of efficiently synthesizing SAM with cheap substrates by microbial cells without adding ATP.

[0092] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. An S-adenosylmethionine synthetase mutant, characterized in that, The S-adenosylmethionine synthase mutant is a mutant in which serine at position 150 and / or isoleucine at position 195 of the S-adenosylmethionine synthase having the amino acid sequence shown in SEQ ID NO.1 is mutated; The S-adenosylmethionine synthase is one of the following mutants: Serine at position 150 of the S-adenosylmethionine synthase having the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine; Isoleucine at position 195 of the S-adenosylmethionine synthase having the amino acid sequence shown in SEQ ID NO.1 is mutated to valine; Serine at position 150 of the S-adenosylmethionine synthase having the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine; and isoleucine at position 195 is mutated to valine.

2. A gene encoding the S-adenosylmethionine synthase mutant according to claim 1.

3. An expression vector carrying the gene according to claim 2.

4. A recombinant bacterium expressing the S-adenosylmethionine synthase mutant described in claim 1, characterized in that, The recombinant bacterium also expresses adenosine kinase and polyphosphate kinase.

5. The recombinant bacterium according to claim 4, wherein The recombinant bacterium uses Escherichia coli E.coli BL21(DE3) as the host and pET28a plasmid and pACYC plasmid as the expression vectors; Using the pET28a plasmid as an expression vector to express MAT I195V / S150A ; using the pACYC plasmid as an expression vector to express AK and PPK.

6. Use of the S-adenosylmethionine synthetase mutant according to claim 1 or the recombinant bacterium according to claim 5 in the production of S-adenosylmethionine, characterized in that, The application uses the S-adenosylmethionine synthase mutant, adenosine kinase and polyphosphate kinase as catalysts, and a buffer solution containing 5-10 g / L methionine, 5-10 g / L adenosine, 10-30 g / L sodium hexametaphosphate, 10-30 mM magnesium chloride, and 100-200 mM potassium chloride as a reaction system to catalytically produce S-adenosylmethionine; Alternatively, the application uses the cell lysate of the recombinant bacterium as a catalyst, and a buffer solution containing 5-10 g / L methionine, 5-10 g / L adenosine, 10-30 g / L sodium hexametaphosphate, 10-30 mM magnesium chloride, and 100-200 mM potassium chloride as a reaction system to catalytically produce S-adenosylmethionine.

7. The application according to claim 6, wherein In the cell lysate, the cell mass OD 600 is 5 - 20; the buffer is 50 - 200 mM Tris-HCl buffer.

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