S-adenosylmethionine synthetase mutants and use thereof

Abstract

Provided are S-adenosylmethionine synthetase mutants and the use thereof, belonging to the technical field of biological enzyme engineering. Site-directed combinatorial mutations are performed on positions 189, 200, 234, 266 and 371 of S-adenosylmethionine synthetase MAT to obtain a series of mutants with improved enzyme activity, wherein the specific enzyme activity of I189V / V266H and V266H / N371R is increased by more than 10 times relative to the parent. The yields of S-adenosylmethionine synthesized by whole cells of recombinant bacteria expressing mutants I189V / V266H / N371R, V266H / Q234N / N371R and I189V / V266H / Q234N / N371R all reach 1550 mg / L or more. The provided enzyme mutants have broad application prospects in the production of S-adenosylmethionine and the construction of genetically engineered microorganisms for S-adenosylmethionine.

Description

An S-adenosylmethionine synthase mutant and its application Technical Field

[0001] This invention relates to an S-adenosylmethionine synthase mutant and its applications, belonging to the field of bioenzyme engineering technology. Background Technology

[0002] S-Adenosylmethionine (SAM) is an important metabolite in organisms, participating in a variety of biochemical reactions within the body.

[0003] SAM is involved in almost all cellular metabolic processes. In vivo, it primarily functions as a methyl donor and participates in various biochemical reactions involving transsulfation, transamination, and transribosylation. It also acts as a signaling molecule to regulate microbial metabolism. Furthermore, SAM is related to the activity of many enzymes; a deficiency in SAM can affect the normal metabolic function of the entire organism. It has significant clinical applications, currently mainly used in the treatment of liver and nervous system diseases, osteoarthritis, and anti-tumor therapies. Due to its aforementioned biological functions and clinical applications, the preparation methods of SAM have attracted increasing research attention.

[0004] Currently, microbial fermentation has become the mainstream method for industrial production of SAM due to its advantages such as ease of scale-up and lower raw material costs. However, this method does not have advantages in terms of fermentation cycle and subsequent product extraction and purification. Whole-cell catalysis not only has the advantages of easy scale-up of microbial fermentation but also eliminates the need for cell disruption, reducing downstream separation and purification costs. However, currently known S-adenosylmethionine synthetases (MAT) all suffer from low enzyme activity and poor stability, resulting in lower substrate conversion levels and product yields far lower than those of microbial fermentation. Furthermore, common MATs exhibit severe substrate and product inhibition. While whole-cell catalysis for SAM synthesis offers advantages such as ease of scale-up, shorter production cycle, simple preparation, and elimination of the cumbersome enzyme purification process, the current yield of whole-cell SAM synthesis is low and cannot reach industrial-scale levels. Summary of the Invention

[0005] To address the problems of low catalytic efficiency and poor stability of S-adenosylmethionine synthase in current industrial production, this invention aims to provide a mutant with enhanced S-adenosylmethionine synthase activity and improve the yield of S-adenosylmethionine synthase via whole-cell catalysis, which has general significance for promoting the synthesis of S-adenosylmethionine.

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

[0007] The S-adenosylmethionine synthase MAT encoded by the sam2 gene from Saccharomyces cerevisiae was cloned. A protein model was established using the Swiss-Model method, and its protein structure was analyzed. The protein was molecularly docked with the substrate, and site-directed mutagenesis was performed at selected sites. The activity of the mutated enzyme was measured to identify sites that have an important impact on enzyme activity. Then, combined mutagenesis was performed to superimpose the advantages of the mutations and obtain mutants with improved activity and stability compared to wild type.

[0008] This invention provides an S-adenosylmethionine synthase mutant, based on the amino acid sequence shown in SEQ ID NO.1, by performing any of the following mutations:

[0009] (1) Mutate isoleucine at position 189 to valine;

[0010] (2) Mutate valine at position 266 to histidine;

[0011] (3) Mutate asparagine at position 371 to arginine;

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

[0013] (5) Mutate isoleucine at position 189 to valine and valine at position 266 to histidine;

[0014] (6) Mutate isoleucine at position 189 to valine and asparagine at position 371 to arginine;

[0015] (7) Mutate isoleucine at position 189 to valine and aspartic acid at position 200 to glutamic acid;

[0016] (8) Mutate isoleucine at position 189 to valine and glutamine at position 234 to asparagine;

[0017] (9) Mutate valine at position 266 to histidine and asparagine at position 371 to arginine;

[0018] (10) Mutate asparagine at position 371 to arginine and aspartic acid at position 200 to glutamic acid;

[0019] (11) Mutate asparagine at position 371 to arginine and glutamine at position 234 to asparagine;

[0020] (12) Mutate valine at position 266 to histidine, asparagine at position 371 to arginine, and glutamine at position 234 to asparagine;

[0021] (13) Mutate isoleucine at position 189 to valine, valine at position 266 to histidine, and asparagine at position 371 to arginine;

[0022] (14) Mutate valine at position 266 to histidine, asparagine at position 371 to arginine, isoleucine at position 189 to valine, and glutamine at position 234 to asparagine.

[0023] The present invention also provides a gene encoding the mutant.

[0024] The present invention also provides an expression vector carrying the gene.

[0025] The present invention also provides microbial cells that express the mutant or carry the expression vector.

[0026] The present invention also provides a method for producing S-adenosylmethionine, wherein the mutant or the microbial cell is used as a catalyst for the reaction.

[0027] In one embodiment of the present invention, the microbial cells are cultured in a culture system to OD. 600 The concentration was 0.6–0.8. An inducer was added, and the cells were induced at 25°C for 24 hours. The cells were then collected and added to a reaction system containing the substrate for 2 hours.

[0028] In one embodiment of the present invention, the recombinant monoclonal antibody is inoculated into 10 mL of LB liquid medium containing kanamycin and cultured at 37°C and 220 rpm for 10-12 h. Then, 2% of the culture medium is transferred to a new 50 mL LB liquid medium containing kanamycin and cultured for about 2-3 h to allow OD to develop. 600 =0.6-0.8, add IPTG (a sterile inducer) to a final concentration of 0.1mM, then incubate at 25℃ and 220rpm for 12-24h, and collect the bacterial cells by centrifugation.

[0029] In one embodiment of the present invention, the substrate includes L-methionine, adenosine-5'-triphosphate disodium salt, potassium chloride, and magnesium chloride. The substrate concentration in the reaction system is 20–60 mM, and the bacterial OD... 600 = (3.2) ± 0.2, reacted at pH 8 ± 1 and 37℃.

[0030] This invention also provides a method for improving the catalytic activity of S-adenosylmethionine synthase, by performing any of the following mutations based on the amino acid sequence shown in SEQ ID NO.1:

[0031] (1) Mutate isoleucine at position 189 to valine;

[0032] (2) Mutate valine at position 266 to histidine;

[0033] (3) Mutate asparagine at position 371 to arginine;

[0034] (4) Mutate glutamine at position 234 to asparagine;

[0035] (5) Mutate isoleucine at position 189 to valine and valine at position 266 to histidine;

[0036] (6) Mutate isoleucine at position 189 to valine and asparagine at position 371 to arginine;

[0037] (7) Mutate isoleucine at position 189 to valine and aspartic acid at position 200 to glutamic acid;

[0038] (8) Mutate isoleucine at position 189 to valine and glutamine at position 234 to asparagine;

[0039] (9) Mutate valine at position 266 to histidine and asparagine at position 371 to arginine;

[0040] (10) Mutate asparagine at position 371 to arginine and aspartic acid at position 200 to glutamic acid;

[0041] (11) Mutate asparagine at position 371 to arginine and glutamine at position 234 to asparagine;

[0042] (12) Mutate valine at position 266 to histidine, asparagine at position 371 to arginine, and glutamine at position 234 to asparagine;

[0043] (13) Mutate isoleucine at position 189 to valine, valine at position 266 to histidine, and asparagine at position 371 to arginine;

[0044] (14) Mutate valine at position 266 to histidine, asparagine at position 371 to arginine, isoleucine at position 189 to valine, and glutamine at position 234 to asparagine.

[0045] The present invention also provides the use of the mutant or the microbial cell in the preparation of S-adenosylmethionine or products containing S-adenosylmethionine.

[0046] In one embodiment of the present invention, the product includes food, medicine, or daily chemical products. Beneficial effects:

[0047] (1) Mutations at one or more sites (189, 200, 234, 266, and 371) of S-adenosylmethionine synthase (MAT) yielded a series of mutants with increased enzyme activity. Mutation of isoleucine at position 189 to valine (I189V) increased the specific enzyme activity by 60.67% compared to the parent; mutation of valine at position 266 to histidine (V266H) increased the specific enzyme activity by 272.63% compared to the parent; mutation of asparagine at position 371 to arginine (N371R) increased the specific enzyme activity by 391.81% compared to the parent; and mutation of isoleucine at position 189 to valine and valine at position 266 to histidine (I189V / V266H) increased the specific enzyme activity by [missing information]. The specific activity was increased by 1353.08% compared to the parent; mutating isoleucine at position 189 to valine and asparagine at position 371 to arginine (I189V / N371R) increased the specific activity by 342.82% compared to the parent; mutating isoleucine at position 189 to valine and aspartic acid at position 200 to glutamic acid (I189V / D200E) increased the specific activity by 159.00% compared to the parent; mutating isoleucine at position 189 to valine and glutamine at position 234 to asparagine (I189V / Q234N) increased the specific activity by 147.45% compared to the parent; mutating valine at position 266 to histidine and asparagine at position 371 to... Arginine (V266H / N371R) showed a 1060.38% increase in specific enzyme activity compared to the parent. Mutating asparagine at position 371 to arginine and aspartic acid at position 200 to glutamic acid (N371R / D200E) increased specific enzyme activity by 239.90% compared to the parent. Mutating asparagine at position 371 to arginine and glutamine at position 234 to asparagine (N371R / Q234N) increased specific enzyme activity by 401.05% compared to the parent. Mutating isoleucine at position 189 to valine, valine at position 266 to histidine, and asparagine at position 371 to arginine (I189V / V266H / N371R) showed... The specific enzyme activity was increased by 98.18% compared to the parent. Mutating valine at position 266 to histidine, asparagine at position 371 to arginine, and glutamine at position 234 to asparagine (V266H / Q234N / N371R) increased the specific enzyme activity by 340.84% ​​compared to the parent. Mutating valine at position 266 to histidine, asparagine at position 371 to arginine, isoleucine at position 189 to valine, and glutamine at position 234 to asparagine resulted in a mutant (I189V / V266H / Q234N / N371R) with a specific enzyme activity increased by 294.57% compared to the parent. The crude enzyme solution activity of the corresponding combination of mutations was also significantly increased compared to the parent.Furthermore, the single-point mutations at positions 189, 200, 234, 266, and 371 all showed varying degrees of increased enzyme activity compared to the parent, indicating that the enzyme activity of this mutant was greatly enhanced.

[0048] (2) The catalytic performance was compared using whole-cell catalysis. After 2 hours of catalysis, the concentration of S-adenosylmethionine synthesized by the parent (WT) was 276.32 mg / L, that of E. coli BL21-pET28a-sam2-I189V was 479.91 mg / L, that of E. coli BL21-pET28a-sam2-V266H was 509.03 mg / L, that of E. coli BL21-pET28a-sam2-N371R was 475.17 mg / L, and that of E. coli BL21-pET28a-sam2-I189V / V266H was 550.57 mg / L. The concentration of S-adenosylmethionine catalyzed by BL21-pET28a-sam2-I189V / N371R was 1119.34 mg / L; the concentration catalyzed by E. coli BL21-pET28a-sam2-I189V / D200E was 695.81 mg / L; the concentration catalyzed by E. coli BL21-pET28a-sam2-I189V / Q234N was 581.65 mg / L; and the concentration catalyzed by E. coli BL21-pET28a-sam2-V266H / N371R was 1087.26 mg / L. The concentration of S-adenosylmethionine catalyzed by BL21-pET28a-sam2-N371R / D200E was 738.50 mg / L; the concentration of S-adenosylmethionine catalyzed by E. coli BL21-pET28a-sam2-N371R / Q234N was 841.597 mg / L; the concentration of S-adenosylmethionine catalyzed by E. coli BL21-pET28a-sam2-I189V / V266H / N371R was 1552.81 mg / L; and the concentration of S-adenosylmethionine catalyzed by E. coli BL21-pET28a-sam2-V266H / Q234N / N371R was 1572.81 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. Attached Figure Description

[0049] Figure 1 shows the relative enzyme activity of mutants obtained by site-directed mutagenesis with wild-type enzyme as control and enzyme activity set at 100%.

[0050] Figure 2 shows a comparison of whole-cell catalysis between the wild-type enzyme and the mutant. Detailed Implementation

[0051] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0052] In the following examples, enzyme activity units (U) are defined as the amount of MAT enzyme required to produce 1 μmol SAM per minute, expressed as specific enzyme activity (U·mg). -1 The SAM enzyme activity assay system (based on final concentration) is as follows: 100 mM Tris-HCl (pH 8.0), 20 mM L-Met, 8 mM reduced glutathione, 50 mM KCl, 20 mM MgCl2, 20 mM ATP. Add 400 μL of crude enzyme solution to each 1 mL of the reaction system, incubate at 37°C for 30 min, remove 500 μL and mix with an equal volume of 20% perchloric acid solution to terminate the reaction, incubate on ice for 30 min, and then incubate at 12000 r·min. -1 Centrifuge for 10 min, collect the supernatant, and determine the SAM concentration by HPLC.

[0053] The method for determining S-adenosylmethionine (SAM) content was as follows: High-performance liquid chromatography (HPLC) was used to determine the SAM concentration. The fermentation supernatant was filtered through a 0.22 μm aqueous filter membrane. The HPLC column was a Diamonsil C18 column (250 x 4.6 mm, 5 μm). The mobile phase was V(2 mM sodium heptanesulfonate and 40 mM ammonium dihydrogen phosphate solution):V(methanol) = 82:18. 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.

[0054] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of S-adenosylmethionine (SAM) per minute under certain conditions, which is one unit of enzyme activity (1 U). In the examples below, crude enzyme activity refers to the enzyme activity per unit volume of crude enzyme solution, expressed in U·mL. -1 .

[0055] Specific enzyme activity is defined as the number of enzyme activity units per unit weight (mg) of protein. The formulas for calculating enzyme activity and specific enzyme activity are as follows:

[0056] In the formula, A SAM V 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 .

[0057] Example 1 Construction of mutants

[0058] 1. Construction of wild-type strain E. coli BL21 / pET-28a(+)-sam2

[0059] The S-adenosylmethionine synthase gene sam2, derived from Saccharomyces cerevisiae and with codon optimization for Escherichia coli, was artificially synthesized by GENEWIZ (Suzhou Genewiz Biotechnology Co., Ltd.). The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.1.

[0060] The target fragment sam2 was amplified using the primers in Table 1, and then ligated with plasmid pET-28a, which had been digested with restriction endonucleases (EcoR I, HindIII), using the homologous recombinase ClonExpress II One Step Cloning Kit in a 37℃ metal bath for 30 min to obtain the recombinant plasmid pET28a-sam2. The recombinant plasmid pET28a-sam2 was transformed into E. coli BL21(DE3), plated on LB plates containing Kan, and after single colonies grew, transformants were picked for colony PCR verification. If the bands matched, they were sent to Tianlin Biotechnology (Shanghai) Co., Ltd. for sequencing to obtain recombinant E. coli E. coli BL21-pET28a-sam2 expressing wild-type S-adenosylmethionine synthase.

[0061] Table 1 Primers used for constructing recombinant strains Note: The lowercase sequence is the recombination sequence added at the 5' end.

[0062] 2. Construction of mutant strains

[0063] (1) Screening of mutation sites

[0064] We used AlphaFold 2 and SWISS MODEL (https: / / swissmodel.expasy.org / ) to predict the three-dimensional model of the MAT protein and evaluated the model. We used AutoDock Vina to dock the substrate and protein and selected the optimal docking site based on docking analysis. We then used Pymol software to perform visualization analysis of the protein.

[0065] Construction of single point mutants:

[0066] Site-directed mutagenesis sites were introduced using reverse PCR. The recombinant plasmid pET28a-sam2 obtained in Example 1 was used as a template for single-point mutagenesis, with primers shown in Table 2. The methylation sites were then recognized by Dpn I enzyme, which digested the template, transforming it into *E. coli* BL21(DE3). The transformed bacteria were plated on LB agar plates containing Kans, and single colonies were allowed to grow. Finally, bacterial selection and sequencing were performed to verify the recombinant *E. coli* expressing the mutant. Taking the construction of recombinant *E. coli* expressing the mutant I189V as an example, using the recombinant plasmid pET28a-sam2 as a template and I189V-F and I189V-R as primers, reverse PCR was performed to obtain the recombinant plasmid pET28a-sam2-I189V. Then, the template was digested by Dpn I enzyme to recognize the methylation site and transformed into *E. coli* BL21(DE3). The transformed bacteria were plated on LB agar plates containing Kans, and after single colonies grew, they were selected and sequenced for verification, yielding the recombinant *E. coli* BL21-pET28a-sam2-I189V expressing the mutant I189V. The same method was used to obtain *E. coli* BL21-pET28a-sam2-V266H, *E. coli* BL21-pET28a-sam2-N371R, and other recombinant *E. coli* expressing single mutants.

[0067] Table 2 Primers used for site-directed mutagenesis

[0068] Construction of multi-site mutants:

[0069] Taking strain E. coli BL21-pET28a-sam2-I189V / V266H / N371R as an example, using plasmid pET28a-sam2-I189V as a template, primers V266H-F and V266H-R were selected for reverse PCR to construct the recombinant plasmid pET28a-sam2-I189V / V266H. Then, using plasmid pET28a-sam2-I189V / V266H as a template, primers N371R-F and N371R-R were used to introduce a site-directed mutation site via reverse PCR to construct the recombinant plasmid pET28a-sam2-I189V / V266H / N371R. Finally, Dpn was used... Enzyme I recognizes methylation sites and digests the template, transforming it into E. coli BL21(DE3). The transformed bacteria are then plated onto LB agar plates containing Kan and single colonies are allowed to grow. Finally, bacteria are picked and sequenced for verification, yielding recombinant E. coli BL21-pET28a-sam2-I189V / V266H / N371R expressing the mutant I189V / V266H / N371R. Using the same method, we obtained E. coli BL21-pET28a-sam2-I189V / V266H, E. coli BL21-pET28a-sam2-I189V / N371R, E. coli BL21-pET28a-sam2-I189V / D200E, E. coli BL21-pET28a-sam2-I189V / Q234N, E. coli BL21-pET28a-sam2-V266H / N371R, E. coli BL21-pET28a-sam2-N371R / D200E, E. coli BL21-pET28a-sam2-N371R / Q234N, and E. coli... BL21-pET28a-sam2-V266H / Q234N / N371R, E. coli BL21-pET28a-sam2-I189V / V266H / Q234N / N371R.

[0070] Mutant screening:

[0071] 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 15 constructed mutant strains and WT were induced at 25℃ for 28 h, and their enzyme activities were then measured. The culture conditions were as follows: the seed culture, which had been cultured at 37℃ and 220 rpm for 12 h, was transferred to the fermentation medium at a 2% inoculum size and cultured for approximately 1-2 h to allow the OD to reach optimal levels. 600After adjusting the concentration to 0.6-0.8, add IPTG to a final concentration of 0.1 mM and incubate at 25℃ and 220 rpm for 24 h. At the end of the incubation, control the cell concentration in the fermentation broth to OD. 600 It is approximately 3.3 ± 0.3.

[0072] Take 2 mL of fermentation broth and incubate at 4℃ and 8000 r·min -1 Centrifuge for 5 min, resuspend and wash the bacterial cells with 50 mM Tris-HCl (pH 8.0) buffer, centrifuge for 5 min, and repeat the washing twice. Take 1 mL of buffer to suspend the bacterial cells, sonicate to disrupt, and then incubate at 4℃ and 12000 rpm. -1 Centrifuge for 10 min, and the supernatant is the crude enzyme solution. Its crude enzyme activity is then measured. The mutant crude enzyme is further purified using nickel column affinity chromatography, and its specific enzyme activity is measured under optimal conditions.

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

[0074] Table 3 Enzyme activity of mutants

[0075] The results are shown in Figure 1 and Table 3. Compared with the original enzyme activity, the specific enzyme activity was 0.2466 ± 0.0022 U·mg. -1 The specific enzyme activities of the mutants I189V, V266H, N371R, I189V / V266H, I189V / N371R, I189V / D200E, I189V / Q234N, V266H / N371R, N371R / D200E, N371R / Q234N, I189V / V266H / N371R, V266H / Q234N / N371R, and I189V / V266H / Q234N / N371R all showed varying degrees of improvement. The crude enzyme activity of mutant I189V was 0.0451 U·ml. -1 The specific enzyme activity was 0.3962 ± 0.0581 U·mg. -1 The WT of the mutant V266H was increased by 60.67% compared to the parental strain. The crude enzyme activity of the mutant V266H was 0.0863 U·ml. -1 The specific enzyme activity was 0.9189 ± 0.0166 U·mg. -1 The WT of the mutant was increased by 272.63% compared to the parental strain. The crude enzyme activity of the mutant N371R was 0.1234 U·ml. -1 The specific enzyme activity was 1.2128 ± 0.0423 U·mg. -1 The WT of the mutant I189V / V266H was increased by 391.81% compared to the parental mutant. The crude enzyme activity of the mutant I189V / V266H was 0.3247 U·mL⁻¹. -1The specific enzyme activity was 3.5833 ± 0.1373 U·mg. -1 The WT of the mutant I189V / N371R was increased by 1353.08% compared to the parent. The crude enzyme activity of the mutant I189V / N371R was 0.1012 U·ml. -1 The specific enzyme activity was 1.092 ± 0.0497 U·mg. -1 The WT of the mutant I189V / D200E was increased by 342.82% compared to the parental strain. The crude enzyme activity of the mutant I189V / D200E was 0.0541 U·ml. -1 The specific enzyme activity was 0.6387 ± 0.0798 U·mg. -1 The WT of the mutant I189V / Q234N was increased by 159.00% compared to the parent. The crude enzyme activity of the mutant I189V / Q234N was 0.0661 U·ml. -1 The specific enzyme activity was 0.6102 ± 0.0282 U·mg. -1 The WT of the mutant V266H / N371R was increased by 147.45% compared to the parent. The crude enzyme activity of the mutant V266H / N371R was 0.2602 U·ml. -1 The specific enzyme activity was 2.8615 ± 0.1585 U·mg. -1 Compared to the parental WT, the activity was increased by 1060.38%. The crude enzyme activity of the mutant N371R / D200E was 0.0868 U·ml. -1 The specific enzyme activity was 0.8382 ± 0.0368 U·mg. -1 The WT of the mutant N371R / Q234N was increased by 239.90% compared to the parent. The crude enzyme activity of the mutant N371R / Q234N was 0.1194 U·ml. -1 The specific enzyme activity was 1.2356 ± 0.032 U·mg. -1 The WT of the mutant V266H / Q234N / N371R was increased by 401.05% compared to the parent. The crude enzyme activity of the mutant V266H / Q234N / N371R was 0.1063 U·ml. -1 The specific enzyme activity was 1.0871 ± 0.0694 U·mg. -1 The WT of the strain was increased by 340.84% ​​compared to the parent strain. The crude enzyme activity of I189V / V266H / N371R was 0.0412 U·ml. -1 The specific enzyme activity was 0.4887 ± 0.0325 U·mg. -1 The WT of the strain was increased by 98.18% compared to the parent strain. The crude enzyme activity of I189V / V266H / Q234N / N371R was 0.0887 U·ml. -1 The specific enzyme activity was 0.973 ± 0.0601 U·mg. -1 Compared to the parent's WT, it increased by 294.57%.

[0076] The similar increase in enzyme activity and specific enzyme activity indicates that the mutation mainly improved catalytic efficiency without changing the expression level and solubility; the increase in enzyme activity was much greater than the increase in specific enzyme activity, indicating that the mutation significantly increased the expression level, but the improvement in catalytic efficiency was limited; the increase in enzyme activity was much smaller than the increase in specific enzyme activity, indicating that the mutation significantly improved catalytic efficiency, but may have reduced the expression level.

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

[0078] Wild-type E. coli BL21-pET28a-sam2 and mutant strains E. coli BL21-pET28a-sam2-I189V, E. coli BL21-pET28a-sam2-V266H, E. coli BL21-pET28a-sam2-N371R, E. coli BL21-pET28a-sam2-I189V / V266H, E. coli BL21-pET28a-sam2-I189V / N371R, E. coli BL21-pET28a-sam2-I189V / D200E, E. coli BL21-pET28a-sam2-I189V / Q234N, E. coli BL21-pET28a-sam2-V266H / N371R, E. coli BL21-pET28a-sam2-N371R / D200E, E. coli BL21-pET28a-sam2-N371R / Q234N, E. coli BL21-pET28a-sam2-V266H / Q234N / N371R, E. coli BL21-pET28a-sam2-I189V / V266H / N371R, and E. coli BL21-pET28a-sam2-I189V / V266H / Q234N / N371R were activated by streaking on plates. Single colonies were picked and transferred to 10 mL of a solution containing Kan. In LB liquid medium, cultured at 37℃ and 220 rpm for 12 h, then transferred at a 2% inoculum to 50 mL TB medium containing Kan, and cultured at 37℃ and 220 rpm for approximately 1.5-2.5 h until the biomass OD... 600 When the concentration of the bacterial cell reached 0.6-0.8, IPTG was added to a final concentration of 0.1 mM, and the cells were incubated at 25°C and 220 rpm for 24 h. The cells were collected and washed twice with 50 mM Tris-HCl (pH 8.0) buffer for 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, reacted at 220 rpm and 37℃ for 2 h, then the reaction was terminated by adding an equal volume of 20% perchloric acid, centrifuged and the supernatant was collected. The yield of SAM was determined by HPLC. The specific results are shown in Figure 2.

[0080] Figure 2 shows that after 2 hours of whole-cell reaction, the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V was 479.91 mg / L, which was approximately 73.68% higher than that of wild-type WT (276.32 mg / L); the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-V266H was 509.03 mg / L, which was approximately 84.22% higher than that of wild-type WT (276.32 mg / L); and the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-N371R was 475.174 mg / L, which was approximately 71.97% higher than that of wild-type WT (276.32 mg / L). The concentration of S-adenosylmethionine synthesized by BL21-pET28a-sam2-I189V / V266H was 550.57 mg / L, which was approximately 99.25% higher than that of wild-type WT (276.32 mg / L); the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / N371R was 1119.34 mg / L, which was approximately 305.09% higher than that of wild-type WT (276.32 mg / L); the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / D200E was 695.81 mg / L, which was approximately 151.81% higher than that of wild-type WT (276.32 mg / L); E. coli The concentration of S-adenosylmethionine synthesized by BL21-pET28a-sam2-I189V / Q234N was 581.65 mg / L, which was approximately 110.51% higher than that of wild-type WT (276.32 mg / L); the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-V266H / N371R was 1087.26 mg / L, which was approximately 293.84% higher than that of wild-type WT (276.32 mg / L); the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-N371R / D200E was 738.50 mg / L, which was approximately 167.39% higher than that of wild-type WT (276.32 mg / L); E. coli The concentration of S-adenosylmethionine synthesized by BL21-pET28a-sam2-N371R / Q234N was 841.597 mg / L, which was about 204.71% higher than that of wild-type WT (276.32 mg / L); the highest concentration of SAM synthesized by E. coli BL21-pET28a-sam2-I189V / V266H / N371R was 1552.81 mg / L, which was about 461% higher than that of wild-type WT (276.32 mg / L).The concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-V266H / Q234N / N371R was 1572.81 mg / L, which was approximately 469.57% higher than that of wild-type WT (276.32 mg / L); the concentration of S-adenosylmethionine synthesized by E. coli BL21-pET28a-sam2-I189V / V266H / Q234N / N371R was 1751.01 mg / L, which was approximately 534.42% higher than that of wild-type WT (276.32 mg / L).

[0081] In summary, this invention heterologously expresses S-adenosylmethionine synthase from *Saccharomyces cerevisiae* to construct recombinant *E. coli*, and modifies the S-adenosylmethionine synthase protein through site-directed mutagenesis to improve enzyme activity. Through whole-cell catalysis, the mutants I189V / V266H / N371R, V266H / Q234N / N371R, and I189V / V266H / Q234N / N371R showed the best performance, achieving S-adenosylmethionine synthesis yields of 1552.81 mg / L, 1572.81 mg / L, and 1751.01 mg / L, respectively. This invention successfully constructs an exogenous expression pathway for S-adenosylmethionine, further improves enzyme activity, saves raw materials and costs, improves economic efficiency, and lays the foundation for industrial production.

[0082] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An S-adenosylmethionine synthase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, perform any of the following mutations: (1) Mutate isoleucine at position 189 to valine; (2) Mutate valine at position 266 to histidine; (3) Mutate asparagine at position 371 to arginine; (4) Mutate glutamine at position 234 to asparagine; (5) Mutate isoleucine at position 189 to valine and valine at position 266 to histidine; (6) Mutate isoleucine at position 189 to valine and asparagine at position 371 to arginine; (7) Mutate isoleucine at position 189 to valine and aspartic acid at position 200 to glutamic acid; (8) Mutate isoleucine at position 189 to valine and glutamine at position 234 to asparagine; (9) Mutate valine at position 266 to histidine and asparagine at position 371 to arginine; (10) Mutate asparagine at position 371 to arginine and aspartic acid at position 200 to glutamic acid; (11) Mutate asparagine at position 371 to arginine and glutamine at position 234 to asparagine; (12) Mutate valine at position 266 to histidine, glutamine at position 234 to asparagine, and asparagine at position 371 to arginine; (13) Mutate isoleucine at position 189 to valine, valine at position 266 to histidine, and asparagine at position 371 to arginine; (14) Mutate isoleucine at position 189 to valine, valine at position 266 to histidine, glutamine at position 234 to asparagine, and asparagine at position 371 to arginine.

2. The gene encoding the mutant of claim 1.

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

4. The expression of the mutant of claim 1, or the expression vector of claim 3 carried by the microbial cell.

5. A recombinant Escherichia coli, characterized in that, Using Escherichia coli BL21(DE3) as the host and pET28a as the expression vector, the mutant described in claim 1 was expressed.

6. A method for producing S-adenosylmethionine, characterized in that, The reaction is carried out using the mutant of claim 1, the microbial cell of claim 4, or the recombinant Escherichia coli of 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, characterized in that, The reaction should be carried out at pH 7–9 and 37–60°C for at least 2 hours.

8. A method for improving the catalytic activity of S-adenosylmethionine synthase, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, perform any of the following mutations: (1) Mutate isoleucine at position 189 to valine; (2) Mutate valine at position 266 to histidine; (3) Mutate asparagine at position 371 to arginine; (4) Mutate glutamine at position 234 to asparagine; (5) Mutate isoleucine at position 189 to valine and valine at position 266 to histidine; (6) Mutate isoleucine at position 189 to valine and asparagine at position 371 to arginine; (7) Mutate isoleucine at position 189 to valine and aspartic acid at position 200 to glutamic acid; (8) Mutate isoleucine at position 189 to valine and glutamine at position 234 to asparagine; (9) Mutate valine at position 266 to histidine and asparagine at position 371 to arginine; (10) Mutate asparagine at position 371 to arginine and aspartic acid at position 200 to glutamic acid; (11) Mutate asparagine at position 371 to arginine and glutamine at position 234 to asparagine; (12) Mutate valine at position 266 to histidine, glutamine at position 234 to asparagine, and asparagine at position 371 to arginine; (13) Mutate isoleucine at position 189 to valine, valine at position 266 to histidine, and asparagine at position 371 to arginine; (14) Mutate isoleucine at position 189 to valine, valine at position 266 to histidine, glutamine at position 234 to asparagine, and asparagine at position 371 to arginine.

9. The use of the mutant of claim 1, or the microbial cell of claim 4, or the recombinant Escherichia coli of claim 5 in the preparation of S-adenosylmethionine or products containing S-adenosylmethionine.

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

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