Methyltransferase mutant for efficiently biocatalytically synthesizing S-adenosylmethionine and analogues thereof and method for applying methyltransferase mutant to alkylation modification

Abstract

The invention discloses a methyltransferase mutant for efficient biocatalytic synthesis of S-adenosylmethionine and analogues thereof and a method for alkylation modification by using the methyltransferase mutant, and belongs to the technical field of bioengineering. According to the present invention, the optimal three mutants M11F / L30I / P209R are screened by using the wild type AteHMT as the template by using the directed evolution means, the S-adenosylmethionine (SAM) and the analogue thereof are synthesized by using the mutants through the biological catalysis, and the catalytic activity of the mutants is 1.5-23.5 times higher than the catalytic activity of the wild type AteHMT; the mutant, carbon methyltransferase CouO from streptomyces reesei and 4 '-O-methyltransferase (Mentha x piperea) FL-4 from mentha flavonoids are subjected to cascade reaction to form a series of alkylate products. The method has the advantages of mild conditions, environmental friendliness, high stereoselectivity, high yield and the like.

Description

Technical Field

[0001] This invention relates to a methyltransferase mutant for the efficient biocatalytic synthesis of S-adenosylmethionine and its analogues, and a method for its alkylation modification, belonging to the field of bioengineering. Background Technology

[0002] Alkylation of hydroxyl, amino, thiol, and activated carbon atoms is an important synthetic reaction. Drug active molecules often contain multiple O, S, C, and N atoms. Chemical alkylation modifications often face challenges such as harsh reaction conditions, complex products, and difficulty in removing byproducts. However, methylation is a ubiquitous reaction in all organisms. This transformation is largely catalyzed by S-adenosylmethionine (SAM, AdoMet)-dependent methyltransferases (MTS). Generally, methylation is a crucial step in the biosynthetic pathway for diversifying the structures of natural products and can be used for chemical, regio, and stereospecific synthesis. Therefore, methyltransferases (MTS) have great potential for the synthesis of active pharmaceutical ingredients (APIs).

[0003] S-adenosylmethionine (SAM)-dependent methyltransferases (MTS) use SAM as a methyl donor to methylate compounds. SAM is an important metabolic intermediate widely found in animals, plants, and microorganisms. Its structure was elucidated in 1951 (The Journal of Biological Chemistry, 1951, 189(1):203-216). It plays the role of a methyl donor in most biological methylation reactions and is the main source of methyl groups (The Journal of Biological Chemistry, 1953, 204(1):403-416). SAM is the second largest cofactor in living organisms after ATP, and its metabolism and balance are related to various aspects of growth, health, and aging. (AccountS of Chemical ReSearch, 2012, 45(4): 555-564.) Many methyltransferases are insensitive to the size of the alkyl substituents in SAM, and if necessary SAM analogues are available, they can catalyze other alkylation reactions. This substrate heterogeneity can be used to expand the structural and functional diversity of chemicals and enable a variety of applications. The structural formulas of SAM and its analogues are as follows:

[0004]

[0005] Currently, the main chemical synthesis method for SAM and its analogues is to use S-adenosine-L-homocysteine ​​(SAH) as a methyl acceptor and directly alkylate it with trifluoroalkyl esters or alkyl bromides (Angewandte Chemie International Edition, 2014, 53(15):3965-3969.). The specific reaction process is as follows:

[0006]

[0007] However, due to the asymmetric substitution of the sulfonium structure in SAM and its analogues, which gives them chiral centers, SAM and its analogues have two epimers: S,S and S,R. The S,S epimers are biologically active, while the S,R epimers inhibit methyltransferases, which is detrimental to the principle of atom economy.

[0008] Enzymatic synthesis has attracted much attention due to its advantages such as mild reaction conditions, high stereoselectivity, and regioselectivity. The technology for synthesizing SAM using enzymes is becoming increasingly mature. However, there are few reports on the enzymatic synthesis of SAM analogs, and the yields are relatively low. Therefore, finding a method for synthesizing SAM and its analogs with high stereoselectivity and high yield is of great significance.

[0009] Conventional chemical alkyl modification often faces challenges such as regioselectivity and stereoselectivity in the synthesis of compounds with multiple nucleophilic attack sites. Therefore, this invention, based on a highly efficient synthetic platform for SAM and its analogues, utilizes a cascade reaction with methyltransferases to obtain alkyl-modified products that are difficult to synthesize using conventional chemical methods, which has significant implications and application prospects. Summary of the Invention

[0010] To address the shortcomings of the aforementioned technologies, this invention aims to modify the halogenated methyltransferase AteHMT derived from Aspergillus terreus using protein engineering techniques to improve the synthesis efficiency of S-adenosylmethionine (SAM) and its analogues. Based on this, a method for alkylation modification is constructed by cascading with methyltransferases, thereby solving the technical problem of lacking a method for synthesizing SAM and its analogues with high stereoselectivity and high yield.

[0011] The first technical solution provided by this invention is a mutant of the halomethyltransferase AteHMT, wherein the mutant is a wild-type halomethyltransferase AteHMT with the amino acid sequence as shown in SEQ ID NO.1, subjected to one or more of the following mutations:

[0012] (1) Methionine M at position 11 is mutated to phenylalanine F;

[0013] (2) The leucine L at position 30 is mutated to isoleucine I;

[0014] (3) Proline P at position 209 is mutated to arginine R.

[0015] In some embodiments, the mutant is a wild-type AteHMT with methionine M at position 11 mutated to phenylalanine F, leucine L at position 30 mutated to isoleucine I, and proline P at position 209 mutated to arginine R.

[0016] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0017] In some embodiments, the nucleotide sequence encoding the halomethyltransferase mutant is shown in SEQ ID NO. 2.

[0018] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0019] In some embodiments, the recombinant vector uses plasmid pET28a or pBad as the expression vector.

[0020] The fourth technical solution provided by the present invention is to express the mutant described in the first technical solution, or to contain the gene described in the second technical solution, or to transform a recombinant cell with the recombinant vector described in the third technical solution.

[0021] In some embodiments, the recombinant cells use fungi or bacteria as host cells.

[0022] In some embodiments, the bacteria include Escherichia coli.

[0023] Optionally, the Escherichia coli is Escherichia coli DH5α-Tir-7.

[0024] The fifth technical solution provided by the present invention is a method for improving the catalytic activity of the halomethyltransferase AteHMT. The method involves mutating the 11th methionine M of the wild-type halomethyltransferase AteHMT, as shown in SEQ ID NO.1, to phenylalanine F, the 30th leucine L to isoleucine I, and the 209th proline P to arginine R.

[0025] The sixth technical solution provided by the present invention is a method for biocatalytic synthesis of S-adenosylmethionine or its analogues. The method uses S-adenosyl-L-homocysteine ​​(SAH) and iodoalkane as substrates, and uses the mutant described in the first technical solution as a catalyst to form a reaction system to catalytically synthesize S-adenosylmethionine or its analogues.

[0026] In some embodiments, the iodoalkane includes, but is not limited to, iodomethane, iodoethane, and iodopropane.

[0027] In some embodiments, the final concentration of SAH is 1 / 100 to 1 mM, and the final concentration of the iodoalkane is 0.01 mM.

[0028] In some embodiments, the reaction time is 4-24 hours and the reaction temperature is 10-30°C.

[0029] In some embodiments, the amount of mutant added is OD. 600 =40.

[0030] The seventh technical solution provided by the present invention is a method for alkylation modification using the mutant described in the first technical solution. The method uses SAM and its analogues synthesized by the mutant described in the first technical solution as alkyl donors, 2,7-dihydroxynaphthalene or flavonoids as acceptors, and combines C-methyltransferase or O-methyltransferase to generate 2,7-dihydroxynaphthalene or flavonoid alkyl compounds.

[0031] In some embodiments, the amino acid sequence of the C-methyltransferase is shown in SEQ ID NO.3, and the amino acid sequence of the O-methyltransferase is shown in SEQ ID NO.4.

[0032] In some embodiments, the substrates for the catalytic synthesis of SAM and its analogues are S-adenosyl-L-homocysteine ​​(SAH) and iodoalkane.

[0033] In some embodiments, the iodoalkane includes, but is not limited to, iodomethane, iodoethane, and iodopropane.

[0034] In some embodiments, the flavonoids include quercetin, kaempferol, and apigenin.

[0035] In some embodiments, the final concentration of SAH is 1 / 100 to 1 mM, and the final concentration of the iodoalkane is 0.01 mM.

[0036] In some embodiments, the final concentration of the receptor is 1 / 100 to 1 mM.

[0037] In some embodiments, the amounts of the mutant, C-methyltransferase, and O-methyltransferase added are respectively OD 600 =40.

[0038] In some embodiments, the reaction time is 4-24 hours and the reaction temperature is 10-30°C.

[0039] The technical effects of this invention are as follows:

[0040] This invention uses wild-type AteHMT as a template and employs directed evolution to screen for the optimal triple mutant M11F / L30I / P209R. Biocatalytic synthesis of S-adenosylmethionine (SAM) and its analogues using this mutant demonstrates catalytic activity 1.5–23.5 times higher than that of wild-type AteHMT. This mutant is then used in a progressive cascade with a carbon methyltransferase CouO from *Streptomyces rishiriensis* and a 4'-O-methyltransferase FL-4 from peppermint flavonoids to synthesize a series of alkylated products. This method offers advantages such as mild conditions, environmental friendliness, high stereoselectivity, and high yield. Attached Figure Description

[0041] Figure 1 The relative activity of S-adenosylalanine synthesis for single mutants, double mutants, and triple mutants compared to wild type was determined.

[0042] Figure 2 High-performance liquid chromatography (HPLC) chromatograms of the reactions of S-adenosine homocysteine ​​and iodomethane with AteHMT wild-type and AteHMT mutant M11F / L30I / P209R, respectively.

[0043] Figure 3 This is the mass spectrum of the product S-adenosylmethionine.

[0044] Figure 4 High-performance liquid chromatography (HPLC) chromatograms of the reactions of S-adenosine homocysteine ​​and iodoethane with AteHMT wild-type and AteHMT mutant M11F / L30I / P209R, respectively.

[0045] Figure 5 The mass spectrum of the product S-adenosylethionine is shown.

[0046] Figure 6 High-performance liquid chromatography (HPLC) chromatograms of the reactions of S-adenosine homocysteine ​​and iodopropane with AteHMT wild-type and AteHMT mutant M11F / L30I / P209R, respectively.

[0047] Figure 7 The mass spectrum of the product S-adenosylalanine is shown.

[0048] Figure 8 The high-performance liquid chromatography (HPLC) chromatogram shows the reaction of whole-cell lysate of carbon methyltransferase CouO with whole-cell lysate of AteHMT mutant M11F / L30I / P209RSAH catalyzed by a cascade reaction.

[0049] Figure 9The mass spectrum of the product is methylated 2,7-dihydroxynaphthalene.

[0050] Figure 10 The mass spectrum of the product is ethylated 2,7-dihydroxynaphthalene.

[0051] Figure 11 The mass spectrum of the product is propylated 2,7-dihydroxynaphthalene.

[0052] Figure 12 The high-performance liquid chromatography (HPLC) chromatogram shows the cascade reaction of luteolin alkylation catalyzed by whole-cell lysate of FL-4 oxymethyltransferase and whole-cell lysate of AteHMT mutant M11F / L30I / P209RSAH.

[0053] Figure 13 This is the mass spectrum of the product, methylated luteolin.

[0054] Figure 14 This is the mass spectrum of the product, ethylated luteolin.

[0055] Figure 15 This is the mass spectrum of the product, propylated luteolin.

[0056] Figure 16 This diagram illustrates the alkylation modification of different compounds via a methyltransferase cascade on the efficient synthetic SAM and analog mutant platform constructed in this invention. Detailed Implementation

[0057] Reference Appendix Figures 1-16 The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0058] Test method:

[0059] Product analysis methods: High performance liquid chromatography (HPLC) and mass spectrometry (MS / MS). The corresponding HPLC detection method used a 5 μm C18 column with a PDA detector. The mobile phase consisted of phase A (H₂O containing 0.1% formic acid) and phase B (acetonitrile). The gradient elution program was: 1% B for 10 min, 1%-10% B for 1 min, 10%-25% B for 7 min, 25%-90% B for 4 min, 90%-1% B for 2 min, and 1% B for 6 min. Materials used in the examples:

[0060] Solid culture medium formula (1L): 5g yeast extract, 10g peptone, 10g sodium chloride, 15g agar, bring to volume with deionized water, and autoclave.

[0061] LB medium formula (1L): 5g yeast extract powder, 10g peptone, 10g sodium chloride, bring to volume with deionized water, and autoclave.

[0062] TB culture medium formula (1L): 24g yeast extract, 12g peptone, 4mL glycerol. Add 900mL deionized water, dissolve, and autoclave. Then add 100mL of a 0.17M KH2PO4 / 0.72M K2HPO4 solution that has undergone the same sterilization process. (Note: Sodium chloride was purchased from Aladdin, and all other components were purchased from Sangon Biotech Co., Ltd.)

[0063] CouO whole cells were prepared from Escherichia coli heterologously expressing C-methyltransferases from Streptomyces spheroides, with reference to the literature "CouO and NovO: C-methyltransferases for tailoring the aminocoumarin scaffold in coumermycin and novobiocin antibiotic biosynthesis" (Biochemistry 44.45(2005):14969-14976.).

[0064] FL-4 whole cells were prepared from Escherichia coli with O-methyltransferase from Mentha x piperita, with reference to the literature "Restricting Promiscuity of Plant Flavonoid 3'-Hydroxylase and 4'-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli" (JAgric Food Chem..2023Jun28;71(25):9826-9835.).

[0065] Example 1: Engineering of the halomethyltransferase AteHMT protein

[0066] (1) Design and preparation of mutants:

[0067] Using the gene sequence shown in SEQ ID NO.2 as a template, primers were designed to perform site-directed mutagenesis at the pocket 7 angstrom sites M11, L30, L38, W40F, and P209, yielding M11F, L30I, L38D, W40F, P209R, and M11F / L30I.

[0068] The coding gene of the M11F / L30I / P209RP mutant. Table 1. Primer design for the three mutants.

[0069]

[0070]

[0071] Using the commercially available Vazyme MultiSOneStepCloning Kit, the wild-type gene shown in SEQ ID NO.2 and the mutant coding gene prepared in step (1) were ligated with the pBad vector to obtain recombinant vectors pBad-WT, pBad-M11F, pBad-L30I, pBad-L38D, pBad-W40F, pBad-P209R, pBad-M11F / L30I, and pBad-M11F / L30I / P209RP (pBad-AteHMT mutant). Specific steps are detailed in the instruction manual. Large fragment amplification: 12.5 μL of KOD one was added to the wild-type template and 1 μL of the primers mentioned above, then sterilized water was added to a total volume of 25 μL. Large fragment amplification program: The PCR program was 98℃ for 2 min, 98℃ for 10 s, 55℃ for 5 s, 68℃ for 10 s, 34X, 68℃ for 10 min, and 12℃ for 10 min. Circular plasmid amplification: Add 12.5 μL of KODone to 1 μL of the amplified fragment from the previous step and 1 μL of wild-type template, then add sterile water to a total volume of 25 μL. Circular plasmid program: The PCR program is 98℃ for 2 min, 98℃ for 10 s, 55℃ for 5 s, 68℃ for 1 min, 34X, 68℃ for 10 min, and 12℃ for 10 min.

[0072] Example 2: Expression of the AteHMT mutant of halomethyltransferase

[0073] The recombinant vectors pBad-WT, pBad-M11F, pBad-L30I, pBad-L38D, pBad-W40FF, pBad-P209R, pBad-M11F / L30I, and pBad-AteHMT mutant constructed in Example 1 were electroporated into competent DH5α-Tir-7 cells. A suitable amount of bacterial culture was plated onto a solid medium containing ampicillin and cultured at 37°C for 16 h. A single colony containing the transformed recombinant plasmid was picked from each culture and placed in LB medium (containing 100 μg / mL ampicillin) and cultured overnight at 37°C. 1% of the LB culture was inoculated into TB medium (containing 100 μg / mL ampicillin) and subsequently cultured at 37°C until the logarithmic growth phase (OD). 600 The OD value was 0.6–0.8. 10 g / L arabinose was added as an inducer, and expression was carried out at 25°C and 160 rpm for 18–20 h. The expression cells were collected separately using a centrifuge pre-cooled to 4°C, washed twice with 100 mM (pH = 7.0) NaH₂PO₄-Na₂HPO₄ buffer solution, and the OD value was determined. 600 When the value is 40, add a small spoonful of lysozyme and store at -80℃.

[0074] Example 3: Sonic lysis of the halomethyltransferase AteHMT and its mutant

[0075] The bacterial cells collected in Implementation Case 2 were thawed overnight on ice. Centrifuge tubes containing bacterial culture were placed in an ice-water bath. The ultrasonic disruptor parameters were set (200W, duration 2s, cooling time 2s) and ultrasonicated for 30 minutes to obtain crude enzyme solutions of wild type (WT) and mutants (M11F, L30I, L38D, W40F, P209R, M11F / L30I, M11F / L30I / P209RP).

[0076] Example 4: Activity assay of single, double, and triple mutations of the halogenated methyltransferase AteHMT mutant

[0077] In a 500 μL reaction system, 430 μL of cell lysate containing the halomethyltransferase AteHMT wild-type or mutant M11F, L30I, L38D, W40F, P209R, M11F / L30I, or M11F / L30I / P209RP was used as a catalyst. SAH and iodomethane were used as substrates, with SAH concentration of 1 mM and iodomethane concentration of 10 mM. 100 mM NaH2PO4-Na2HPO4 (pH = 7.0) and DMSO (v / v, 10%) were added. Wild-type enzyme was used as a control. The reaction was carried out at 25 °C for 30 min to obtain the corresponding products. The reaction was terminated by adding one volume of methanol. High-performance liquid chromatography (HPLC) analysis was performed. See [link to HPLC analysis]. Figure 1 .

[0078] Specially by Figure 1 It can be seen that the mutant M11F / L30I / P209R is the best mutant, with the mutant activity being 23.5 times higher than that of the wild type.

[0079] Example 5: The AteHMT mutant of halomethyltransferase catalyzes the synthesis of SAM from SAH

[0080] In a 500 μL reaction system, 430 μL of cell lysate containing the wild-type or mutant AteHMT M11F / L30I / P209RP halomethyltransferase was used as a catalyst. SAH and iodomethane were used as substrates, with SAH concentration of 1 mM and iodomethane concentration of 10 mM. 100 mM NaH₂PO₄-Na₂HPO₄ (pH = 7.0) and DMSO (v / v, 10%) were also added. Wild-type enzyme was used as a control. The reaction was carried out at 25 °C for 30 min to obtain the corresponding product. The reaction was terminated by adding one volume of methanol. High-performance liquid chromatography and mass spectrometry analysis were performed. See [link to results]. Figure 2 and Figure 3 .

[0081] Specially by Figure 2 It can be seen that both the mutant M11F / L30I / P209R and the wild type can produce SAM by catalyzing SAH with iodomethane, but the mutant activity is 1.5 times higher than that of the wild type.

[0082] Example 6: The AteHMT mutant of halomethyltransferase catalyzes the synthesis of SAE from SAH

[0083] In a 500 μL reaction system, 430 μL of cell lysate containing wild-type or M11F / L30I / P209RP mutant halomethyltransferase AteHMT was used as a catalyst. SAH and iodoethane were used as substrates (SAH concentration: 1 mM, iodoethane concentration: 10 mM), 100 mM NaH₂PO₄-Na₂HPO₄ (pH = 7.0), and DMSO (v / v, 10%) was added. Wild-type enzyme was used as a control. The reaction was carried out at 25 °C for 4 h to obtain the corresponding product. The reaction was terminated by adding one volume of methanol. High-performance liquid chromatography and mass spectrometry analysis were performed. See [link to results]. Figure 4 and Figure 5 .

[0084] Depend on Figure 4 It can be seen that both the mutant M11F / L30I / P209R and the wild type can produce SAE by catalyzing SAH with iodoethane, but the mutant activity is 12.7 times higher than that of the wild type.

[0085] Example 7: The AteHMT mutant of halomethyltransferase catalyzes the synthesis of SAP from SAH

[0086] In a 500 μL reaction system, 430 μL of cell lysate containing the wild-type or M11F / L30I / P209RP mutant of the halomethyltransferase AteHMT was used as a catalyst. SAH and iodopropane were used as substrates (SAH concentration: 1 mM, iodopropane concentration: 10 mM), with 100 mM PBS buffer (pH = 7.0) and DMSO (v / v, 10%). The wild-type enzyme was used as a control. The reaction was carried out at 25 °C for 8 h to obtain the corresponding product. The reaction was terminated by adding one volume of methanol. High-performance liquid chromatography and mass spectrometry analysis were performed. See [link to results]. Figure 6 and Figure 7 .

[0087] Depend on Figure 6 and Figure 7 It can be seen that both the mutant M11F / L30I / P209R and the wild type can catalyze SAH with iodopropane to obtain SAE, but the mutant activity is 23.5 times higher than that of the wild type.

[0088] Example 8: Synthesis of 2,7-dihydroxynaphthalene alkylation catalyzed by the AteHMT mutant of halomethyltransferase and C-methyltransferase cascade.

[0089] In a 1 mL reaction system, the lysis buffer of the AteHMT mutant M11F / L30I / P209RP was coupled with CouO whole-cell lysis buffer, with SAH added at a concentration of 0.01 mM, iodoalkane at a concentration of 10 mM, 100 mM PBS (pH = 7.0), 2,7-dihydroxynaphthalene at 1 mM, and DMSO (v / v, 10%). The reaction was carried out at 25 °C for 24 h to obtain the corresponding product. The reaction was terminated by adding 1 volume of methanol. High performance liquid chromatography and mass spectrometry analysis were performed, and the results are as follows: Figures 8 to 11 As shown, 2,7-dihydroxynaphthylmethyl, ethyl and propylated products were successfully synthesized via cascade, with yields of 74.2%, 52.6% and 9.7%, respectively.

[0090] Example 9: Alkylation of flavonoids catalyzed by the AteHMT mutant of halomethyltransferase and the O-methyltransferase cascade.

[0091] In a 1 mL reaction system, the concentration of 0.01 mM lysis buffer of the AteHMT mutant M11F / L30I / P209RP and FL-4 whole-cell lysis buffer catalyzing SAH was 10 mM iodoalkane, with 100 mM PBS buffer (pH = 7.0), 1 mM luteolin, and DMSO (v / v, 10%). The reaction was carried out at 25 °C for 24 h to obtain the corresponding product. The reaction was terminated by adding 1 volume of methanol. High performance liquid chromatography and mass spectrometry analysis were performed, and the results are as follows: Figures 12 to 15 As shown, the methyl, ethyl and propylated products of luteolin were successfully synthesized via cascade synthesis, with yields of 41.7%, 22.4% and 8.3%, respectively.

[0092] 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. A mutant of the halomethyltransferase AteHMT, characterized in that, The mutant is a wild-type halogenated methyltransferase AteHMT with the amino acid sequence shown in SEQ ID NO.1, subjected to one or more of the following mutations: (1) Methionine at position 11 is mutated to phenylalanine; (2) Leucine at position 30 is mutated to isoleucine; (3) Proline at position 209 is mutated to arginine.

2. The mutant of the halomethyltransferase AteHMT according to claim 1, characterized in that, The mutant is formed by mutating methionine at position 11 of the wild-type halogenated methyltransferase AteHMT to phenylalanine, leucine at position 30 to isoleucine, and proline at position 209 to arginine.

3. The gene encoding the mutant of claim 1 or 2.

4. A recombinant vector carrying the gene of claim 3.

5. A recombinant cell expressing the mutant of claim 1 or 2, or containing the gene of claim 3, or transformed with the recombinant vector of claim 4.

6. A method for improving the catalytic activity of the halomethyltransferase AteHMT, characterized in that, The method involves mutating methionine at position 11 to phenylalanine, leucine at position 30 to isoleucine, and proline at position 209 to arginine in the wild-type halogenated methyltransferase AteHMT with the amino acid sequence shown in SEQ ID NO.

1.

7. A method for biocatalytic synthesis of S-adenosylmethionine or its analogues, wherein the method comprises using S-adenosyl-L-homocysteine ​​and iodoalkane as substrates, and using the mutant described in claim 1 or 2 as a catalyst to form a reaction system for catalytic synthesis of S-adenosylmethionine or its analogues; wherein the iodoalkane includes, but is not limited to, iodomethane, iodoethane, and iodopropane.

8. The method according to claim 7, characterized in that, The final concentration of S-adenosyl-L-homocysteine ​​is 0.01–1 mM, the final concentration of the iodoalkane is 5–15 mM, and the amount of the mutant added is OD. 600 =40; The reaction time is 4-24 hours and the reaction temperature is 10-30℃.

9. A method for alkylation modification using the mutant according to claim 1 or 2, characterized in that, The method involves using SAM and its analogues synthesized by the mutant described in claim 1 or 2 as alkyl donors, 2,7-dihydroxynaphthalene or flavonoids as acceptors, and combining C-methyltransferase or O-methyltransferase to generate 2,7-dihydroxynaphthalene or flavonoid alkyl compounds. The amino acid sequence of the C-methyltransferase is shown in SEQ ID NO.3, and the amino acid sequence of the O-methyltransferase is shown in SEQ ID NO.4; The iodoalkanes include, but are not limited to, iodomethane, iodoethane, and iodopropane, and the flavonoids include quercetin, kaempferol, and apigenin.

10. The method according to claim 9, characterized in that, The final concentration of the receptor is 0.01–1 mM; the amounts of the mutant, C-methyltransferase, and O-methyltransferase added are respectively OD 600 =40; The reaction time is 4-24 hours and the reaction temperature is 10-30℃.

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