A mutant of s-adenosylmethionine synthetase and its use in the production of s-adenosylmethionine

Abstract

The application belongs to the technical field of genetic engineering and enzyme engineering, and discloses a S-adenosyl methionine synthetase mutant and application thereof in S-adenosyl methionine production. I22M The mutant SAM2 is obtained by replacing the 22th amino acid from isoleucine (I) to methionine (M) through site-specific mutation on wild-type S-adenosyl methionine synthetase from Saccharomyces cerevisiae. The yield of S-adenosyl methionine is analyzed by high performance liquid chromatography (HPLC). The results show that, compared with the wild-type enzyme, the mutant SAM2 I22M significantly improves the synthesis efficiency of S-adenosyl methionine by 36%, which indicates that the mutant has potential superiority in producing S-adenosyl methionine.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the fields of genetic engineering and enzyme engineering, and particularly relates to an S-adenosylmethionine synthase mutant and its application in the production of S-adenosylmethionine. Background Technology

[0002] S-adenosyl-L-methionine (SAM), also known as adenosylmethionine, is an important bioactive metabolite in living organisms. It participates extensively in transmethylation, transsulfation, and transaminopropylation reactions, playing a crucial role in the synthesis and metabolism of proteins, nucleic acids, and phospholipids. Due to its applications in liver protection, joint health, and mood regulation, the demand for SAM continues to increase in the pharmaceutical, health food, and related biomanufacturing fields.

[0003] Currently, the main methods for preparing SAM include chemical synthesis, microbial fermentation, enzymatic synthesis, and whole-cell catalysis. Among these, whole-cell catalysis eliminates the need for complex enzyme separation and purification, and leverages intracellular coenzyme regeneration and metabolic maintenance systems to achieve substrate transformation. It features a relatively simplified process and good catalytic system stability, making it an important research direction for SAM biomanufacturing.

[0004] In whole-cell catalytic systems, SAM is typically generated by the reaction of ATP and L-methionine catalyzed by S-adenosylmethionine synthase. Existing research has focused on enzyme source screening, heterologous expression, and optimization of expression conditions for this catalytic step. For example, by comparing S-adenosylmethionine synthases from different sources, some studies have found that SAM2 from Saccharomyces cerevisiae has good application potential in relevant catalytic systems, and further improvements in enzyme yield and overall conversion levels can be achieved through host construction, expression regulation, or optimization of fermentation parameters.

[0005] However, the aforementioned improvements mainly focus on increasing SAM2 expression levels, improving the cellular catalytic environment, or enhancing the supply of the enzyme in the system. For whole-cell synthesis systems with SAM2 as the core catalytic unit, when the supply of substrates ATP and L-methionine is constant, the intrinsic catalytic capacity of SAM2 remains a crucial factor determining SAM production efficiency. If the intrinsic enzyme activity of natural SAM2 is insufficient, even if increasing expression levels increases enzyme protein accumulation, there may still be limitations in the conversion efficiency per enzyme molecule and the potential for further synergistic effects on the catalytic system, thus affecting SAM yield improvement and process economics.

[0006] Therefore, it is still necessary to modify the structure of the SAM2 enzyme itself to obtain S-adenosylmethionine synthase variants with improved catalytic performance, thereby providing new technical solutions for improving SAM synthesis efficiency and reducing production costs. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides an S-adenosylmethionine synthase mutant and its application in the production of S-adenosylmethionine.

[0008] This invention is achieved as follows: an S-adenosylmethionine synthase mutant, SAM2. I22M The amino acid sequence is shown in SEQ ID NO.4.

[0009] Furthermore, the fusion protein obtained by fusing the mutant protein described in SEQ ID NO.4 with a protein tag.

[0010] Furthermore, the gene encoding the mutant protein or fusion protein described in SEQ ID NO.4.

[0011] Furthermore, the gene-encoding cassette, recombinant vector, recombinant microorganism, or in vitro recombinant cell may be used.

[0012] Furthermore, the application of the mutant, fusion protein, encoding gene or expression cassette having the above-mentioned encoding gene, recombinant vector, recombinant microorganism or ex vivo recombinant cell in the preparation of S-adenosylmethionine synthase.

[0013] Another object of the present invention is to provide a method for increasing the activity of S-adenosylmethionine synthase, comprising the following steps:

[0014] The S-adenosylmethionine synthase was mutated as follows: isoleucine I at position 22 of SEQ ID NO.2 was mutated to methionine M.

[0015] Furthermore, the polynucleotide encoding the gene is shown in SEQ ID NO.3.

[0016] Furthermore, the application of mutants, fusion proteins, encoding genes or expression cassettes containing the above-mentioned encoding genes, recombinant vectors, recombinant microorganisms or ex vivo recombinant cells in the preparation of S-adenosylmethionine.

[0017] Furthermore, the application process includes preparing S-adenosylmethionine by whole-cell catalysis of recombinant microorganisms, wherein the recombinant microorganisms are obtained by transferring the recombinant vector encoding the gene into engineered bacteria BL10;

[0018] The engineered bacterium BL10 described in this scheme has a clear origin, originating from the wild-type Bacillus licheniformis strain WX-02. This WX-02 strain has been deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M208065, on April 24, 2008. Based on this wild-type WX-02 strain, and following the modification origin described in Zhou Yinhua's "Modification of Bacillus licheniformis WX-02 for High-Efficiency Expression of Exogenous Proteins" (Master's Thesis, Huazhong Agricultural University, 2019), 10 genes related to its extracellular secretion expression were further knocked out to obtain the engineered bacterium BL10. Therefore, BL10 is not a naturally occurring strain obtained through re-screening, but rather an engineered strain formed by gene knockout modification based on the already deposited Bacillus licheniformis WX-02, and can be used as a host bacterium for high-efficiency expression of exogenous proteins.

[0019] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0020] This invention utilizes site-directed mutagenesis to mutate isoleucine at position 22 of S-adenosylmethionine synthase to methionine (this invention names the mutant SAM2). I22M Compared to the control strain expressing wild-type SAM2, the expression mutant SAM2... I22M The recombinant strain produced 3.99 mM of SAM, a significant increase of 36%, suggesting it is more suitable for industrial production. Experiments show that the recombinant Bacillus licheniformis strain expressing the S-adenosylmethionine synthase mutant has excellent SAM production capacity and better prospects for industrial application compared to recombinant strains containing the unmutated S-adenosylmethionine synthase gene.

[0021] The S-adenosylmethionine synthase mutant SAM2 provided by this invention I22M This mutant is a modified SAM2 protein with specific amino acid sites, as shown in SEQ ID NO.4. By directionally adjusting key sites of the SAM2 protein, this mutant alters the local structural state of the enzyme molecule, enhancing its reactivity in the catalytic conversion of ATP and L-methionine. Compared to the unmodified SAM2 enzyme, this mutant exhibits superior SAM synthesis performance under the same catalytic system, thereby improving the efficiency of substrate-to-target product conversion. Fusion proteins, encoding genes, expression cassettes, recombinant vectors, and recombinant cells constructed based on this mutant can further integrate these catalytic advantages into enzyme preparation and whole-cell catalytic systems, providing more suitable biocatalytic elements for improving SAM production efficiency and reducing unit product preparation costs, thus possessing significant industrial application value.

[0022] Existing technologies for increasing SAM yield typically focus on screening enzyme sources, increasing SAM2 expression levels, or optimizing host and culture conditions. Their underlying logic remains primarily based on "increasing enzyme supply" or "improving the external reaction environment," failing to directly overcome the limitation of natural SAM2's inherent catalytic capacity. This invention, however, employs a site-specific modification approach targeting the SAM2 protein itself to obtain SAM2... I22M This mutant improves the catalytic performance of enzymes at the molecular structural level. The approach is not a simple optimization of conventional factors such as expression levels and culture parameters, but rather establishes a new structure-function relationship through specific amino acid substitutions, directly corresponding to increased enzyme activity and enhanced SAM synthesis. For those skilled in the art, existing technology alone cannot predict that mutations at the I22M site will produce a technical effect beneficial to SAM synthesis, nor does it provide clear guidance on using this specific site as an effective target for modification. Therefore, this invention possesses outstanding substantive features and significant progress.

[0023] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are as follows: Compared with the unmodified SAM2 enzyme, the mutant of the present invention can exhibit better SAM synthesis performance under the same catalytic system, with a 36% increase in yield and improved efficiency of substrate to target product conversion. In industrial production, it can improve SAM production efficiency and reduce unit product preparation cost, and has good industrial application value.

[0024] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad: The present invention provides a new idea for industrial production of SAM. By modifying the enzyme molecules with protein engineering, the efficiency of SAM production in whole-cell catalysis is improved and the production cost is reduced.

[0025] (3) The technical solution of the present invention solves the technical problem that people have been eager to solve but have never been able to achieve: by modifying the SAM2 protein, the SAM2 protein’s reaction ability in the conversion of ATP and L-methionine is improved.

[0026] (4) The technical solution of the present invention overcomes technical bias: Existing technologies mainly improve SAM yield by screening enzyme sources, upregulating gene expression, and optimizing host and culture conditions, but have not yet addressed the fundamental bottleneck of the limited catalytic activity of SAM2 itself. The present invention modifies the SAM2 protein at specific sites to improve its catalytic performance at the level of enzyme molecular structure. Attached Figure Description

[0027] Figure 1 This is a flowchart of a method for improving the activity of S-adenosylmethionine synthase provided in an embodiment of the present invention.

[0028] Figure 2This is a comparison chart of the whole-cell catalytic SAM synthesis yields of the S-adenosylmethionine synthase mutant and the wild-type strain provided in the embodiments of the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] The applicant replaced the 22nd amino acid of the wild-type S-adenosylmethionine synthase derived from Saccharomyces cerevisiae with methionine (M) instead of isoleucine (I), thus obtaining the S-adenosylmethionine synthase mutant SAM2 of this invention. I22M As shown in SEQ ID NO.4.

[0031] The scope of protection of this invention also includes:

[0032] The fusion protein obtained by fusing the mutant protein described in SEQ ID NO.4 with a protein tag.

[0033] The gene encoding the mutant protein or fusion protein described in SEQ ID NO.4.

[0034] Expression cassettes, recombinant vectors, recombinant microorganisms, or in vitro recombinant cells containing the above-mentioned coding genes.

[0035] The above-mentioned mutants, fusion proteins, mutants or fusion protein encoding genes described in SEQ ID NO.4, expression cassettes having the above-mentioned encoding genes, recombinant vectors, recombinant microorganisms or ex vivo recombinant cells in the preparation of S-adenosylmethionine synthase.

[0036] like Figure 1 As shown in the embodiment of the present invention, a method for improving the activity of S-adenosylmethionine synthase includes the following steps:

[0037] S101, the S-adenosylmethionine synthase was mutated as follows: isoleucine I at position 22 of SEQ ID NO.2 was mutated to methionine M.

[0038] The coding gene of the mutant described in SEQ ID NO.4 is preferably that shown in SEQ ID NO.3.

[0039] The above-mentioned mutants, fusion proteins, mutants or fusion proteins encoding genes described in SEQ ID NO.4, expression cassettes having the above-mentioned encoding genes, recombinant vectors, recombinant microorganisms or ex vivo recombinant cells in the preparation of S-adenosylmethionine.

[0040] The preferred application described above includes the following steps: preparing S-adenosylmethionine by whole-cell catalysis of recombinant microorganisms, wherein the recombinant microorganisms are obtained by transferring a recombinant vector containing the above-mentioned encoding gene into engineered bacteria BL10;

[0041] Wild-type Bacillus licheniformis WX-02 was isolated from salt mine soil samples in Yingcheng City, Hubei Province, China. The engineered strain BL10 is a strain of Bacillus licheniformis that can be used to efficiently express exogenous proteins after 10 genes that are expressed in the wild-type WX-02 strain are knocked out.

[0042] Specific implementation of the present invention:

[0043] The culture media involved in the following examples are as follows:

[0044] LB liquid medium: yeast extract 5 g·L -1 10 g / L of peptone -1 NaCl 10 g·L -1 pH 7.0.

[0045] LB solid medium: yeast extract 5 g·L -1 10 g / L of peptone -1 NaCl 10 g·L -1 15 g·L agar powder -1 .

[0046] Fermentation medium: 5 g·L -1 24 g·L yeast powder -1 Tryptone 12 g·L -1 K2HPO4·3H2O 16.4 g·L -1 KH2PO4 2.3 g·L -1 pH 7.0, 115 o Sterilize at 20 min.

[0047] Whole-cell catalytic reaction system: ATP 10 mM, Met 10 mM, Tris 50 mM, KCl 10 mM, MgCl2·6H2O 20 mM, Tween 80 1%.

[0048] Example 1:

[0049] Construction of wild-type S-adenosylmethionine synthase (SAM2):

[0050] Primer pair SAM2-F / SAM2-R was designed to clone an S-adenosylmethionine synthase gene, SAM2 (GenBank: NC_001136.10, gene sequence shown in SEQ ID NO.1, protein encoded by SEQ ID NO.2), from *Saccharomyces cerevisiae* (GenBank: GCA_000146045.2). Using the *Bacillus subtilis* 168 genome as a template, the P43 promoter was amplified using primers P43-F and P43-R; the amylase terminator TamyL was amplified using primers TamyL-F and TamyL-R. SOE-PCR was then performed using primers P43-F and TamyL-R to obtain the fusion fragment P43-SAM2-TamyL. Using plasmid pHY300PLK as a template, and primers 300-amp-T5-F and 300-amp-T5-R, full-plasmid PCR amplification was performed to obtain the linearized pHY300PLK vector. After electrophoresis, the amplified products were purified and recovered using an Omega DNA agarose gel extraction kit. The fusion fragment was then fused with the linearized pHY300PLK vector using the ClonExpress II one-step cloning kit to obtain the recombinant plasmid.

[0051] P43-F: GCTCATGAGACAATAACCCGCGGAATTTCCAATTTCA

[0052] P43-R:AGTTTTGCTCTTGGACATTATATATTCCTCCTTTCT

[0053] SAM2-F: AGAAAGGAGGAATATATAATGTCCAAGAGCAAAACT

[0054] SAM2-R:TCCGTCCTCTCTGCTCTTTTAAAATTCCAATTTCTT

[0055] TamyL-F: AAGAAATTGGAATTTTAAAAGAGCAGAGAGGACGGA

[0056] TamyL-R:GAGTAAACTTGGTCTGACAGTAATGATGACGGTCCAGC

[0057] 300-amp-T5-F:CTGTCAGACCAAGTTTACTCATA

[0058] 300-amp-T5-R: GGGTTATTGTCTCATGAGCG

[0059] The fused recombinant plasmid was transformed into E. coli DH5α competent cells, and positive colonies were screened using LB agar plates containing tetracycline. 37 o After overnight culture on a C shaker, the plasmid was extracted and sequenced for verification. The recombinant plasmid with correct sequencing was named pHY300-P43-SAM2.

[0060] The correctly sequenced plasmid pHY300-P43-SAM2 was electroporated into wild-type Bacillus licheniformis WX-02 cells at 2.4 kV and 4.8-5.2 ms using a BIO-RAD Pulse controller (GenePulser™). This plasmid, combined with the knockout of 10 genes involved in its extracellular secretion expression, was then used to create the engineered strain BL10 competent cells for high-efficiency expression of exogenous proteins. 800 μL of recovery medium was rapidly added, and the mixture was incubated at 37°C. o After resuscitation at 110 rpm for 3 h, the sample was spread onto selection medium containing tetracycline resistance and incubated at 37°C. o After incubating overnight at C, transformants were selected, and colony PCR was performed to confirm their correctness. The cultures were then inoculated into LB medium containing 20 μg / mL tetracycline resistance and incubated at 37°C. o Wild-type S-adenosylmethionine synthase (SAM2) was obtained by culturing the bacteria in a shaker for 14 h.

[0061] The preparation method of the engineered bacteria BL10 is as follows:

[0062] First, taking the knockout of flagellin Hag as an example, the recombinant knockout vector T2-hag was constructed.

[0063] Based on the whole genome information of *B. licheniformis* WX-02, the sequence of the *hag* gene was found on the NCBI website. Primers T2-hag-F1 and T2-hag-R1 were designed approximately 500 bp upstream of the *hag* gene, and primers T2-hag-F2 and T2-hag-R2 were designed approximately 500 bp downstream of the *hag* gene. Using the genomic DNA of *B. licheniformis* WX-02 as a template, approximately 500 bp fragments of each of the upstream and downstream homologous arms (A, B) were amplified. Then, using primers T2-hag-F1 and T2-hag-R2, SOE-PCR was performed to ligate the upstream and downstream homologous arms, yielding fragment A+B, approximately 1000 bp in size. A+B was double-digested with restriction endonucleases XbaⅠ and SacⅠ. The resulting *hag* (A+B) fragment was ligated to the temperature-sensitive knockout plasmid T2(ori), which had undergone the same double-digestion treatment. The ligation product was transformed into *E. coli* DH5α. After the transformant was confirmed as a positive clone by PCR, the plasmid was extracted and sequenced for verification. The resulting recombinant knockout plasmid was T2-hag.

[0064] The correctly sequenced plasmid T2-hag was electroporated into *B. licheniformis* WX-02 competent cells using a bio-pulse converter (BIO-RAD) at 2.4 kV and a pulse duration of 4.8–5.2 ms. The cells were then incubated at 37°C. o Cultured overnight at C, transformants were selected and verified by colony PCR. Positive clones were selected for single / double crossover subculturing.

[0065] Single exchange: Select the successfully electroporated transformants from the previous step and inoculate them into 5 mL of liquid LB medium (containing 20 μg / mL Kan antibiotic), incubate at 45°C. o C. Incubate at 180 rpm for 10–12 h for the first generation. Dilute the third-generation bacterial culture 10–6 times, and spread 100 μL of the diluted culture evenly on agar plates containing 20 μg / mL Kan antibiotic. Incubate at 45°C. o Culture under C conditions; after single colonies grow on the plate, pick a small number of single colonies and streak them on the corresponding antibiotic plate. After 5-6 hours, pick an appropriate amount of cells for single crossover verification; if the verification is correct, perform double crossover passage.

[0066] Double exchange: Take an appropriate amount of single colony that has been verified by single exchange and add it to 5 mL of liquid LB medium. Incubate at 37°C. o C. Subculture at 180 rpm for a total of 3 generations; dilute the culture medium after the third subculture by 10. -6 Take 100 μL and spread it evenly on an LB solid medium plate, incubate at 37°C. oCultured at C; after single colonies grew on the plates, single colonies were picked and inoculated onto LB agar plates and LB agar plates containing 20 μg / mL Kan antibiotic, respectively, and cultured at 37°C. o Cultured at C for 12 h; single colonies that did not grow on LB agar plates containing 20 μg / mL Kan antibiotic were selected for double-exchange colony PCR identification, and the strains that were verified to be double-exchange were named BL1.

[0067] Following the above method, knockout plasmids for mpr, vpr, aprX, epr, bpr, wprA, aprE, amyL, and bprA were constructed. These nine genes were then sequentially knocked out on the engineered strain BL1 to obtain strain BL10.

[0068] Example 2:

[0069] Construction of S-adenosylmethionine synthase SAM2 mutant strain:

[0070] Site-directed mutagenesis was performed on the SAM2 catalytic active site, followed by reverse PCR amplification. The mutation was then inserted into the gene sequence. Specifically, isoleucine was mutated to methionine at position 22 of the catalytic domain of the S-adenosylmethionine synthase molecule. Using primers I22M-F and I22M-R, and with the plasmid from Example 1 as a template, the linear plasmid of the entire SAM2 mutant sequence I22M was amplified (the S-adenosylmethionine synthase SAM2 gene after point mutation is shown in SEQ ID NO.3, and the encoded protein is shown in SEQ ID NO.4).

[0071] I22M-F: GGTCACACATCTTGTCTGGGTGACCTTCACCGA

[0072] I22M-R:CAGACAAGATGTGTGACCAAGTTTCTGATGCTAT

[0073] The plasmid was recovered and purified according to the method described in Example 1, and then processed using the ClonExpress II one-step cloning kit to transform it into E. coli DH5α competent cells. Positive colonies were screened using LB agar plates containing tetracycline. After verification of correctness using specific primers, the colonies were inoculated into LB medium containing tetracycline resistance and incubated at 37°C. o After overnight culture on a C-type shaker, plasmid pHY300-P43-SAM2 was extracted. I22M Sequencing was performed to verify the sequence; the correctly sequenced recombinant plasmid was named pHY300-P43-SAM2. I22M .

[0074] The plasmid was electroporated into Bacillus licheniformis BL10 and plated onto a selection medium containing tetracycline resistance. The plasmid was then heated to 37°C. o Cultured overnight at C, transformants were selected, and verified transformants were inoculated into LB medium containing 20 μg / mL tetracycline resistance, and incubated at 37°C. o After culturing in a shaker for 14 h, a mutant strain of S-adenosylmethionine synthase SAM2 was obtained.

[0075] Example 3:

[0076] like Figure 2 Comparison of SAM production yields between S-adenosylmethionine synthase (SAM2) mutant strain and wild-type strain via whole-cell catalysis:

[0077] S-adenosylmethionine synthase SAM2 mutant strain and S-adenosylmethionine synthase SAM2 wild-type strain were activated from preserved glycerol tubes and inoculated into LB medium containing 20 μg / mL tetracycline resistance. The culture was then incubated at 37°C. o C, cultured for 14 h. Transferred to fermentation medium, inoculum size 3%, at 37°C. o Cultured at C for 24 h. The fermentation broth (containing 20 OD cells) was centrifuged at 7500 rpm for 5 min, the supernatant was removed, and the cells were collected. The cells were then washed with PBS buffer and centrifuged again to collect the supernatant (repeated twice) to obtain the whole-cell catalyst. The cells were resuspended in 10 mL of the reaction mixture and the catalytic reaction was carried out in a 50 mL Erlenmeyer flask at 37°C. o The reaction was carried out at a constant temperature of C for 12 h in a shaker. After the reaction was completed, 1 mL of the reaction solution was taken, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. The supernatant was diluted 20 times and the amount of SAM generated was determined by high performance liquid chromatography.

[0078] HPLC conditions: Agilent HPLC system (equipped with a UV detector), injection volume 10 μL, mobile phase: 0.01 mol / L ammonium formate:methanol = 97:3, pH = 3.0, column: Hypersil ODS C18 column (4.6 mm × 250 mm, 2.5 μm film), column temperature: 30 °C o C.

[0079] The results showed that the expression mutant SAM2 I22M The recombinant strain achieved a whole-cell catalytic production of SAM of 3.99 mM, which was significantly increased by 36% compared with the control.

[0080] SEQ ID NO.1

[0081]

[0082] SEQ ID NO.2

[0083] MSKSKTFLFTSESVGEGHPDKICDQVSDAILDACLEQDPFSKVACETAAKTGMIMVFGEITTKARLDYQQIVRDTIKKIGYDDSAKGFDYKTCNVLVAIEQQSPDIAQGLHYEKSLEDLGAGDQGIMFGYATDETPEGLPLTILLAHKLNMAMADARRDGSLPWLRPDTKTQVTVEYEDDNGRWVPKRIDTVVISAQHADEISTADLRTQLQKDIVEKVIPKDMLDENTKYFIQPSGRFVIGGPQGDAGLTGRKIIVDAYGGASSVGGGAFSGKDYSKVDRSAAYAARWVAKSLVAAGLCKRVQVQFSYAIGIAEPLSLHVDTYGTATKSDDEIIEIIKKNFDLRPGVLVKELDLARPIYLPTASYGHFTNQEYSWEKPKKLEF

[0084] SEQ ID NO.3

[0085]

[0086] SEQ ID NO.4

[0087] MSKSKTFLFTSESVGEGHPDKMCDQVSDAILDACLEQDPFSKVACETAAKTGMIMVFGEITTKARLDYQQIVRDTIKKIGYDDSAKGFDYKTCNVLVAIEQQSPDIAQGLHYEKSLEDLGAGDQGIMFGYATDETPEGLPLTILLAHKLNMAMADRDGSLPWLRPDTKTQVTVEYEDDNGRWVPKRIDTV VISAQHADEISTADLRTQLQKDIVEKVIPKDMLDENTKYFIQPSGRFVIGGPQGDAGLTGRKIIVDAYGGASSVGGGAFSGKDYSKVDRSAAYAARWVAKSLVAAGLCKRVQVQFSYAIGIAEPLSLHVDTYGTATKSDDEIIEIIKKNFDLRPGVLVKELDLARPIYLPTASYGHFTNQEYSWEKPKKLEF

[0088] In the description of this invention, unless otherwise stated, "a plurality of" means two or more; the terms "upper," "lower," "left," "right," "inner," "outer," "front end," "rear end," "head," "tail," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first," "second," "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0089] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An S-adenosylmethionine synthase mutant, characterized in that, The S-adenosylmethionine synthase mutant SAM2 I22M The amino acid sequence is shown in SEQ ID NO.

4.

2. The S-adenosylmethionine synthase mutant as described in claim 1, characterized in that, The fusion protein obtained by fusing the mutant protein described in SEQ ID NO.4 with a protein tag.

3. The S-adenosylmethionine synthase mutant as described in claim 1, characterized in that, The gene encoding the mutant protein or fusion protein described in SEQ ID NO.

4.

4. The S-adenosylmethionine synthase mutant as described in claim 3, characterized in that, The expression cassette encoding the gene, recombinant vector, recombinant microorganism, or in vitro recombinant cell.

5. The use of the mutant of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, or the expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant cell having the above-mentioned encoding gene as described in claim 4 in the preparation of S-adenosylmethionine synthase.

6. A method for improving the activity of S-adenosylmethionine synthase using the S-adenosylmethionine synthase mutant as described in any one of claims 1-5, comprising the following steps: Step 1: The S-adenosylmethionine synthase is mutated as follows: the isoleucine I at position 22 of SEQ ID NO.2 is mutated to methionine M.

7. The S-adenosylmethionine synthase mutant as described in claim 3, characterized in that, The polynucleotide encoding the gene is shown in SEQ ID NO.

3.

8. The use of the mutant of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, or the expression cassette, recombinant vector, recombinant microorganism or ex vivo recombinant cell having the above-mentioned encoding gene as described in claim 4 in the preparation of S-adenosylmethionine.

9. The application according to claim 8, wherein the application process includes preparing S-adenosylmethionine by whole-cell catalysis of recombinant microorganisms, wherein the recombinant microorganism is obtained by transferring a recombinant vector having the encoding gene of claim 3 into engineered bacteria BL10; Bacillus licheniformis wild-type WX-02 was isolated from salt mine soil samples in Yingcheng City, Hubei Province, China. It can grow in 10% sodium chloride and exhibits high salt tolerance. The engineered strain BL10 is a strain that can be used to efficiently express exogenous proteins after knocking out 10 genes that are expressed in its extracellular secretion on Bacillus licheniformis wild-type WX-02.

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