A methionine adenosyltransferase mutant and use thereof

Abstract

The application discloses a methionine adenosyltransferase mutant and application thereof, and the methionine adenosyltransferase mutant is obtained by performing at least one site mutation on the amino acid sequence of wild-type Escherichia coli methionine adenosyltransferase shown in SEQ ID NO. 1. Rational protein modification technology is used to modify the Escherichia coli methionine adenosyltransferase, and a methionine adenosyltransferase mutant is obtained. The mutant can reduce the inhibition of product S-adenosylmethionine (SAM) on the methionine adenosyltransferase, enlarge the product release opening, make the product SAM more easily release, improve the substrate conversion rate, and improve the yield of SAM. Meanwhile, through semi-rational protein modification technology, the optimal reaction pH value of the enzyme is reduced, the generation of by-products in the reaction is reduced, the enzyme is more suitable for large-scale industrial production, and the enzyme has more market competitiveness.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a methionine adenosine transferase mutant and its applications. Background Technology

[0002] S-Adenosylmethionine (SAM) is an important metabolic intermediate present in all organisms. It is a cofactor involved in group transfer reactions in various metabolic processes, serving as a major methyl donor for the methylation of DNA, proteins, and secondary metabolites. It plays a crucial role in transmethylation, transsulfation, and transaminopropylation processes, and is also an intermediate metabolite in the synthesis of spermine and spermidine in vivo. SAM-dependent group transfer reactions have wide applications in chemical biology, synthetic biology, and metabolic engineering; in particular, methylation-dependent reactions are a key step in the production of pharmaceuticals and fine chemicals. Furthermore, SAM has various beneficial effects on human health and has been used as a functional nutrient or drug for the prevention and treatment of liver disease, osteoarthritis, and depression.

[0003] Due to the increasing demand for SAM, numerous attempts have been made to develop more economical and efficient SAM production methods. SAM production can be achieved through chemical synthesis, microbial fermentation, and in vitro enzymatic synthesis. Chemical synthesis is not widely used due to difficulties in product purification, low yield, and environmental pollution. Microbial fermentation of SAM suffers from drawbacks such as long fermentation cycles, low conversion rates, and complex extraction processes. In contrast, in vitro enzymatic synthesis offers advantages such as short reaction intervals, high conversion rates, high product concentrations, ease of separation and purification, and minimal environmental pollution. SAM is synthesized from L-methionine (L-Met) and adenosine triphosphate (ATP) as substrates via methionine adenosine transferase (EC 2.5.1.6). Methionine adenosine transferase derived from *E. coli* exhibits high specific activity, low Km value, and high expression efficiency, making it suitable for in vitro enzymatic reactions.

[0004] However, in industrial production, efficiency and cost are the most critical considerations for the application of this enzyme. The main drawback of this enzyme is that the product SAM forms an enzyme complex with methionine adenosine transferase, affecting the binding of the substrate to the enzyme. Although the inhibition can be relieved by adding a high concentration of p-toluenesulfonate, this introduces new impurities, which inevitably increases the cost of raw material input and subsequent purification. Furthermore, the optimal pH for this enzyme is 8.0-9.0. During prolonged reactions under alkaline pH conditions, the product SAM is easily decomposed into 5'-deoxy-5'-methionine (MTA) and S-adenosine homocysteine ​​(SAH), see [link to relevant documentation]. Figure 1 This increases the cost of separating impurities later and also affects product purity.

[0005] Therefore, in the process of industrial application of this enzyme, two problems urgently need to be solved: first, to relieve or reduce the feedback inhibition of the enzyme by the product SAM; and second, to lower the optimal reaction pH of the enzyme, thereby improving enzyme activity, reducing the generation of by-products, and increasing SAM yield. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a methionine adenosine transferase mutant and its application, which solves the problems of low efficiency and high cost of existing methionine adenosine transferase in industrial production. By creating a mutant, the inhibitory effect of the product SAM is reduced, and the optimal pH value of the enzyme is adjusted to improve catalytic activity and yield, and reduce the generation of by-products.

[0007] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0008] The primary objective of this application is to provide a methionine adenosine transferase mutant, which is obtained by mutating the amino acid sequence of the wild-type Escherichia coli methionine adenosine transferase shown in SEQ ID NO.1 at at least one of the following sites, wherein the mutation sites include:

[0009] (1) The isoleucine at position 103 is mutated to valine (I103V);

[0010] (2) The isoleucine at position 303 is mutated to valine (I303V);

[0011] (3) The lysine at position 37 is mutated to arginine (K37R);

[0012] (4) The threonine at position 58 is mutated to serine or glycine (T58S or T58G);

[0013] (5) The valine at position 107 is mutated to threonine (V107T);

[0014] (6) The aspartic acid at position 108 is mutated to glycine (D108G);

[0015] (7) Glutamine at position 115 is mutated to asparagine (Q115N);

[0016] (8) The alanine at position 302 is mutated to serine, valine or cysteine ​​(A302S or A302V or A302C).

[0017] Preferably, in the above technical solution, the methionine adenosine transferase mutant includes at least one mutation site as described in (1)-(8) above, and at least one of the following mutation sites:

[0018] (9) The isoleucine at position 211 is mutated to threonine (I211T);

[0019] (10) The glutamine at position 298 is mutated to asparagine or threonine (Q298N or Q298T);

[0020] (11) The methionine at position 312 is mutated to serine, glycine, or alanine (M312S, M312G, or M312A).

[0021] (12) The valine at position 321 is mutated to methionine (V321M);

[0022] (13) The arginine at position 337 is mutated to histidine or glutamine (R337H or R337Q);

[0023] (14) The lysine at position 370 is mutated to arginine (K370R);

[0024] (15) The lysine at position 373 is mutated to isoleucine (K373I);

[0025] (16) The leucine at position 376 is mutated to glutamine (L376Q).

[0026] The second objective of this application is to provide a gene encoding the aforementioned methionine adenosine transferase mutant.

[0027] A third objective of this application is to provide a recombinant vector containing the aforementioned methionine adenosine transferase mutant gene.

[0028] The fourth objective of this application is to provide a recombinant microorganism, which is constructed by introducing the above-mentioned gene into Escherichia coli via plasmid or by integrating it into Escherichia coli through genetic engineering.

[0029] The fifth objective of this application is to provide the application of the above-mentioned recombinant vector or the above-mentioned recombinant microorganism in the production of S-adenosylmethionine.

[0030] The sixth objective of this application is to provide a method for synthesizing SAM by adding methionine, ATP, and the aforementioned methionine adenosine transferase mutant to a reaction system to carry out an enzymatic reaction to synthesize SAM.

[0031] Preferably, in the above technical solution, the pH value of the reaction system is 6.8-8.0, and the reaction temperature is 30-40℃.

[0032] Preferably, in the above technical solution, the pH value of the reaction system is 7.0 and the reaction temperature is 40°C.

[0033] Preferably, in the above technical solution, the reaction system further includes magnesium salt and potassium salt.

[0034] The above-described technical solution of the present invention has the following beneficial effects:

[0035] This application utilizes semi-rational protein modification technology to modify Escherichia coli methionine adenosine transferase, obtaining a methionine adenosine transferase mutant. This mutant reduces the inhibitory effect of the product SAM on methionine adenosine transferase, and by enlarging the product release orifice, makes the product SAM easier to release, thereby increasing the substrate conversion rate and increasing the yield of SAM. At the same time, through semi-rational protein modification technology, the optimal reaction pH of the enzyme is lowered, reducing the generation of by-products in the reaction, making it more suitable for large-scale industrial production and more competitive in the market. Attached Figure Description

[0036] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0037] Figure 1 A schematic diagram of the structure of impurities resulting from SAM degradation.

[0038] Figure 2 This is a schematic diagram of the tetramer structure of wild-type methionine adenosine transferase in Example 1.

[0039] Figure 3 This is a schematic diagram of the amino acid residues at the release port of SAM, the product of wild-type methionine adenosine transferase in Example 1.

[0040] Figure 4 This is an SDS-PAGE electrophoresis image of the methionine adenosine transferase mutant protein expression in Example 2.

[0041] Figure 5 The HPLC chromatogram for detecting SAM is shown in Example 3.

[0042] Figure 6 This is a schematic diagram of the activity of the single-point mutant of methionine adenosine transferase in Example 3.

[0043] Figure 7 This is a schematic diagram of the mutation site described in Example 5.

[0044] Figure 8 This is a schematic bar chart showing the activity of the methionine adenosine transferase mutant in Example 5 at pH 7 and 8.

[0045] Figure 9 The HPLC chromatogram for the detection of SAM is shown in Example 7. Detailed Implementation

[0046] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0047] Example 1: Molecular Dynamics Simulation and Structure Analysis

[0048] The steps for molecular dynamics simulation are as follows:

[0049] (i) The amino acid residue sequence (SEQ ID No.1) of the wild-type protein was used to perform three-dimensional structural modeling to obtain a complete three-dimensional model of the protein.

[0050] (ii) The molecules of substrate ATP, L-methionine, product SAM and byproduct pyrophosphate, etc., were constructed using Avogardro software to build pdb format model files, and the corresponding molecular topology was calculated using AmberTools19 software and the accompanying BCC force field.

[0051] (III) Molecular dynamics simulations of protein-ligand binding were performed using Gromacs software combined with the Amber03 force field. During the simulation, the protein and ligand molecules were placed in a cubic box-shaped space, with a minimum distance of 1.0 nm between the protein and the box boundary. The box was filled with TIP3P-type water molecules and sodium ions to ensure electroneutrality. The steepest descent method was used to minimize the energy of the system. Subsequently, the system temperature was increased from 0 K to 313 K to achieve isothermal and isochoric equilibrium, followed by isothermal and isobaric equilibrium. Next, a 60-nanosecond dynamics simulation was performed in the isothermal and isobaric equilibrium system, with a simulation step size of 1 femtosecond, and the data was saved every 5 nanoseconds.

[0052] Structural analysis was performed on the simulated protein-ligand docking model, and the results are as follows:

[0053] Escherichia coli methionine adenosine transferase has a homotetrameric structure with four active sites, see [link to relevant documentation]. Figure 2The enzyme is severely inhibited by the product SAM during in vitro enzymatic reactions. Structural analysis of the protein's active site revealed a hydrophobic region near the product SAM, where sites I103 and I303 exhibit hydrophobic interactions with the product SAM. To mitigate these interactions, one of the two sites was mutated from I to V. Molecular dynamics simulations clearly showed that the distance between the methyl group and the amino acid side chain of SAM increased, thus weakening the hydrophobic interaction. Further analysis of the pores released by SAM revealed that amino acid residues such as K37, R39, T58, Q99, V107, D108, Q115, and A302 easily clog the pore openings. These residues could be modified to have shorter side chains and smaller volumes. Molecular dynamics simulations clearly showed that K37R, T58S, T58G, V107T, D108G, Q115N, A302S, A302V, and A302C positively contribute to pore enlargement. The R39 and Q99 sites likely play important roles in enzyme catalysis and should not be modified. The locations of the relevant amino acids near the active site described above are listed below. Figure 3 .

[0054] Example 2: Upstream construction and protein expression of methionine adenosine transferase and its mutants

[0055] (1) Construction of wild-type methionine adenosine transferase genetically engineered bacteria

[0056] Based on the gene sequence encoding E. coli methionine adenosine transferase published in GenBank (SEQ ID No. 2), primers were designed to amplify the E. coli methionine adenosine transferase gene. Using E. coli DH5α genomic DNA as a template, the positive primer was: 5'- TAAGAAGGAGATATACAT ATGGCAAAACACCTTTTTACGT-3', the antisense primer is: 5'- T TAGCAGCCGGATCTCAgtggtggtggtggtggtg The underlined part of CTTCAGACCGGCAGCAT-3' represents a homologous fragment near the NdeI and XhoI sites on plasmid pET22b(+). After the amplified PCR fragment is recovered by gel electrophoresis, it is mixed with the pET22b(+) fragment that has been double-digested with NdeI and XhoI. The fragment is then ligated using a seamless cloning kit. The ligation product is transformed into E. coli DH5α competent cells. After the positive clone is correctly sequenced, the plasmid is named pET22b-metK. Plasmid pET22b-metK is then transformed into protein expression strains E. coli BL21(DE3) or Rosetta(DE3) to construct a genetically engineered bacterium expressing wild-type methionine adenosine transferase (with a His-Tag tag at the C-terminus).

[0057] (2) Construction of genetically engineered bacteria with single-point mutant of methionine adenosine transferase

[0058] Mutation primers were designed for the mutation sites in Example 1, as shown in Table 1. Using plasmid pET22b-metK as a template, whole-plasmid PCR amplification was performed to construct the mutant recombinant plasmid.

[0059] The full plasmid PCR reaction system (50 μl) consisted of: 30 μl ddH2O, 4 μl dNTPs (2.5 mM), 1.5 μl positive primer (10 mM), 1.5 μl antisense primer (10 mM), 1 μl plasmid template, 10 μl 5×PrimerSTAR GXL buffer, and 2 μl PrimerSTAR GXL DNA polymerase. The PCR reaction conditions were: 98℃ for 10 s, 55℃ for 15 s, 68℃ for 90 s, for 30 cycles, and stored at 4℃.

[0060] After the PCR reaction, the PCR product needs to be treated with DpnI enzyme. After being treated at 37°C for 2 hours, it is transformed into E. coli DH5α competent cells. After the positive clone is correctly sequenced, the corresponding plasmid is transformed into the protein expression strain E. coli BL21(DE3) or Rosetta(DE3) to construct a genetically engineered bacterium that expresses a single-point mutant of methionine adenosine transferase efficiently.

[0061] Table 1. List of primers for single-point mutation

[0062]

[0063]

[0064] (3) Protein expression of methionine adenosine transferase mutant

[0065] The genetically engineered bacteria containing the constructed methionine adenosine transferase mutant were cultured overnight at 37°C, and then transferred at a 1% inoculum to 500mL Erlenmeyer flasks containing 100mL LB broth. The cultures were then incubated at 37°C until the OD (oxidative stress) was achieved. 600 ≈0.6; Add 1mM isopropyl-β-D-thiogalactoside (IPTG) to start induction, collect the bacterial cells by centrifugation after 5 hours, and then perform SDS polyacrylamide protein electrophoresis to detect the protein expression level. Figure 4 This is an SDS-polyacrylamide protein electrophoresis image. Lane 1 contains protein markers ranging from 15-150 kDa (commercially available); Lane 2 contains wild-type methionine adenosine transferase protein, approximately 42 kDa; Lanes 3-9 contain some methionine adenosine transferase mutant proteins, all approximately 42 kDa.

[0066] Example 3 Activity detection of methionine adenosine transferase mutant

[0067] The activity assay system for the methionine adenosine transferase mutant was as follows: 10 mM ATP, 10 mM methionine, 20 mM MgCl2, 100 mM KCl and 100 mM Tris-HCl buffer (pH 8.0).

[0068] The bacterial cells of the methionine adenosine transferase mutant were sonicated at low temperature, and the supernatant was collected by centrifugation to obtain a crude enzyme solution. 2 mg / ml of the crude enzyme solution was added to the above system, and the reaction was carried out at 40°C. After 30 minutes of reaction, 20% perchloric acid was immediately added to terminate the reaction. The reaction solution was aspirated with a syringe, filtered through a 0.22 μm membrane, and the amount of SAM product generated was analyzed by HPLC.

[0069] The HPLC conditions for SAM detection were as follows: mobile phase consisted of a buffer solution containing 1% glacial acetic acid, 10 mM sodium heptanesulfonate, and 15% acetonitrile; the column was a Kromasil C18 column; the flow rate was 1 mL / min; the detection wavelength was 260 nm; and the detection temperature was 30 °C. See the activity detection chromatogram below. Figure 5 The retention time of SAM is 13-14 minutes. In addition, the reaction generates impurities SAH (retention time 4.1 minutes) and MTA (retention time 10.5 minutes).

[0070] The activity is defined as the amount of enzyme that converts to generate 1 μM SAM within 1 minute under conditions of 40℃ and pH 8.0, which is one unit of activity.

[0071] The activity of wild-type and various mutants in generating SAM is shown in the figure. Figure 6 Based on the above data on the activity of wild-type and mutant methionine adenosine transferase, it can be seen that T58S, I103V, A302S, and I303V are relatively high-activity mutation sites that can significantly enhance the enzyme's activity in catalyzing the production of SAM.

[0072] Example 4: Simulation and Selection of Multipoint Mutants

[0073] The highly active mutation sites screened in Example 3 were combined in two or more locations, and protein structure simulation was performed according to the method described in Example 1. The ligand and protein binding morphology, active site pore size and catalytic site distance were evaluated, and suitable sites that could be combined were selected. Multi-site combination mutation was performed according to the method described in Example 2 (2).

[0074] The activity of the mutant protein was tested according to the method described in Example 3, and the combined mutants T58S / I103V / A302S, T58S / I103V, I103V / A302S, and T58S / A302S were selected. The above mutants increased the activity of wild-type protein by 153%, 125%, 90%, and 86%, respectively.

[0075] Example 5: Further Simulation Optimization of the Mutant

[0076] During the synthesis reaction, it was also found that in the reaction system at pH 8.0, in addition to the product SAM, impurities SAH and MTA were also generated. Lowering the pH of the reaction system buffer to 7.0 significantly reduced the formation of impurities SAH and MTA, but at the same time, the yield of SAM also decreased.

[0077] Further molecular dynamics simulations were performed on the mutant T58S / I103V / A302S (SEQ ID No. 3) under the following conditions: ① neutral and ② slightly acidic solutions.

[0078] (a) The mutant T58S / I103V / A302S was modeled in three dimensions using AlphaFold2 to obtain a complete three-dimensional model of the protein.

[0079] (II) Molecular dynamics simulations of the mutant protein T58S / I103V / A302S binding to the ligand (calculation method described in Example 1) were performed using Gromacs software combined with the Amber03 force field. During the dynamics simulation, the protein and ligand molecules were placed in a cubic box-shaped space, with a minimum distance of 1.0 nm between the protein and the box boundary. ① The box was filled with TIP3P-type water molecules and sodium ions to make the system electrically neutral, or ② hydrated hydrogen ions were used to replace some sodium ions, and some chloride ions were added to fill the box to make the system weakly acidic while maintaining electrical neutrality. The steepest descent method was used to minimize the energy of the system, and then the system temperature was increased from 0 K to 313 K to achieve isothermal and isochoric equilibrium, followed by isothermal and isobaric equilibrium. Next, a 60-nanosecond dynamics simulation was performed in the isothermal and isobaric equilibrium system, with a simulation step size of 1 femtosecond, and the system data was saved every 5 nanoseconds.

[0080] Structural analysis was performed on the simulated mutant protein T58S / I103V / A302S and its ligand docking model. The morphology of the protein was compared under ① neutral and ② weakly acidic conditions. It was found that certain amino acid residues are crucial to the protein's stability under slightly acidic conditions, especially some surface amino acid residues near the C-terminus. (See...) Figure 7 It includes the following loci: I211, Q298, M312, V321, R337, K370, K373, and L376.

[0081] Following the method described in Example 2(2), saturation mutations were performed on the above-mentioned sites, mutation primers were designed, and a mutant library was constructed, as shown in Table 2. Protein expression was then performed according to the method described in Example 2(3).

[0082] Table 2. List of primers for saturation mutations

[0083]

[0084]

[0085] The mutant activity was detected according to the method described in Example 3, and the pH value of the buffer was reduced. The buffer system was modified to contain 10 mM ATP, 10 mM methionine, 20 mM MgCl2, 100 mM KCl and 100 mM Tris-HCl (pH 7.0).

[0086] Enzyme activity assays confirmed that, based on the mutant T58S / I103V / A302S, mutants containing the following sites maintained good catalytic activity even at pH 7.0: I211T, Q298N, Q298T, M312S, M312G, M312A, V321M, R337H, R337Q, K370R, K373I, and L376Q. (See [link to relevant documentation]). Figure 8 .

[0087] The mutant protein was further screened using two- or multi-site combination simulations under slightly acidic conditions, following the methods described above. The ligand and protein binding morphology, active site pore size, and catalytic site distance were evaluated, and suitable sites were selected for multi-site combination mutations. Enzyme activity assays determined the optimal mutant to be T58S / I103V / Q298T / A302S / M312S / K373I (SEQ ID No. 4).

[0088] Example 6: Synthesis of SAM using a methionine adenosine transferase mutant

[0089] The strain expressing the methionine adenosine transferase mutant T58S / I103V / A302S (SEQ ID No. 3) was inoculated into LB medium and cultured overnight at 37°C. Then, at a 1% inoculum, it was transferred to a 2L Erlenmeyer flask containing 400mL of LB liquid medium and cultured at 37°C until OD580 reached. 600 ≈0.6; Add 1mM IPTG to start induction, and collect the bacterial cells by low-temperature centrifugation after 5 hours. Add 20mM Tris-HCl (pH 8.0) buffer to the bacterial cells, homogenize, and then use an ultrasonic disruptor to disrupt the bacterial cells. The liquid after disruption is the crude enzyme solution containing the methionine adenosine transferase mutant.

[0090] The reaction solution and conditions for synthesizing SAM were as follows: 100 mM KCl, 50 mM MgCl2, 80 mM methionine, and 80 mM ATP. The reaction temperature was set at 40°C, and the pH was controlled at approximately 8.0. A crude enzyme solution of 10 g / L was added for the reaction. The amount of SAM produced was determined by HPLC after the reaction was terminated; the SAM yield reached over 60 mM. The HPLC detection conditions were the same as in Example 3. Small amounts of impurities MTA and SAH were generated and need to be removed in subsequent processes.

[0091] After the reaction was completed, the enzyme solution was recovered by ultrafiltration, and the activity was still more than 80% of the original.

[0092] Example 7: Synthesis of SAM using a methionine adenosine transferase mutant

[0093] The strain expressing the methionine adenosine transferase mutant T58S / I103V / Q298T / A302S / M312S / K373I (SEQ ID No. 4) was inoculated into LB medium and cultured overnight at 37°C. Then, 1% of the inoculum was transferred to 2L Erlenmeyer flasks containing 400mL of LB liquid medium and cultured at 37°C until OD580 reached. 600 ≈0.6; Add 1mM IPTG to start induction, and collect the bacterial cells by low-temperature centrifugation after 5 hours. Add 20mM Tris-HCl (pH 7.0) buffer to the bacterial cells, homogenize, and then use an ultrasonic disruptor to disrupt the bacterial cells. The liquid after disruption is the crude enzyme solution containing the methionine adenosine transferase mutant.

[0094] The reaction solution composition and conditions for synthesizing SAM were as follows: 100 mM KCl, 50 mM MgCl2, 80 mM methionine, and 80 mM ATP. The reaction temperature was set at 40°C, and the pH was controlled at approximately 7.0. A crude enzyme solution of 10 g / L was added for the reaction. The amount of SAM produced was determined by HPLC after the reaction was terminated; the SAM yield reached over 63 mM. The HPLC detection conditions were the same as in Example 3, see [link to Example 3]. Figure 9 The generation of impurities MTA and SAH is low, which simplifies the subsequent purification process.

[0095] After the reaction was completed, the enzyme solution was recovered by ultrafiltration, and the activity was still more than 80% of the original.

[0096] Comparative Example 1: Experiment on the synthesis of SAM using wild-type methionine adenosine transferase

[0097] The strain expressing wild-type methionine adenosine transferase was inoculated into LB medium and cultured overnight at 37°C. Then, at a 1% inoculum, it was transferred to a 2L Erlenmeyer flask containing 400mL of LB liquid medium and cultured at 37°C until OD500 was reached. 600≈0.6; Add 1mM IPTG to start induction, and collect the bacterial cells by low-temperature centrifugation after 5 hours. Add 20mM Tris-HCl (pH 8.0) buffer to the bacterial cells, homogenize, and then use an ultrasonic disruptor to disrupt the bacterial cells. The liquid after disruption is the crude enzyme solution containing wild-type methionine adenosine transferase.

[0098] The reaction solution and conditions for synthesizing SAM were as follows: 100 mM KCl, 50 mM MgCl2, 80 mM methionine, and 80 mM ATP. The reaction temperature was set at 40℃, and the pH was controlled at approximately 8.0. A crude enzyme solution of 10 g / L was added for the reaction. The amount of SAM produced was determined by HPLC after the reaction was terminated. The SAM yield was 25 mM, with impurities MTA and SAH also present.

[0099] It is evident that the methionine adenosine transferase mutant T58S / I103V / A302S (SEQ ID No. 3) can increase the catalytic activity of wild-type protein by more than 2 times, resulting in a significant increase in yield; while the mutant T58S / I103V / Q298T / A302S / M312S / K373I (SEQ ID No. 4) can further enhance the catalytic activity of wild-type protein, and reduce the pH value of the reaction, thereby reducing the generation of impurities.

[0100] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A methionine adenosine transferase mutant, characterized in that, The mutant was obtained by mutating the amino acid sequence of the wild-type Escherichia coli methionine adenosine transferase shown in SEQ ID NO.1 at at least one of the following sites, wherein the mutation sites include: (1) The isoleucine at position 103 is mutated to valine (I103V); (2) The isoleucine at position 303 is mutated to valine (I303V); (3) The lysine at position 37 is mutated to arginine (K37R); (4) The threonine at position 58 is mutated to serine or glycine (T58S or T58G); (5) The valine at position 107 is mutated to threonine (V107T); (6) The aspartic acid at position 108 is mutated to glycine (D108G); (7) Glutamine at position 115 is mutated to asparagine (Q115N); (8) The alanine at position 302 is mutated to serine, valine or cysteine ​​(A302S or A302V or A302C).

2. The methionine adenosine transferase mutant according to claim 1, characterized in that, The methionine adenosine transferase mutant contains at least one of the mutation sites described in (1)-(8) above, and at least one of the following mutation sites: (9) The isoleucine at position 211 is mutated to threonine (I211T); (10) The glutamine at position 298 is mutated to asparagine or threonine (Q298N or Q298T); (11) The methionine at position 312 is mutated to serine, glycine, or alanine (M312S, M312G, or M312A). (12) The valine at position 321 is mutated to methionine (V321M); (13) The arginine at position 337 is mutated to histidine or glutamine (R337H or R337Q); (14) The lysine at position 370 is mutated to arginine (K370R); (15) The lysine at position 373 is mutated to isoleucine (K373I); (16) The leucine at position 376 is mutated to glutamine (L376Q).

3. A gene encoding a methionine adenosine transferase mutant as described in claim 1 or 2.

4. A recombinant vector comprising the methionine adenosine transferase mutant gene of claim 3.

5. A recombinant microorganism, characterized in that, The recombinant microorganism is constructed by introducing the gene described in claim 3 into Escherichia coli via plasmid or by integrating it into Escherichia coli through genetic engineering.

6. The use of the mutant according to any one of claims 1-2, the recombinant vector according to claim 4, or the recombinant microorganism according to claim 5 in the production of S-adenosylmethionine.

7. A method for synthesizing S-adenosylmethionine, characterized in that, Methionine, ATP, and the methionine adenosine transferase mutant as described in claim 1 or 2 are added to the reaction system to carry out an enzymatic reaction to synthesize SAM.

8. The method according to claim 7, characterized in that, The pH value of the reaction system is 6.8-8.0, and the reaction temperature is 30-40℃.

9. The method according to claim 7, characterized in that, The pH value of the reaction system is 7.0, and the reaction temperature is 40℃.

10. The method according to claim 7, characterized in that, The reaction system also includes magnesium salts and potassium salts.

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