Process for the preparation of s-adenosyl-methyl cysteine and its use

Abstract

The application discloses a preparation method and application of S-adenosyl-methyl cysteine, and the preparation method comprises the following reaction: S-adenosyl-L-cysteine is methylated by using a first methyltransferase or a first methyltransferase mutant and a methyl donor to obtain S-adenosyl-methyl cysteine; or S-adenosyl-L-homocysteine is methylated by using a methyltransferase or a first methyltransferase mutant and a methyl donor to obtain S-adenosyl-methionine. The method has the characteristics of low cost and simple operation, and enables S-adenosyl-methyl cysteine to replace expensive S-adenosyl methionine (SAM) as a coenzyme and a methyl donor in a methylation reaction, and is applied to methylation modification of natural products based on a coenzyme regeneration system.

Description

Technical Field

[0001] This application relates to the field of biocatalysis, specifically to a method for preparing S-adenosine-methylcysteine ​​and its application. Background Technology

[0002] S-Adenosylmethionine (SAM) is an important physiologically active substance widely found in plants, animals, and microorganisms. It is the second most common component involved in enzymatic reactions, second only to adenosine triphosphate (ATP). In methyltransferases, it plays a crucial role as a cofactor and key methyl donor. However, SAM is a complex, relatively unstable, and expensive compound. Its preparation mainly involves three methods: biotechnology (preparing SAM in living cells followed by isolation), chemical synthesis, enzymatic reactions, and microbial synthesis. However, SAM prepared using these techniques remains expensive and cannot be recycled. Therefore, researchers have developed a simpler and more powerful SAM recycling system. This system uses halogenated methyltransferases (HMTs) or thiopurine methyltransferases (TPMTs), employs commercially available reagents MeI or MeOTs as methyl donors, and SAH as a methyl transfer carrier. Only a catalytic amount of SAH or SAM is needed to initiate the reaction, achieving preparative-grade specific methylation of natural products. Based on this cyclic system, researchers have further developed SAM analogs, mainly by replacing the methyl group on the sulfur atom with other groups, such as ethyl, propyl, allyl, and fluoromethyl, but all of these are based on SAH as a carrier. Therefore, SAH that can be produced or supplied on a large scale may become an important foundation for the development and industrial application of methyltransferase biocatalysis technology. Currently, SAH can be prepared by chemical synthesis or by the condensation reaction of adenosine and L-homocysteine ​​(L-Hcy) acid catalyzed by SAHH, but both methods suffer from the problem of high L-Hcy costs. To solve this problem, researchers have developed an economical and efficient one-step method to synthesize SAH from racemic homocysteine ​​thiolactone. This process is completed through the synergistic catalysis of recombinant α-amino-3-caprolactam racemic enzyme (ACLR), bleomycin hydrolase (BLH), and SAH hydrolase (SAHH). Although this system solves the problem of high raw material costs, it requires the use of a multi-enzyme cascade system, which is relatively complex and also increases production costs to some extent. The cost problem of SAH or SAM analog applications has not been fundamentally solved. Summary of the Invention

[0003] This invention provides a method for preparing S-adenosyl-methylcysteine ​​and its application. This method is characterized by low cost and simple operation, and allows S-adenosyl-methylcysteine ​​to replace the expensive S-adenosylmethionine (SAM) as a coenzyme and methyl donor in methylation reactions. Based on a coenzyme regeneration system, it is applied to the methylation modification of natural products.

[0004] The first aspect of this invention provides a method for preparing S-adenosyl-methylcysteine ​​or S-adenosyl-methionine, the method comprising the following reactions: S-adenosyl-L-cysteine ​​was obtained by methylating S-adenosyl-L-cysteine ​​with a methyl donor using a promethyltransferase or a promethyltransferase mutant; or, S-adenosyl-L-homocysteine ​​was obtained by methylating S-adenosyl-L-homocysteine ​​with a methyl donor using a methyltransferase or a mutant of the first methyltransferase. Wherein, the amino acid sequence of the first methyltransferase is as shown in SEQ ID NO:2 or SEQ ID NO:3; or, the amino acid sequence of the first methyltransferase mutant has at least 85% identity with SEQ ID NO:2 or SEQ ID NO:3, and maintains or has a first methyltransferase mutant with better efficacy than the first methyltransferase.

[0005] In some embodiments, the present invention provides a method for preparing S-adenosyl-methylcysteine, the method comprising the following reaction: S-adenosyl-L-cysteine ​​was obtained by methylating S-adenosyl-L-cysteine ​​with a methyl donor using a promethyltransferase or a promethyltransferase mutant; or, S-adenosyl-L-homocysteine ​​was obtained by methylating S-adenosyl-L-homocysteine ​​with a methyl donor using a methyltransferase or a mutant of the first methyltransferase. Wherein, the amino acid sequence of the first methyltransferase is as shown in SEQ ID NO:2 or SEQ ID NO:3; or, the amino acid sequence of the first methyltransferase mutant has at least 85% identity with SEQ ID NO:2 or SEQ ID NO:3, and maintains or has a first methyltransferase mutant with better efficacy than the first methyltransferase.

[0006] In some embodiments, the present invention provides a method for preparing S-adenosylmethionine, the method comprising the following reaction: S-adenosyl-L-cysteine ​​was obtained by methylating S-adenosyl-L-cysteine ​​with a methyl donor using a promethyltransferase or a promethyltransferase mutant; or, S-adenosyl-L-homocysteine ​​was obtained by methylating S-adenosyl-L-homocysteine ​​with a methyl donor using a methyltransferase or a mutant of the first methyltransferase. Wherein, the amino acid sequence of the first methyltransferase is as shown in SEQ ID NO:2 or SEQ ID NO:3; or, the amino acid sequence of the first methyltransferase mutant has at least 85% identity with SEQ ID NO:2 or SEQ ID NO:3, and maintains or has a first methyltransferase mutant with better efficacy than the first methyltransferase.

[0007] In some embodiments, the first methyltransferase mutant differs from the amino acid sequence shown in SEQ ID NO: 2 at one or more sites of G84, T165, and R43, wherein the positions of G84, T165, and R43 are referenced to the amino acid residue numbers in SEQ ID NO: 2; and / or, The first methyltransferase mutant differs from the amino acid sequence shown in SEQ ID NO: 2 by no more than 3 amino acid residues in total.

[0008] In some specific implementations, the total number of differences in the amino acid residues is 1, 2, or 3.

[0009] In some preferred embodiments, the first methyltransferase mutant contains one or more of the following differences compared to the amino acid sequence shown in SEQ ID NO: 2: (1) G84A or G84S; (2) One of T165E, T165G, T165M, T165Q, T165H, T165R, T165K, T165I, T165F, T165L, T165W, T165A, T165P, T165C, T165N, T165V, T165S, T165Y and T165D; (3) R43A.

[0010] In some preferred embodiments, the difference between the first methyltransferase mutant and the amino acid sequence shown in SEQ ID NO: 2 includes one or more of G84A / S, T165E / G / M / Q, and R43A.

[0011] In some embodiments, the first methyltransferase mutant has one of the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 2: (1) G84A; (2) G84S; (3) T165E; (4) T165G; (5) T165M; (6) G84S and T165Q; (7) R43A and T165M; (8) G84A and R43A; In some preferred embodiments, the first methyltransferase mutant has mutations R43A and T165M compared to the amino acid sequence shown in SEQ ID NO: 2.

[0012] In some embodiments, the first methyltransferase mutant differs from the amino acid sequence shown in SEQ ID NO: 3 at one or more sites in V140 and Q49, wherein the positions of V140 and Q49 are referenced to the amino acid residue numbers in SEQ ID NO: 3; and / or, The first methyltransferase mutant differs from the amino acid sequence shown in SEQ ID NO: 3 by no more than two amino acid residues in total. In some specific implementations, the number of amino acid residue differences is 1, 2, or 3.

[0013] In some embodiments, the first methyltransferase mutant contains one or more of V140A / M and Q49A compared to the amino acid sequence shown in SEQ ID NO: 3; In some preferred embodiments, the first methyltransferase mutant contains mutations in Q49A and V140M compared to the amino acid sequence shown in SEQ ID NO: 3.

[0014] In some implementations, the reaction includes the following steps: (i) Dissolve S-adenosyl-L-cysteine ​​and the methyl donor in PBS; (ii) Add the first methyltransferase or the first methyltransferase mutant to the system to initiate the reaction; (iii) Quenching reaction; In some preferred embodiments, the final concentration of the S-adenosyl-L-cysteine ​​is 0.1-4.0 mM; and / or, The methyl donor is MeI or MeOTs; and / or, The PBS concentration is 50 mM; and / or, The reaction concentration of the first methyltransferase or the first methyltransferase mutant is 1-1000 μM; and / or, The reaction conditions are 10-50 degrees Celsius, 0-1000 rpm, for example, 25 degrees Celsius, 180 or 800 rpm; and / or, The quenching reaction is carried out using twice the volume of methanol or an equal volume of 1% TFA.

[0015] In some preferred embodiments, the reaction concentration of MeI is 1-10 mM, and the reaction concentration of MeOTs is 1-10 mM.

[0016] In some specific embodiments, the final concentration of the S-adenosyl-L-cysteine ​​is 1 mM or 2 mM; and / or, The reaction concentration of MeI is 8 mM, and the reaction concentration of MeOTs is 3 mM or 8 mM; and / or, The reaction concentration of the first methyltransferase or the first methyltransferase mutant is 1 μM, 2 μM, 10 μM, 25 μM, 250 μM, 60 μM, 300 μM, 400 μM, 600 μM, 200 μM or 30 μM.

[0017] In some embodiments, the stock solution of the S-adenosine-L-cysteine ​​is a 200 mM stock solution prepared with dilute hydrochloric acid; and / or, The MeI mother liquor is a 400 mM mother liquor prepared with DMSO; the MeOTs mother liquor is a 200 mM or 1000 mM mother liquor prepared with MeCN or acetonitrile.

[0018] In some preferred embodiments, step (iii) further includes quenching the reaction followed by centrifugation, filtration, rotary evaporation, and dissolution to obtain the product.

[0019] In some specific embodiments, step (iii) further includes centrifuging at 10,000 rpm for 20 minutes after quenching the reaction, collecting the supernatant, filtering and drying to obtain a white residue, and dissolving it in a small amount of water; and / or, using HPLC to collect product peaks or estimate the concentration of the final product.

[0020] In some embodiments, the preparation method further includes the synthesis of S-adenosine-L-cysteine, the synthesis comprising the following steps: Using hydrolytic enzymes, L-cysteine, adenine nucleoside and NAD + S-adenosine-L-cysteine ​​was prepared.

[0021] In some preferred embodiments, the hydrolase is selected from S-adenosyl homocysteine ​​hydrolases; and / or, The reaction concentration of L-cysteine ​​is 10 mM; and / or, The reaction concentration of adenosine Ado is 10 mM; and / or, The NAD + The reaction concentration was 2 mM.

[0022] In some preferred embodiments, the S-adenosine homocysteine ​​hydrolase is SacSAHH or SsoSAHH.

[0023] In some specific embodiments, the S-adenosine homocysteine ​​hydrolase is SacSAHH.

[0024] In some embodiments, the amino acid sequence of the SacSAHH is as shown in SEQ ID NO: 2; and / or, L-cysteine, adenosine Ado and NAD + Both are soluble in 50 mM Tris-HCl at pH 7.5; In some preferred embodiments, the synthesis further includes quenching the reaction with 3 times the volume of methanol after the reaction is completed, centrifuging at 10,000 rpm for 20 min, filtering the supernatant and evaporating to obtain a white residue, washing the residue multiple times with ice-cold water and methanol, and drying the collected white solid in a vacuum drying oven to obtain the final product.

[0025] A second aspect of the present invention provides a transferase mutant based on SEQ ID NO:2, which is defined in the preparation method described in the first aspect of the present invention.

[0026] A third aspect of the present invention provides a transferase mutant based on SEQ ID NO:3, which is defined in the preparation method described in the first aspect of the present invention.

[0027] A fourth aspect of the present invention provides an isolated nucleic acid molecule that encodes an enzyme mutant as described in the second or third aspect of the present invention.

[0028] A fifth aspect of the present invention provides a recombinant expression vector comprising isolated nucleic acid molecules as described in the fourth aspect of the present invention.

[0029] In some preferred embodiments, the recombinant expression vector is a plasmid.

[0030] In some preferred embodiments, the backbone of the plasmid is pET-22b, pET28a, pET-30a, pET-32a, pET-43.1a, or pET-DEST51.

[0031] In some specific implementations, the backbone of the plasmid is pET28a.

[0032] A sixth aspect of the present invention provides a transformant comprising an isolated nucleic acid molecule as described in the fourth aspect of the present invention, or a recombinant expression vector as described in the fifth aspect of the present invention.

[0033] In some preferred embodiments, the host cell used in the construction of the transformant is *Escherichia coli* (E. coli). Escherichia coli ).

[0034] In some preferred embodiments, the Escherichia coli is Escherichia coli BL21 dmtn(DE3).

[0035] The seventh aspect of the present invention provides a method for preparing a transferase mutant as described in the second or third aspect of the present invention, comprising culturing a transformant as described in the sixth aspect of the present invention to obtain a fermentation product.

[0036] In some preferred embodiments, the culture medium used for the culture is selected from LB liquid medium or TB liquid medium, and / or the culture conditions are: shaking culture at a temperature of 37±1°C.

[0037] In some preferred embodiments, the method further includes the step of purifying the fermentation product to obtain the first methyltransferase mutant.

[0038] The eighth aspect of the present invention provides the use of a first methyltransferase having an amino acid sequence as shown in SEQ ID NO: 2 or 3, a first methyltransferase mutant as described in the second or third aspect of the present invention, an isolated nucleic acid molecule as described in the fourth aspect of the present invention, a recombinant expression vector as described in the fifth aspect of the present invention, or a transformant as described in the sixth aspect of the present invention in the preparation of S-adenosyl-methylcysteine ​​and / or S-adenosyl-methionine.

[0039] A ninth aspect of the present invention provides a reaction end product system for preparing S-adenosyl-methylcysteine, the reaction end product system comprising: (1) S-adenosyl-methylcysteine; and / or, S-adenosyl-L-cysteine; (2) Methyl donor; and, (3) A first methyltransferase having the amino acid sequence shown in SEQ ID NO: 2 or 3 and any one of the transferase mutants described in the second or third aspect of the present invention; In some preferred embodiments, the reaction end product system further includes one or more of the following: (i) L-cysteine; (ii) Adenine nucleotides; (iii) NAD + Or NADH; and (iv) S-adenosine homocysteine ​​hydrolase.

[0040] In some preferred embodiments, the S-adenosine homocysteine ​​hydrolase is SacSAHH or SsoSAHH.

[0041] In some specific embodiments, the S-adenosine homocysteine ​​hydrolase is SacSAHH.

[0042] The tenth aspect of this invention provides a method for methylating a compound, the method comprising the following reaction: Methylation modification of substrates was performed using coenzymes and second methyltransferases; The coenzyme is S-adenosyl-methylcysteine ​​or S-adenosyl-L-cysteine; the second methyltransferase is an oxymethyltransferase, a carbon methyltransferase, a thiomethyltransferase or a protein methyltransferase.

[0043] In some preferred embodiments, the method further includes a method for preparing and / or regenerating S-adenosine-methylcysteine.

[0044] In some specific embodiments, the preparation method is as defined in the preparation method described in the first aspect of the present invention; the regeneration method is to convert S-adenosyl-L-cysteine ​​into S-adenosyl-methylcysteine.

[0045] In some embodiments, the methylation modification sites on the substrate include nucleophilic atoms in the compound or groups formed therefrom.

[0046] In some specific embodiments, the nucleophilic atom is an oxygen atom, a nitrogen atom, a sulfur atom, or a carbon atom; the group is a hydroxyl group, a carboxyl group, an amino group, or a thiol group.

[0047] In some preferred embodiments, the substrate is a flavonoid compound.

[0048] In some specific implementations, the substrate is luteolin, scutellarin, myricetin, hesperidin, or kaempferol.

[0049] In some implementations, the second methyltransferase is an oxymethyltransferase.

[0050] In some preferred embodiments, the oxymethyltransferase is ScOMT or NfOMT.

[0051] In some specific embodiments, the amino acid sequence of ScOMT is shown in SEQ ID NO: 4, and the amino acid sequence of NfOMT is shown in SEQ ID NO: 5.

[0052] In some embodiments, when the initial material added to the system is S-adenosyl-methylcysteine, the concentration of S-adenosyl-methylcysteine ​​is 0.01-100 mM, for example, 1 mM; or, When the initial material added to the system is S-adenosyl-L-cysteine, the system satisfies one or more of the following conditions: (A) The concentration of the S-adenosyl-L-cysteine ​​is 0.01-10 mM; (B) The system includes a methyl donor; (C) The system comprises a first methyltransferase as shown in SEQ ID NO:2 or SEQ ID NO:3 or a first methyltransferase mutant as described in the second or third aspect of the present invention.

[0053] In some specific embodiments, the concentration of S-adenosyl-L-cysteine ​​is 0.1 mM.

[0054] In some preferred embodiments, when the initial material introduced into the system is S-adenosyl-L-cysteine, the method includes the following steps: a. Add the S-adenosyl-L-cysteine ​​and the methyl donor to a container, then add PBS and mix well; b. Add the second methyltransferase lysis buffer and the first methyltransferase or the first methyltransferase mutant lysis buffer; c. Add the substrate and react at 20-50℃ and 0-1000rpm for 1-48 hours.

[0055] In some specific implementations, step c is: reacting at 25 or 30°C and 180 or 900 rpm for 1-48 hours, or 16 or 20 hours.

[0056] In some implementations, the system also satisfies one or more of the following conditions: (I) The substrate concentration is 0.01-100 mM; (II) The concentration of the second methyltransferase is 1-100 mg / mL; (III) The methyl donor is a MeOT; (IV) The concentration of the first methyltransferase or the first methyltransferase mutant is 0.01-100 mg / mL.

[0057] In some specific implementations, the substrate concentration is 1 mM; The concentration of the second methyltransferase was 32.61 mg / mL; The methyl donor is a MeOTs at a concentration of 3 mM; The concentration of the first methyltransferase or the first methyltransferase mutant was 6.52 mg / mL; The first methyltransferase or the first methyltransferase mutant is a first methyltransferase mutant containing R43A and T165M compared to the amino acid sequence shown in SEQ ID NO: 2.

[0058] In some preferred embodiments, the method further includes step d: adding cold methanol to the reaction solution to terminate the reaction, centrifuging at 10,000 rpm for 30 min, collecting the supernatant, filtering and concentrating it, dissolving the residue in a mixture of DMSO and methanol, centrifuging and filtration through a membrane, and purifying it by preparative liquid chromatography (Pre-HPLC) to obtain the product.

[0059] The eleventh aspect of this invention provides a reaction end product system for methylation modification of a compound, the reaction end product system comprising: (1) S-adenosyl-methylcysteine; and / or, S-adenosyl-L-cysteine; and, (2) Second methyltransferase; the second methyltransferase is an oxymethyltransferase, a carbon methyltransferase, a thiomethyltransferase, or a protein methyltransferase; and, (3) Substrate; and / or, product; In some specific implementations, the second methyltransferase is an oxymethyltransferase.

[0060] In some preferred embodiments, the oxymethyltransferase is ScOMT or NfOMT.

[0061] In some preferred embodiments, the reaction end product system further includes: The first methyltransferase having the amino acid sequence shown in SEQ ID NO: 2 or 3 and any one of the first methyltransferase mutants as described in the second or third aspect of the present invention; And / or, methyl donor.

[0062] In some preferred embodiments, the substrate is a flavonoid.

[0063] In some specific embodiments, the flavonoids are luteolin, scutellarin, myricetin, hesperidin, or kaempferol.

[0064] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0065] The reagents and raw materials used in this invention are all commercially available.

[0066] The positive and progressive effects of this invention are as follows: This invention utilizes sources Sulfolobus acidocaldariusThe synthesis of SAH hydrolase SacSAHH was modified by replacing L-Hcy with inexpensive and readily available L-Cys, enabling the one-step, efficient synthesis of the SAH analog S-adenosyl-L-cysteine ​​(SAC) with adenosine. Directed evolution of the thiopurine methyltransferase acl and rational design of the halide methyltransferase AtHMT yielded mutants acl_R43A-T165M and AtHMT_Q49A-V140M, capable of high-conversion methylation of SAC to synthesize S-Me-SAC. The SAC / S-Me-SAC synthesis method developed in this invention has a significantly lower cost than the traditional SAH / SAM method and can be applied to coenzyme regeneration systems to achieve methylation of natural products, further reducing the cost of large-scale preparation of methylated products. Attached Figure Description

[0067] Figure 1 This is a technical overview diagram of the present invention.

[0068] Figure 2 Nuclear magnetic resonance (NMR) of compound 1 obtained in Example 9 1 H spectrum.

[0069] Figure 3 Nuclear magnetic resonance (NMR) of compound 1 obtained in Example 9 13 C-spectrum.

[0070] Figure 4 The image shows the HSQC nuclear magnetic resonance spectrum of compound 1 obtained in Example 9.

[0071] Figure 5 The image shows the nuclear magnetic resonance HMBC spectrum of compound 1 obtained in Example 9.

[0072] Figure 6 This is a high-performance liquid chromatogram of the catalytic methylation reaction in Example 9. Detailed Implementation

[0073] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below: In some embodiments, the sequences of the variants described in this disclosure may have at least 85% identity with their source sequences. Sequence identity described in this disclosure can be measured using sequence analysis software, such as the computer program BLAST with default parameters, particularly BLASTP or TBLASTN.

[0074] In this disclosure, sequence identity between sequences can be calculated as follows. To determine the percentage of identity between two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in the first and second amino acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being compared is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, the molecules are identical at that position. Sequence comparison and the calculation of the percentage of identity between two sequences can be performed using mathematical algorithms. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program, which is integrated into the GCG software package. The algorithm uses a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. A particularly preferred set of parameters (and one set of parameters that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. The percentage of identity between two amino acid sequences can also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17), which has been incorporated into the ALIGN program (version 2.0). Additionally or alternatively, the protein sequence described in this disclosure can be further used as a "query sequence" to perform a search against public databases to, for example, identify other family member sequences or related sequences.

[0075] The terms "polynucleotide," "nucleic acid," or "nucleotide sequence" in this invention refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0076] The term "isolated" nucleic acid molecule or polynucleotide in this invention refers to a nucleic acid molecule, DNA, or RNA that has been separated from its natural environment. In this invention, the recombinant polynucleotide encoding a polypeptide contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules typically found in cells containing the polynucleotide molecule, but which are located extrachromosomally or at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this invention, in positive and negative strand forms, and in double strand forms. The isolated polynucleotides or nucleic acids of this invention further include synthetically generated molecules of this type. Additionally, the polynucleotide or nucleic acid may be or may include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0077] The terms "vector" or "expression vector" and "expression construct" are used interchangeably in this invention, referring to a DNA molecule to which a specific gene operatively linked is introduced into a target cell and directed for expression. The vector comprises a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vector of this invention comprises an expression cassette. The expression vector can undergo transcription of a large amount of stable mRNA. Once the expression vector is in the target cell, a ribonucleic acid molecule or protein encoded by the gene is generated by cellular transcription and / or translation mechanisms. In one embodiment, the expression vector of this invention comprises an expression cassette containing a polynucleotide sequence encoding a fragment of the bispecific antigen-binding molecule of this invention.

[0078] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including primary transformed cells and their derived progeny. The nucleic acids of the progeny may not be completely identical to those of the parent cells and may contain mutations. Host cells are any type of cell that can be used to generate the bispecific antigen-binding molecule of this invention. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0079] The terms "pET-22b", "pET28a", "pET-30a", "pET-32a", "pET-43.1", and "pET-DEST51" refer to pET vectors, all of which use the T7 promoter / T7 RNA polymerase expression system as their core building block. This series of vectors shares the following characteristics: they employ a T7 lac promoter to drive target gene transcription, contain a T7 terminator sequence, carry ampicillin or kanamycin resistance selection markers, and include a multiple cloning site (MCS) for target gene insertion expression. All of the above-mentioned pET series vectors can achieve high-level transcription and translation controlled by the T7 promoter in the field of recombinant protein expression, can achieve effective selection through corresponding antibiotic resistance markers, can induce target gene expression in corresponding strains, and the N-terminal or C-terminal fusion tag of the expression product can be flexibly regulated according to the vector selection. Therefore, those skilled in the art generally agree that vectors such as pET-22, pET-28a, pET-30a, pET-32a, and pET-43.1a are equivalent in achieving high-level expression of recombinant proteins using the T7 expression system.

[0080] The term "BL21 dmtn(DE3)" refers to a strain derived from BL21(DE3), the core of which is the knockout of BL21(DE3). dcm and mcr It contains relevant methyltransferase genes and retains the DE3 lysogenized T7 expression system, specifically designed for the expression of recombinant proteins that need to avoid DNA methylation interference, and expresses methylation-sensitive exogenous genes.

[0081] The term "acl" refers to a bacterium derived from Achromobacterium obeliskinea. Achromobacter obae α-amino-ε-caprolactam racemases (acl).

[0082] The term "AtHMT" is derived from Arabidopsis thaliana. Arabidopsis thaliana Halide methyltransferase (HMT).

[0083] The terms "SacSAHH" and "SsoSAHH" are derived from thermophilic archaea, respectively. Sulfolobus acidocaldarius and Saccharolobus solfataricus S-Adenosyl-L-Homocysteine ​​Hydrolase (SAHH).

[0084] The terms "ScOMT" and "NfOMT" respectively refer to Streptomyces cinnamonii ( Streptomyces cinnamoneus ) and Nostoc fachob-152 ( Nostoc sp. FACHB-152 O-methyltransferase (O-Methyltransferase).

[0085] The S-adenosyl-L-cysteine ​​(SAC) described herein has the chemical structure shown in Formula 1:

[0086] Formula 1

[0087] The S-adenosyl-methylcysteine ​​(S-Me-SAC) described herein has the chemical structure shown in Formula 2:

[0088] Formula 2

[0089] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0090] Example 1: Expression and purification of SacSAHH

[0091] The plasmid SacSAHH / pET-28a was chemically transformed into E. coli BL21 dmtn(DE3) competent cells. E. coli BL21-dmtn(DE3) is an engineered strain obtained by knocking out the mtn gene from E. coli BL21(DE3) according to the method described in the literature (Angew. Chem. Int. Ed. 2021, 60, 1524-1527). SacSAHH / E. coli Monoclonal clones of BL21 dmtn(DE3) were inoculated into 10 mL LB medium containing 50 μg / mL Kana and incubated overnight at 37 °C and 220 rpm. Then, 20 mL of the pre-culture was inoculated into 1 L TB medium containing 50 μg / mL Kana resistance and cultured at 37 °C and 220 rpm until the OD600 reached approximately 0.6–0.8. After cooling the culture to 20 °C, 1 mL of 0.2 M IPTG (final concentration 0.2 mM) was added, and expression was induced at 20 °C and 220 rpm for 20 h.

[0092] The method for obtaining a large number of cells by fermentation of SacSAHH is as follows: 10 μL of SacSAHH glycerol bacteria were inoculated into 10 mL of LB medium containing 50 μg / mL Kana, and then cultured at 37 °C and 220 rpm for 12 h. Then, 10 mL of the culture was inoculated into 1.2 L of LB medium containing 50 μg / mL Kana, and then cultured overnight at 37 °C and 220 rpm. 30 L of TB medium was prepared in a 50 L fermenter, and an appropriate amount of sterile antifoaming agent was added. 1.2 L of seed culture was then inoculated, and the mixture was stirred at 37 °C and 400 rpm for approximately 6-8 h. When the OD was approximately 7-8, the temperature was lowered to 20 °C, and 12.5 mL of 0.5 M IPTG (final concentration 0.2 mM) was added to induce expression for 12 h. One hour after adding IPTG, feed was added at a rate of 1.5 L / h for a total of 10 L (the feed medium consisted of 30% glycerol and 15% yeast extract).

[0093] Fermentation broth was centrifuged at 9000 rpm for 15 min at 4 °C to obtain bacterial cells. For small-scale cell lysis, 10 g of cells were added to 20 mL of lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl) and 0.5 mg / mL lysozyme. The mixture was shaken at 220 rpm for 30 min at 28 °C, then vortexed thoroughly and disrupted (600 W, 2 s on, 4 s off, for a total of 12 min). For large-scale cell lysis, an appropriate amount of cells was added to lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl), resuspended completely, and then disrupted using an ultra-high pressure nano-homogenizer (800 bar, 4 cycles). The mixture was then centrifuged at 12000 rpm for 60 min at 4 °C, and 80 μL of 5 M imidazole was added to the supernatant to a final concentration of 20 mM. The protein was then purified using Ni affinity chromatography. The clarified lysis buffer was loaded onto a chromatography column containing an appropriate volume of Ni ion packing material. The column was equilibrated with lysis buffer containing 20 mM imidazole (10 column volumes), followed by equilibration with lysis buffer containing 30-40 mM imidazole (10 column volumes). Finally, the protein was eluted with lysis buffer containing 300 mM imidazole (5-10 column volumes). The protein was desalted using a PD-10 desalting column and equilibrated with 50 mM Tris-HCl (pH 7.5, 300 mM NaCl, 10% glycerol) or dialyzed using a dialysis bag (6.4 cm / mL, 10 kDa, source leaf). Each 50 mL eluent was dialyzed three times with 5 L of storage buffer and 50 mM Tris-HCl (pH 7.5, 300 mM NaCl, 10% glycerol) (the buffer was changed every 6 hours, with the last dialyzed overnight). The concentration of the obtained protein was determined using Nanodrop, and its purity was determined by SDS-PAGE.

[0094] Example 2: Expression and purification of acl, AtHMT, ScOMT and NfOMT and preparation of their lysis buffers

[0095] The plasmids acl / pET-28a and AtHMT / pET-28a were chemically transformed into... E. coliBL21 dmtn(DE3) competent cells. 10 μL of glycerol-containing bacteria (acl, AtHMT, MT-6, and MT-31) were inoculated into 20 mL of LB medium containing 50 μg / mL Kana resistance, and cultured overnight at 37 °C and 220 rpm. Then, 20 mL of the pre-cultures were inoculated into 1 L of LB medium containing 50 μg / mL Kana resistance and cultured at 37 °C and 220 rpm until the OD600 reached approximately 0.8. After cooling the culture to room temperature, 1 mL of 0.2 M IPTG (final concentration 0.2 mM) was added. Acl and AtHMT were induced to express for 24 h at 20 °C and 220 rpm. MT-6 and MT-31 were induced to express for 20 h at 28 °C and 220 rpm. The bacterial culture was centrifuged at 4 °C and 9000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were collected, yielding approximately 5 g of bacterial cells per liter.

[0096] For ScOMT and NfOMT, 200 mL of 50 mM PBS (100 mM NaCl, pH 7.8) was added to every 7.5 g of bacterial cells. After resuspending, the cells were homogenized (800 bar, 3 cycles), centrifuged, and the supernatant was used for the methylation preparation reaction. For the optimal mutants of acl and AtHMT, 50 mL of 50 mM PBS (100 mM NaCl, pH 7.8) was added to every 5 g of mutant bacterial cells. After resuspending, the cells were homogenized (800 bar, 3 cycles), centrifuged, and the supernatant was used for the methylation preparation reaction.

[0097] The purification method for ACIL and AtHMT is as follows: The lysis supernatant is loaded onto a chromatography column containing 1 mL of Ni ion packing material. The column is equilibrated with 10 mL of lysis buffer (20 mM PBS (pH 7.8, 300 mM NaCl)). Impurities are then washed with 10 mL of lysis buffer containing 10 mM and 20 mM imidazole, respectively. Finally, the protein is eluted with 5–10 mL of lysis buffer containing 250 mM imidazole. Desalting is performed using a PD-10 desalting column, and the column is equilibrated with storage buffer (20 mM PBS (pH 7.8, 300 mM NaCl)). Alternatively, dialysis can be performed using a 10 kDa, 4.5 cm wide dialysis bag with 20 mM PBS (pH 7.8, 300 mM NaCl). The dialysis buffer is changed three times within 24 hours, and protein concentration is determined using Nanodrop.

[0098] Thiopurine methyltransferase acl, halide methyltransferase At HMT, SAH hydrolase Sac SAHH, ScOMT and Nf OMT was synthesized by Sangon Biotech Co., Ltd. after codon optimization and then ligated into the vector pET-28a. Sac SAHH's restriction enzyme sites are NdeI and XhoI. At HMT, Sc OMT and Nf OMT has NdeI and HindIII restriction sites, both of which retain the N-terminal His tag; acl has NcoI / XhoI restriction sites, both of which retain the C-terminal His tag.

[0099] Sac The amino acid sequence of SAHH is shown in SEQ ID NO: 1; the amino acid sequence of acl is shown in SEQ ID NO: 2; At The HMT amino acid sequence is shown in SEQ ID NO: 3; Sc The OMT amino acid sequence is shown in SEQ ID NO: 2; Nf The OMT amino acid sequence is shown in SEQ ID NO: 5.

[0100] SacSAHH amino acid sequence (SEQ ID NO: 1)

[0101] MDYRVKDLSLAEQGRKQIEWAELHMPALMEIRKRFNAEKPLDGIRIGAVLHVTKETAVLVETLKAGGAEIALAGSNPLSTQDDVAAGLAKNGIHVYAWRGETE KDYYDNIREILKYEPHVIMDDGGDLHAYVHENNLTSKIVGGTEETTTGVIRLKAMEEEKVLKYPVIAVNNAFTKYLFDNRIGTGQSTIDGILRATNILIAGKVA VVIGYGWVGRGIASRFKGMGARVIVVESSPFRALEALMDGFDVMTMNRASEIGDIFVTATGNLNVVSRDHILRMKDGAVLANSGHFNVEIDVKGLKEISVETRE VRQNLEEYKLRNGKRIYLLADGRLVNLVAAEGHPSEVMDLSFCNQALSVEHLIKNKGKLENKVYNVPIEIDEQVARLKLKALGIEIEELTIEQKEYIKQWKYGT

[0102] acl amino acid sequence (SEQ ID NO: 2)

[0103] MSTPSLIPSGVHEVLAKYKDGNYVDGWAELWDKSKGDRLPWDRGFPNPALEDTLIQKRAIIGGPLGQDAQGKTYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSATAVDVCQEEQAKNGDQYPVRDAEIGQGKITFVQGDFFEDTWLEKLNLTRNCFDVIYDYTFFCALNPSMRPQWALRHTQLLADSPRGHLICLEFPRHKDPSVQGPPWGSASEAYRAHLSHPGEEIPYDASRQCQFDSSKAPSAQGLERVAYWQPERTHEVGKNEKGEVQDRVSIWQRPPQSSL

[0104] Amino acid sequence of AtHMT (SEQ ID NO: 3)

[0105] MAEEQQNSDQSNGGNVIPTPEEVATFLHKTVEEGGWEKCWEEEITPWDQGRATPLIVHLVDTSSLPLGRALVPGCGGGHDVVAMASPERFVVGLDISESALAKANETYGSSPKAEYFSFVKEDVFTWRPTELFDLIFDYVFFCAIEPEMRPAWAKSMYELLKPDGELITLMYPITDHVGGPPYKVDVSTFEEVLVPIGFKAVSVEENPHAIPTRKGKEKLGRWKKIN

[0106] Amino acid sequence of ScOMT (SEQ ID NO: 4)

[0107] MSSAERDTERTPVGWQERSIVMQLAFGQMSTHVLGVAVRLEVFDRVGDGELTAASLADDLGTHPQATHRLLRALAGLRLLTETAPGTFATTPAGDLLRSGVPGTLLSVARVFTDPSMTRGWELLEQSVRTGRTGFDAAFGTDFFGYLKEHPALSAEFNVAMGQATRVAAEVLPAHY DFGRFGTIVDVGGGDGTLLSAVLRAHAGPRGVVYDTAEGLAQAPARLADEGIADRVTLTVGDFFASAPAGGDLYLLKSVIHDWNDEQCVTILRHIRDVIPADGTLLIVEPVLPAVVSGETTELAYLSDLNMLVNVGGRERTADDFAALCSEAGFKLGGITPLPQPNVFQLIEATPA

[0108] NfOMT amino acid sequence (SEQ ID NO: 5)

[0109] MSTPSPALFLNTVNAYQRSAAIKAAVELDIFSVISQGIESSQSLAQKCQTSERGMRILCDYLVIMGFMTKETEGYRLTPDSAMFLARQSKFYLGDTVEFLLSPMITSSFNDLTSAVRTGGTVVSSEGTLSPQNPVWVQFAKAMAPMMANPAQLIAQLVSQNNIKPTK VLDISASHGLFGIAIAQHNPDAEIFGVDWASVLEVAQENARIQGVGSRYHTIAGSAFEVDYGNNYDLVLLPNFLHHFDVATCEQLLRKIKNALVTDGRVIVFDFIPNTDRITPPDAAAFSLVMLATTPSGDAYTFGEYESMFSNAGFSRCQLHPLPPTQQQVIVANK

[0110] Example 3: Synthesis and preparation of S-adenosyl-L-cysteine ​​(SAC)

[0111] The purification method for SacSAHH in this example is the same as in Example 1. The required system is 30 mL. In a 100 mL Erlenmeyer flask, weigh the following: 36.3 mg L-Cys (Biode Pharmaceuticals, 10 mM), 80.2 mg adenosine Ado (Admas, 10 mM), and 39.8 mg NAD. +(Admas, 2 mM) was first partially dissolved in 50 mM Tris-HCl (Admas, pH 7.5), then 65 μM SacSAHH (3 mg / mL, 5.4 mL) was added, and finally, 50 mM Tris-HCl (pH 7.5) was added to bring the volume to 30 mL, for a total of 12 flasks. The mixture was reacted on a shaker at 28 °C and 170 rpm for 8 h, and the remaining amount of Admas was monitored by LC-MS. When the reaction was almost complete, 3 times the volume of methanol was added to quench the reaction. The mixture was centrifuged at 10,000 rpm for 20 min, and the supernatant was filtered and evaporated to dryness to obtain a white residue. The residue was washed repeatedly with ice-cold water and methanol, and the collected white solid was dried in a vacuum drying oven to obtain S-adenosyl-L-cysteine ​​(SAC). The compound was characterized by NMR. SAC, white solid, 82.1% yield. 1H NMR (400 MHz, DeuteriumOxide) δ 8.52 (s, 1H), 8.46 (s, 1H), 6.15 (d, J = 4.8 Hz, 1H), 4.90 (t, J =5.1 Hz, 1H), 4.47 (t, J = 5.1 Hz, 1H), 4.40 – 4.34 (m, 1H), 4.22 (dd, J =7.9, 4.3 Hz, 1H), 3.25 (dd, J = 14.9, 4.3 Hz, 1H), 3.14 – 3.03 (m, 3H). 13CNMR (100 MHz, D2O) δ 170.5, 149.9, 148.3, 144.5, 143.1, 119.0, 88.6, 84.3, 73.3, 72.3, 52.2, 33.6, 32.0.

[0112] Example 4: Directed Evolution of acl and Rational Design of AtHMT

[0113] Table 1 Primers related to acl mutation

[0114] Saturated mutant libraries of acl_G84 and acl_T165 were constructed using primers G84NNK-F / R and T165NNK-F / R as shown in Table 1. The PCR system consisted of: 0.5 μL acl plasmid (0.5 ng / μL), 1 μL each of forward and reverse primers (10 μM stock solution), 10 μL 2×KOD, and 7.5 μL ddH2O to a final volume of 20 μL. The PCR conditions were: 98 °C pre-denaturation for 1 min, followed by 15 cycles of denaturation (98 °C, 10 s) - annealing (60 °C, 5 s) - extension (68 °C, 1 min 40 s), and a final extension of 5 min. After successful PCR verification by nucleic acid gel electrophoresis, 1 μL of DpnI was added to the PCR product and the mixture was digested overnight at 37 °C. Next, 5 μL of the digestion product was transformed into 100 μL of Escherichia coli DH5α. After recovery, no plating was performed. The transformed product was inoculated into 5 mL LB tubes containing 50 μg / mL Kana and cultured overnight at 37 °C and 220 rpm. Plasmid extraction was then performed, followed by transformation into... Escherichia coli In BL21-dmtn(DE3) competent states.

[0115] The obtained single colonies were picked up with sterile toothpicks and placed into shallow-well plates, each well containing 100 μL of LB medium with 50 μg / mL Kana. 192 single colonies were selected from each site and cultured overnight at 37 °C and 800 rpm. Then, 10 μL of the bacterial culture was inoculated into 250 μL of LB medium containing 50 μg / mL Kana as a seed plate and cultured overnight at 37 °C and 800 rpm. The remaining bacterial culture was preserved by adding an equal volume of 40% glycerol. 50 μL of the seed culture was inoculated into 950 μL of LB medium containing 50 μg / mL Kana and 0.1 mM IPTG and induced to express overnight at 20 °C and 950 rpm.

[0116] Remove the plate and centrifuge at 4000 g for 10 min. Discard the supernatant and add 200 μL of lysis buffer (50 mM PBS, pH 7.8, 100 mM NaCl) to each well. Add SAC to a final concentration of 1 mM, 0.5 mg / mL lysozyme, 4 U / mL totipotent nuclease, and 0.3×bugbster. Incubate at 25 °C and 700 rpm for 30 minutes. Add 3 μL of 0.2 M MeOTs to each well and react at 25 °C and 800 rpm for 20 h.

[0117] After the reaction was complete, 50 μL of 4% TFA was added to each well to quench the reaction. The mixture was then centrifuged at 4000 g for 20 min, and the supernatant was transferred to a 96-well filter plate (10 μm, Sangon Biotech, catalog number B615006). After centrifugation at 4000 g for 1 min, the filtrate was transferred to a liquid chromatography vial. The conversion rate was calculated by HPLC based on the reduction in substrate peak area. Mutants with a conversion rate of 30% or higher were sequenced. The HPLC analysis method was as follows: Cation-exchange LC column 150 × 4.6 mm (Luna 5 μm SCX 100 Å); mobile phases were 20 mM H3PO4 (A) and 20 mM H3PO4 containing 1 M NaCl (B). Both phases A and B were filtered through a 0.22 μm filter membrane. The mobile phase gradient was 0–0.5 min, 20% B; 0.5–7.5 min, 20–99% B; 7.5–10 min, 99% B; 10–10.5 min, 99–20% B; 10.5–12 min, 20% B; the flow rate was 1 mL / min, and the UV was 260 nm. Mutants with a transformation rate of over 30% were selected from the NNK saturated mutant library (1–7) for sequencing. Specific mutations and transformation rates are shown in Table 2.

[0118] Table 2. Selected mutants and their corresponding transformation rates

[0119] Other mutations in acl and At The PCR system for the HMT mutation consisted of: plasmid (0.5 ng / μL), 1 μL each of the relevant forward and reverse primers (10 μM stock solution), 10 μL of 2×KOD, and 7.5 μL of ddH2O to a final volume of 20 μL. The PCR conditions were: 98 °C pre-denaturation for 1 min, followed by 15 cycles of denaturation (98 °C, 10 s) - annealing (55 °C, 5 s) - extension (68 °C, 1 min 40 s), and a final extension of 5 min. After successful PCR verification by nucleic acid gel electrophoresis, 1 μL of [unclear text - possibly a typo, should be removed] was added to the PCR product. Dpn I. Digest at 37°C overnight. PCR product transformation also begins with transformation to... Escherichia coli After DH5α is correctly sequenced, the plasmid is then transformed into... Escherichia coli In BL21-dmtn(DE3) competent cells, the expression and screening system and detection method for acl mutants are the same as those described above. At The expression of HMT mutants is the same as acl, and the screening system is also the same as acl, but 3 mM MeOTs need to be replaced with 8 mM MeI.

[0120] Ultimately, the optimal mutant acl_R43A-T165M, capable of efficiently catalyzing SAC methylation, was obtained. At HMT_Q49A-V140M.

[0121] Example 5 acl, At Specific activity assay of HMT and its mutants

[0122] One unit of enzyme activity (U) refers to the amount of enzyme required to catalyze the conversion of 1 nanomolar (nmol) of substrate per minute under optimal reaction conditions. The protein purification method in this example is the same as in Example 2.

[0123] The reaction system of acl and its mutants (200 μL): 1 mM SAC or SAH, 3 mM MeOTs dissolved in 50 mM PBS (pH 7.8, 100 mM NaCl), reaction at 25 °C, 800 rpm metal bath. (1) To determine the specific activity of wild-type acl, 600 μM protein was required for SAC substrates, and the reaction time was 60 min; for SAH substrates, 2 μM protein was required, and the reaction time was 15 min. (2) To determine the specific activity of acl_G84A and acl_G84S, 60 μM protein was required for SAC substrates, and the reaction time was 60 min; for SAH substrates, 2 μM mg / mL protein was required, and the reaction time was 15 min. (3) To determine the specific activity of acl_T165E, acl_T165M and acl_G84S-T165Q, 300 μM protein was required for SAC substrates, and the reaction time was 60 min; for SAH substrates, 10 μM protein was required, and the reaction time was 15 min. (4) To determine the specific activity of acl_T165G, 400 μM protein was required for SAC substrates, and the reaction time was 60 min; for SAH substrates, 10 μM protein was required, and the reaction time was 15 min. (5) Measure the specific activity of acl_R43A-G84A. For SAC substrate, 250 μM protein is required, and the reaction time is 15 min. For SAH substrate, 5 μM protein is required, and the reaction time is 5 min. (6) Measure the specific activity of acl_R43A-T165M. For SAC substrate, 60 μM protein is required, and the reaction time is 30 min. For SAH substrate, 2 μM protein is required, and the reaction time is 15 min. Take 20 μL of reaction solution each time, add 20 μL of 1% TFA to quench the reaction, and perform 3 replicates for each mutant. Calculate the conversion rate by HPLC. The HPLC method is the same as in Example 4.

[0124] At HMT and its mutant reaction system (200 μL): 1 mM SAC or SAH, 8 mM MeOTs dissolved in 50 mM PBS (pH 7.8, 100 mM NaCl), reaction at 25 °C, 800 rpm metal bath. (1) Determination At The specific activity of HMT wild-type requires 200 μM protein for SAC substrate and 10 min for reaction, and 1 μM L protein for SAH substrate and 5 min for reaction; (2) determination At The specific activity of HMT_Q49A-V140M wild-type requires 25 μM protein for SAC substrate and 10 min of reaction time, and 2 μM protein for SAH substrate and 5 min of reaction time. Each time, 20 μL of reaction solution is taken, and 20 μL of 1% TFA is added to quench the reaction. Triple replicates are performed for each mutant. Conversion rate is calculated by HPLC, using the same method as in Example 4. Table 3 shows the acl, At Specific activity data of HMT and its mutants against SAC and SAH.

[0125] Table 3 acl, At Specific activity data of HMT and its mutants against SAC and SAH

[0126] Example 6 acl, At Determination of the kinetic constants of HMT and its mutants

[0127] The kinetic constants were determined by calculating the conversion rate using HPLC. The HPLC analytical method is described in Example 4. Then, GraphPad Prism 9.0 software was used to fit the initial rate of the enzyme (the amount of SAC converted per unit time) at different concentrations to a Michaelis model to determine the kinetic constants. k cat and K m value.

[0128] The kinetics of different concentrations of SAC were determined by acl while maintaining a constant MeOTs concentration of 3 mM. The reaction system for acl kinetic constant determination (200 μL) consisted of 3 mM MeOTs, 600 μM acl, and 50 mM PBS (pH 7.8, 100 mM NaCl), at 25 °C. SAC concentrations of 0.1 mM, 0.3 mM, 0.5 mM, 0.6 mM, 1.0 mM, 1.2 mM, 1.5 mM, 1.8 mM, 2.0 mM, and 2.5 mM were measured. Samples were taken at 15 min, 30 min, and 45 min for each concentration. 40 μL of the reaction solution was taken each time, and 40 μL of 1% TFA was added to quench the reaction before analysis by HPLC. The reaction system for determining the kinetic constant of acl_R43A-T165M (200 μL) consisted of 3 mM MeOTs, 60 μM acl_R43A-T165M, and 50 mM PBS (pH 7.8, 100 mM NaCl). The reaction was carried out at 25 °C and 800 rpm. SAC concentrations of 0.1 mM, 0.3 mM, 0.5 mM, 0.7 mM, 0.9 mM, 1.0 mM, 1.5 mM, 1.8 mM, 2.0 mM, and 2.5 mM were measured. Samples were taken at 3 min, 6 min, and 9 min for the 0.1 mM and 0.3 mM concentrations, and at 10 min, 15 min, and 20 min for the other concentrations. For each reaction, 40 μL of the reaction solution was taken, and 40 μL of 1% TFA was added to quench the reaction. The mixture was then analyzed by HPLC.

[0129] At HMT conducted kinetic measurements on different concentrations of SAC while keeping the MeI concentration constant at 3 mM. At HMT kinetic constant determination reaction system (200 μL): 8 mM MeI, 200 μM At HMT, 50 mM PBS (pH 7.8, 100 mM NaCl), 25 °C, 800 rpm reaction. SAC concentrations were measured at 0.1 mM, 0.3 mM, 0.6 mM, 0.9 mM, 1.2 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, and 4.0 mM, with samples taken at 10 min, 15 min, and 20 min for each concentration. 40 μL of reaction solution was taken each time, and 40 μL of 1% TFA was added to quench the reaction before analysis by HPLC. At HMT_Q49A-V140M kinetic constant determination reaction system (200 μL): 8 mM MeI, 25 μM AtHMT_Q49A-V140M, 50mM PBS (pH 7.8, 100 mM NaCl), 25 °C. SAC concentrations were measured at 0.1 mM, 0.3 mM, 0.6 mM, 0.9 mM, 1.0 mM, 1.2 mM, 1.8 mM, 2.0 mM, 3.0 mM, and 4.0 mM. Samples were taken at 10 min, 15 min, and 20 min for each concentration. 40 μL of the reaction solution was taken each time, and 40 μL of 1% TFA was added to quench the reaction. Analysis was performed by HPLC. Table 4 shows the acl / At Kinetic data of HMT and its mutants.

[0130] Table 4 acl / At Kinetic data of HMT and its mutants

[0131] Example 7 Preparation of S-Adenosyl-Methylcysteine ​​(S-Me-SAC)

[0132] Purification of acl and its mutants is described in Example 2. The reaction system was 30 mL: In a 100 mL shake flask, 5 mL of 50 mM PBS (pH 7.8, 100 mM NaCl) and 2 mM SAC were added, mixed, and the pH was checked. If it was too acidic, it was adjusted to approximately 7.0 with NaOH. Then, 6 mM MeOTs were added, followed by 30 μM acl_R43A-T165M (1 mg / mL). The final volume was then brought to 30 mL with 50 mM PBS (pH 7.8, 100 mM NaCl). The reaction was carried out at 25 °C and 180 rpm, and the conversion was monitored by HPLC until it exceeded 99%. The reaction was quenched with twice the amount of methanol, centrifuged at 10,000 rpm for 20 min, and the supernatant was filtered and evaporated to dryness to obtain a white residue. This residue was dissolved in a small amount of water, and the product peak was collected by HPLC. The collected product was placed on ice and freeze-dried. The obtained solid was dissolved in D2O and identified by NMR. The HPLC method was the same as in Example 4. S-Me-SAC, 1 H NMR (600 MHz, Deuterium Oxide) δ 8.92 (s, 1H), 8.91 (s, 1H), 6.59 (d, J = 3.8 Hz, 1H), 5.25 (dd, J = 5.5, 3.9 Hz, 1H), 5.08 (t, J = 5.8 Hz, 1H), 5.04 -5.01 (m, 1H), 4.92(t, J= 7.2 Hz, 1H), 4.57 (dd, J = 13.7, 9.3 Hz, 1H), 4.45 (dd, J = 13.7, 2.7Hz, 1H), 4.37 (d, J = 7.2 Hz, 2H), 3.54 (s, 3H); 13 C NMR (151 MHz, D2O) δ169.5, 150.0, 148.5, 145.0, 144.3, 119.8, 90.5, 79.3, 73.5, 72.8, 49.6, 45.9,42.2, 26.9; HRMS (ESI-Orbitrap): calcd for [C 14 H 21 O5N6S] + (M + ):385.12887, found:385.12941.

[0133] Example 8: Control experiment verifies the application capability of MTs to S-Me-SAC.

[0134] Based on the synthesis method of Example 7, in a 20 mL reaction system with a SAC concentration of 2 mM, after the SAC had almost completely reacted, 1.5 times the amount of methanol was added to quench the reaction. The mixture was centrifuged at 10,000 rpm for 30 min, the supernatant was collected, the solvent was removed using a vacuum rotary evaporator, and then 1.5 mL of ddH2O was added to dissolve the residue. The approximate concentration of the crude S-Me-SAC product was estimated to be approximately 32 mM based on the concentration corresponding to the SAC peak area. The pH needed to be adjusted to approximately 6.0 before the reaction.

[0135] Sc OMT and Nf OMT induction expression is described in Example 2. Take 5 mL of the expressed bacterial culture, centrifuge at 10000 rpm for 5 min, discard the supernatant, add 1 mL of lysis buffer (1 mg / mL lysozyme, 4 U / mL totipotent nuclease, and 0.3×bugbuster in 50 mM PBS buffer (pH 7.8, 100 mM NaCl), resuspend, and lyse at 30 °C and 600 rpm for 1.0 h. Then centrifuge at 10000 rpm for 15 min, and use the supernatant from the lysis buffer for further reaction.

[0136] The control experimental verification system was 200 μL. Sc OMT and NfAdd approximately 1 mM S-Me-SAC to the OMT1 lysis buffer, and then add 1 mM hesperidin ( Sc OMT validation substrate) or luteolin ( Nf OMT1 (substrate for verification), reacted at 25 °C, 900 rpm for 16 h. A negative control was also performed without S-Me-SAC; all other conditions remained unchanged. After the reaction, an equal volume of methanol was added to quench the reaction, followed by centrifugation at 10000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane and transferred to a HPLC vial for HPLC analysis. The HPLC method was: Thermo Acclaim column. TM The test setup was 120 C18 (5 μm, 120 Å, 4.6 × 250 mm, Lot No. 02020124). The mobile phase consisted of 0.1% trifluoroacetic acid (A) and acetonitrile (B). The mobile phase gradient was 0–15 min, 20%–100% B; 15–20 min, 20% B. The flow rate was 1 mL / min, and the UV was 260 / 280 nm. Table 5 shows the results of the control experiments. The results indicate that… Sc OMT and Nf OMT can utilize S-Me-SAC to achieve methylation of natural products.

[0137] Table 5 Results of the Control Experiment

[0138] Examples 9-13 are Sc OMT and Nf OMT is based on a coenzyme regeneration system and uses SAC to initiate the reaction to prepare methylated products. The SAC was prepared in Example 3. The brands of MeOTs and MeI are both Admas.

[0139] Example 9

[0140] To prepare 5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4 H Taking -chromone-4-one 1 as an example, the raw materials and preparation method are as follows:

[0141] In a 500 mL shake flask, add 0.1 mM SAC, 3 mM MeOTs, and 15 mL of 50 mM PBS (100 mM NaCl, pH 7.8). Mix well; the pH should now be around 7.0. Then add 200 mL of 37.5 mg / mL (wet cell weight / buffer). NfThe lysis buffer of OMT and 15 mL of 100 mg / mL (wet cell weight / buffer) acl_R43A-T165M lysis buffer were added, and finally 1 mM luteolin was added. The reaction was carried out at 30 °C and 180 rpm for 4 h. The conversion rate was monitored by high performance liquid chromatography (HPLC). The analysis method is described in Example 8. After the reaction was completed, 2.5 times the volume of cold methanol was added to the reaction solution to terminate the reaction. The mixture was centrifuged at 10,000 rpm for 30 min, and the supernatant of the collected mixture was filtered and concentrated. The residue was dissolved in a mixture of DMSO and methanol, centrifuged and filtered through a membrane, and purified by preparative liquid chromatography (Pre-HPLC). The obtained product was structurally identified by NMR. The preparative column was a Hypersil PREP HS C18 (10 μm, 10 × 150 mm, Thermo Scientific). The mobile phase was 0.1% trifluoroacetic acid (A) and acetonitrile (B). The chromatographic gradient program was as follows: 0–1 min, 20% B; 1–21 min, 20%–100% B; 21–24 min, 100% B; 24–30 min, 20% B; the flow rate was set to 5 mL / min, and the UV was 260 nm. A yellow solid was obtained, with a conversion rate of 88.3%.

[0142] The above-mentioned yellow solid was characterized by NMR and mass spectrometry, and the results are as follows: Figures 2-6 As shown, where Figure 2 This is the proton NMR spectrum. Figure 3 This is a carbon NMR spectrum. Figure 4 This is an HSQC image from nuclear magnetic resonance imaging. Figure 5 This is an HMBC image from nuclear magnetic resonance imaging. Figure 6 The image shows the high-performance liquid chromatogram after the reaction. The specific data from NMR and high-resolution mass spectrometry are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 12.95 (s, 1H), 7.57 – 7.52 (m, 2H), 6.95(d, J = 8.9 Hz, 1H), 6.87 (s, 1H), 6.52 (d, J = 2.1 Hz, 1H), 6.21 (d, J = 2.1Hz, 1H), 3.88 (s, 3H).

[0143] 13 C NMR (101 MHz, DMSO- d6) δ 181.8, 164.3, 163.7, 161.5, 157.4, 150.8,148.1, 121.5, 120.4, 115.8, 110.2, 103.7, 103.2, 98.9, 94.1, 56.0.

[0144] HRMS (ESI-Orbitrap): calcd for [C 16 H 12 O6] + (M+H + ): 301.07066, found: 301.06972.

[0145] Example 10

[0146] To prepare 5,7-dihydroxy-2-(4-hydroxyphenyl)-3-methoxy-4 H -Crone-4-one 2,3,5-dihydroxy-2-(4-hydroxyphenyl)-7-methoxy-4 H -Crone-4-one 3,5-hydroxy-2-(4-hydroxyphenyl)-3,7-dimethoxy-4 H Taking -chromone-4-one as an example, the raw materials and preparation method are as follows:

[0147] In a 500 mL shake flask, add 0.1 mM SAC, 3 mM MeOTs, and 15 mL of 50 mM PBS (100 mM NaCl, pH 7.8). Mix well; the pH should now be around 7.0. Then add 200 mL of 37.5 mg / mL (wet cell weight / buffer). Nf The OMT lysis buffer and 15 mL of 100 mg / mL (wet cell weight / buffer) acl_R43A-T165M lysis buffer were added, and finally 1 mM kaempferol was added. The reaction was carried out at 30 °C and 180 rpm for 20 h. The remaining operations were the same as in Example 9. After separation and purification, three compounds were obtained as yellow solids. The conversion rates of compound 2 were 43.5%, compound 3 was 11.8%, and compound 4 was 35.7%.

[0148] The above-mentioned yellow solid was characterized by NMR and mass spectrometry, and the specific data are as follows: Compound 2 1 H NMR (400 MHz, DMSO- d6) δ 12.68 (s, 1H), 7.96 – 7.91 (m, 2H), 6.96 – 6.92 (m, 2H), 6.43 (d, J J = 2.0 Hz, 1H), 6.19 (d, J J = 2.1 Hz, 1H), 3.78 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ 177.9, 164.1, 161.3, 160.2, 156.4, 155.6, 137.6, 130.2, 120.6, 115.7, 104.2, 98.6, 93.7, 59.7. HRMS (ESI-Orbitrap): calcd for [C 16 H 12 O6] + (M+H + ): 301.07066, found: 301.0697.

[0149] Compound 3

[0150] 1 H NMR (400 MHz, DMSO- d 6) δ 12.47 (s, 1H), 10.15 (s, 1H), 9.51 (s, 1H), 8.11 – 8.06 (m, 2H), 6.96 – 6.91 (m, 2H), 6.74 (d, J J = 2.2 Hz, 1H), 6.35 (d, J J = 2.2 Hz, 1H), 3.86 (s, 3H).

[0151] 13 C NMR (101 MHz, DMSO- d 6) δ 176.1, 164.9, 160.4, 159.4, 156.1, 147.3, 136.0, 129.6, 121.6, 115.5, 104.1, 97.5, 92.0, 56.1.

[0152] HRMS (ESI-Orbitrap): calcd for [C 16 H 12 O6] + (M+H+ ): 301.07066, found: 301.06996.

[0153] Compound 4

[0154] 1 H NMR (400 MHz, DMSO- d 6) δ 12.67 (s, 1H), 10.30 (s, 1H), 8.00 – 7.95 (m, 2H), 6.98 – 6.93 (m, 2H), 6.74 (d, J = 2.3 Hz, 1H), 6.37 (d, J = 2.2 Hz,1H), 3.86 (s, 3H), 3.80 (s, 3H).

[0155] 13 C NMR (101 MHz, DMSO- d 6) δ 178.1, 165.1, 160.9, 160.3, 156.3, 156.0,137.8, 130.2, 120.5, 115.7, 105.2, 97.8, 92.3, 59.7, 56.1.

[0156] HRMS (ESI-Orbitrap): calcd for [C 17 H 14 O6] + (M+H + ): 315.08631, found: 315.08522.

[0157] Example 11

[0158] To prepare 5,7-dihydroxy-6-methoxy-2-phenyl-4-propane with the following structural formula H -Crone-4-one 5,5-hydroxy-6,7-dimethoxy-2-phenyl-4 H -Crotene-4-one 6, the raw materials and preparation method are as follows:

[0159] In a 500 mL shake flask, add 0.1 mM SAC, 3 mM MeOTs, and 15 mL of 50 mM PBS (100 mM NaCl, pH 7.8). Mix well; the pH should now be around 7.0. Then add 200 mL of 37.5 mg / mL (wet cell weight / buffer). ScThe OMT lysis buffer and 15 mL of 100 mg / mL (wet cell weight / buffer) acl_R43A-T165M lysis buffer were added, and finally 1 mM baicalein was added. The reaction was carried out at 30 °C and 180 rpm for 20 h. The remaining operations were the same as in Example 9. After separation and purification, two compounds in yellow solid form were obtained. The conversion rate of compound 5 was 49.6%, and the conversion rate of compound 6 was 44.2%.

[0160] The above-mentioned yellow solid was characterized by NMR and mass spectrometry, and the specific data are as follows: Compound 5 1 H NMR (400 MHz, DMSO- d 6) δ 12.90 (s, 1H), 8.08 – 8.01 (m, 2H), 7.60 –7.54 (m, 3H), 6.93 (s, 1H), 6.68 (s, 1H), 3.75 (s, 3H).

[0161] 13 C NMR (101 MHz, DMSO- d 6) δ 182.3, 163.3, 157.85, 152.8, 152.6,132.1, 131.66, 130.8, 129.2, 126.4, 104.7, 104.3, 94.5, 60.0.

[0162] HRMS (ESI-Orbitrap): calcd for [C 16 H 12 O5] + (M+H + ): 285.07575, found: 285.07473.

[0163] Compound 6

[0164] 1 H NMR (400 MHz, DMSO- d 6) δ 12.76 (s, 1H), 8.12 – 8.08 (m, 2H), 7.63 –7.55 (m, 3H), 7.03 (s, 1H), 6.97 (s, 1H), 3.93 (s, 3H), 3.74 (s, 3H).

[0165] 13 C NMR (101 MHz, DMSO- d6) δ 182.4, 163.5, 158.9, 152.8, 152.03,132.2, 132.0, 130.6, 129.2, 126.4, 105.4, 105.0, 91.8, 60.1, 56.5.

[0166] HRMS (ESI-Orbitrap): calcd for [C 17 H 14 O5] + (M+H + ): 299.09140, found: 299.09040.

[0167] Example 12

[0168] To prepare 3,5,7-trihydroxy-2-(3,4,5-trimethoxyphenyl)-4 H -Cr-4-one7, the raw materials and preparation method are as follows:

[0169] In a 500 mL shake flask, add 0.1 mM SAC, 3 mM MeOTs, and 15 mL of 50 mM PBS (100 mM NaCl, pH 7.8). Mix well; the pH should now be around 7.0. Then add 200 mL of 37.5 mg / mL (wet cell weight / buffer). Sc The OMT lysis buffer and 15 mL of 100 mg / mL (wet cell weight / buffer) acl_R43A-T165M lysis buffer were added, and finally 1 mM myricetin was added. The mixture was reacted at 30 °C and 180 rpm for 20 h. The remaining operations were the same as in Example 9. After separation and purification, a yellow solid was obtained, with a conversion rate of 82.8%.

[0170] The above-mentioned yellow solid was characterized by NMR and mass spectrometry, and the specific data are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 12.35 (s, 1H), 7.48 (s, 2H), 6.52 (d, J =2.1 Hz, 1H), 6.21 (d, J = 2.1 Hz, 1H), 3.85 (s, 6H), 3.75 (s, 3H).

[0171] 13 C NMR (101 MHz, DMSO-d 6) δ 176.2, 164.2, 160.7, 156.3, 152.8, 145.7,139.2, 136.9, 126.3, 105.6, 103.15, 98.4, 93.8, 60.3, 56.1.

[0172] HRMS (ESI-Orbitrap): calcd for [C 18 H 16 O8] + (M+H + ): 361.09179, found: 361.09052.

[0173] Example 13

[0174] The raw materials and preparation method for preparing 2-(3,4-dimethoxyphenyl)-5,7-dihydroxyflavanone 8 with the following structural formula are as follows:

[0175] In a 500 mL shake flask, add 0.1 mM SAC, 3 mM MeOTs, and 15 mL of 50 mM PBS (100 mM NaCl, pH 7.8). Mix well; the pH should now be around 7.0. Then add 200 mL of 37.5 mg / mL (wet cell weight / buffer). Sc The OMT lysis buffer and 15 mL of 100 mg / mL (wet cell weight / buffer) acl_R43A-T165M lysis buffer were added, and finally 1 mM hesperidin was added. The reaction was carried out at 30 °C and 180 rpm for 4 h. The remaining operations were the same as in Example 9. After separation and purification, a white solid was obtained, with a conversion rate of 98.7%.

[0176] The white solid described above was characterized by NMR and mass spectrometry, and the specific data are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 12.14 (s, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.02 (dd, J = 8.3, 2.0 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 5.96 – 5.88 (m,2H), 5.46 (dd, J= 12.7, 3.0 Hz, 1H), 3.77 (s, 3H), 3.76 (s, 3H), 3.32 – 3.25(m, 1H), 2.71 (dd, J = 17.1, 3.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d 6) δ 196.3, 166.9, 163.6, 162.9, 149.1, 148.8,131.1, 119.4, 111.6, 110.7, 101.8, 96.0, 95.2, 78.6, 55.7, 55.6, 42.2. HRMS (ESI-Orbitrap): calcd for [C 17 H 16 O6] + (M+H + ): 367.11521, found:367.11400。

Claims

1. A method for preparing S-adenosyl-methylcysteine ​​or S-adenosyl-methionine, characterized in that, The preparation method includes the following reaction: S-adenosyl-L-cysteine ​​was obtained by methylating S-adenosyl-L-cysteine ​​with a methyl donor using a promethyltransferase or a promethyltransferase mutant; or, S-adenosyl-L-homocysteine ​​was obtained by methylating S-adenosyl-L-homocysteine ​​with a methyl donor using a methyltransferase or a mutant of the first methyltransferase. Wherein, the amino acid sequence of the first methyltransferase is as shown in SEQ ID NO:2 or SEQ ID NO:3; or, the amino acid sequence of the first methyltransferase mutant has at least 85% identity with SEQ ID NO:2 or SEQ ID NO:3, and maintains or has a first methyltransferase mutant with better efficacy than the first methyltransferase.

2. The preparation method according to claim 1, characterized in that, The first methyltransferase mutant differs from the amino acid sequence shown in SEQ ID NO: 2 in one or more of the following amino acid residues: G84, T165, and R43, wherein the positions of G84, T165, and R43 are as specified in the amino acid residue numbers in SEQ ID NO: 2; and / or, The total number of amino acid residue differences between the first methyltransferase mutant and the amino acid sequence shown in SEQ ID NO: 2 does not exceed 3, for example, 1, 2 or 3; Preferably, the first methyltransferase mutant contains one or more of the following differences compared to the amino acid sequence shown in SEQ ID NO: 2: (1) G84A or G84S; (2) One of T165E, T165G, T165M, T165Q, T165H, T165R, T165K, T165I, T165F, T165L, T165W, T165A, T165P, T165C, T165N, T165V, T165S, T165Y and T165D; (3) R43A; The preferred materials are one or more of G84A / S, T165E / G / M / Q and R43A.

3. The preparation method according to claim 1 or 2, characterized in that, The first methyltransferase mutant has one of the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 2: (1) G84A; (2) G84S; (3) T165E; (4) T165G; (5) T165M; (6) G84S and T165Q; (7) R43A and T165M; (8) G84A and R43A; Preferably, the first methyltransferase mutant has mutations of R43A and T165M compared to the amino acid sequence shown in SEQ ID NO:

2.

4. The preparation method according to claim 1, characterized in that, The first methyltransferase mutant differs from the amino acid sequence shown in SEQ ID NO: 3 in one or more amino acid residues at sites V140 and Q49, wherein the positions of V140 and Q49 are as specified in the amino acid residue numbers in SEQ ID NO: 3; and / or, The total number of amino acid residue differences between the first methyltransferase mutant and the amino acid sequence shown in SEQ ID NO: 3 does not exceed 2, for example, 1 or 2.

5. The preparation method according to claim 4, characterized in that, The first methyltransferase mutant contains one or more of V140A / M and Q49A compared to the amino acid sequence shown in SEQ ID NO: 3; Preferably, the first methyltransferase mutant contains mutations of Q49A and V140M compared to the amino acid sequence shown in SEQ ID NO:

3.

6. The preparation method according to any one of claims 1-5, characterized in that, The reaction includes the following steps: (i) Dissolve S-adenosyl-L-cysteine ​​and the methyl donor in PBS; (ii) Add the first methyltransferase or the first methyltransferase mutant to the system to initiate the reaction; (iii) Quenching reaction; Preferably, the final concentration of the S-adenosyl-L-cysteine ​​is 0.1-4.0 mM, for example, 1 mM or 2 mM; and / or, The methyl donor is MeI or MeOTs; preferably, the reaction concentration of MeI is 1-10 mM, for example, 8 mM, and the reaction concentration of MeOTs is 1-10 mM, for example, 3 mM or 8 mM; and / or, The PBS concentration is 50 mM; and / or, The reaction concentration of the first methyltransferase or the first methyltransferase mutant is 1-1000 μM, for example 1 μM, 2 μM, 10 μM, 25 μM, 250 μM, 60 μM, 300 μM, 400 μM, 600 μM, 200 μM, or 30 μM; and / or, The reaction conditions are 10-50 degrees Celsius, 0-1000 rpm, for example, 25 degrees Celsius, 180 or 800 rpm; and / or, The quenching reaction is carried out using twice the volume of methanol or an equal volume of 1% TFA.

7. The preparation method according to claim 6, characterized in that, The stock solution for S-adenosine-L-cysteine ​​is a 200 mM stock solution prepared with dilute hydrochloric acid; and / or, The MeI mother liquor is a 400 mM mother liquor prepared with DMSO; the MeOTs mother liquor is a 200 mM or 1000 mM mother liquor prepared with MeCN or acetonitrile. Preferably, step (iii) further includes centrifugation, filtration, rotary evaporation, and dissolution to obtain the product after quenching the reaction; for example, centrifuging at 10,000 rpm for 20 minutes after quenching the reaction, collecting the supernatant, filtering and rotary evaporation to obtain a white residue, and dissolving it in a small amount of water; and / or, using HPLC to collect product peaks or estimate the concentration of the final product.

8. The preparation method according to claim 7, characterized in that, The preparation method further includes the synthesis of S-adenosine-L-cysteine, which includes the following steps: S-adenosyl-L-cysteine ​​was prepared using hydrolytic enzymes, L-cysteine, adenine nucleoside, and NAD+. Preferably, the hydrolase is selected from S-adenosyl homocysteine ​​hydrolases, preferably SacSAHH or SsoSAHH, for example SacSAHH; and / or, The reaction concentration of L-cysteine ​​is 10 mM; and / or, The reaction concentration of adenosine Ado is 10 mM; and / or, The NAD + The reaction concentration was 2 mM.

9. The preparation method according to claim 8, characterized in that, The amino acid sequence of the SacSAHH is shown in SEQ ID NO: 2; and / or, L-cysteine, adenosine Ado and NAD + Both are soluble in 50 mM Tris-HCl at pH 7.5; Preferably, the synthesis further includes quenching the reaction with 3 times the volume of methanol after the reaction is completed, centrifuging at 10,000 rpm for 20 min, filtering the supernatant and evaporating to obtain a white residue, washing the residue multiple times with ice-cold water and methanol, and drying the collected white solid in a vacuum drying oven to obtain the final product.

10. A transferase mutant as defined in the preparation method of claim 2 or 3.

11. A transferase mutant as defined in the preparation method of claim 4 or 5.

12. An isolated nucleic acid molecule, characterized in that, The isolated nucleic acid molecule encodes the enzyme mutant as described in claim 10 or 11.

13. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid molecule as described in claim 12; Preferably, the recombinant expression vector is a plasmid, and the backbone of the plasmid is preferably pET-22b, pET28a, pET-30a, pET-32a, pET-43.1a or pET-DEST51, for example pET28a.

14. A transformant, characterized in that, The transformant comprises the isolated nucleic acid molecule as described in claim 12, or the recombinant expression vector as described in claim 13; Preferably, the host cell used in the construction of the transformant is Escherichia coli. More preferably, the Escherichia coli is Escherichia coli BL21 dmtn(DE3).

15. A method for preparing the transferase mutant as described in claim 10 or 11, comprising culturing the transformant as described in claim 14 to obtain a fermentation product; Preferably, the culture medium used for the culture is selected from LB liquid medium or TB liquid medium, and / or the culture conditions are: shaking culture at a temperature of 37±1℃; More preferably, the method further includes the step of purifying the fermentation product to obtain the first methyltransferase mutant.

16. Use of a first methyltransferase having an amino acid sequence as shown in SEQ ID NO: 2 or 3, a first methyltransferase mutant as described in claim 10 or 11, an isolated nucleic acid molecule as described in claim 12, a recombinant expression vector as described in claim 13, or a transformant as described in claim 14 in the preparation of S-adenosyl-methylcysteine ​​and / or S-adenosyl-methionine.

17. A reaction end product system for preparing S-adenosine-methylcysteine, characterized in that, The reaction end product system includes: (1) S-adenosyl-methylcysteine; and / or, S-adenosyl-L-cysteine; (2) Methyl donor; and, (3) A first methyltransferase having the amino acid sequence shown in SEQ ID NO: 2 or 3 and any one of the transferase mutants as described in claim 10 or 11; Preferably, the reaction end product system further includes one or more of the following: (i) L-cysteine; (ii) Adenine nucleotides; (iii) NAD + Or NADH; and; (iv) S-adenosine homocysteine ​​hydrolase, preferably SacSAHH or SsoSAHH, such as SacSAHH.

18. A method for methylating a compound, characterized in that, The method includes the following reaction: Methylation modification of substrates was performed using coenzymes and second methyltransferases; The coenzyme is S-adenosyl-methylcysteine ​​or S-adenosyl-L-cysteine; the second methyltransferase is an oxymethyltransferase, a carbon methyltransferase, a thiomethyltransferase or a protein methyltransferase. Preferably, the method further includes a method for preparing and / or regenerating S-adenosyl-methylcysteine, wherein the preparation method is, for example, as defined in any one of claims 1-9; and the regeneration method is, for example, converting S-adenosyl-L-cysteine ​​into S-adenosyl-methylcysteine.

19. The method as described in claim 18, characterized in that, The methylation modification sites on the substrate include nucleophilic atoms in the compound or groups formed therefrom, such as oxygen, nitrogen, sulfur, or carbon atoms; and such groups include hydroxyl, carboxyl, amino, or thiol groups. Preferably, the substrate is a flavonoid compound, such as luteolin, scutellarin, myricetin, hesperidin, or kaempferol.

20. The method as described in claim 18, characterized in that, The second methyltransferase is an oxymethyltransferase; Preferably, the oxygen methyltransferase is ScOMT or NfOMT; The amino acid sequence of ScOMT is shown in SEQ ID NO: 4 for example, and the amino acid sequence of NfOMT is shown in SEQ ID NO: 5 for example.

21. The method according to any one of claims 18-20, characterized in that, When the initial material added to the system is S-adenosyl-methylcysteine, the concentration of S-adenosyl-methylcysteine ​​is 0.01-100 mM, for example, 1 mM; or, When the initial material added to the system is S-adenosyl-L-cysteine, the system satisfies one or more of the following conditions: (A) The concentration of the S-adenosyl-L-cysteine ​​is 0.01-10 mM, for example 0.1 mM; (B) The system includes a methyl donor; (C) The system comprises a first methyltransferase as shown in SEQ ID NO:4 or SEQ ID NO:3 or a first methyltransferase mutant as described in claim 10 or 11; Preferably, when the initial material introduced into the system is S-adenosyl-L-cysteine, the method includes the following steps: a. Add the S-adenosyl-L-cysteine ​​and the methyl donor to a container, then add PBS and mix well; b. Add the second methyltransferase lysis buffer and the first methyltransferase or the first methyltransferase mutant lysis buffer; c. Add the substrate and react at 20-50°C, e.g., 25 or 30°C, 0-1000 rpm, e.g., 180 or 900 rpm for 1-48 hours, e.g., 16 or 20 hours.

22. The method as described in claim 21, characterized in that, The system also satisfies one or more of the following conditions: (I) The substrate concentration is 0.01-100 mM, for example, 1 mM; (II) The concentration of the second methyltransferase is 1-100 mg / mL, for example, 32.61 mg / mL; (III) The methyl donor is a MeOT, for example, a MeOT at a concentration of 3 mM; (IV) The concentration of the first methyltransferase or the first methyltransferase mutant is 0.01-100 mg / mL, for example 6.52 mg / mL; (V) The first methyltransferase or the first methyltransferase mutant is preferably a first methyltransferase mutant containing R43A and T165M compared to the amino acid sequence shown in SEQ ID NO: 2; Preferably, the method further includes step d: adding cold methanol to the reaction solution to terminate the reaction, centrifuging at 10,000 rpm for 30 min, collecting the supernatant, filtering and concentrating it, dissolving the residue in a mixture of DMSO and methanol, centrifuging and passing it through a membrane, and purifying it by preparative liquid chromatography (Pre-HPLC) to obtain the product.

23. A reaction end product system for methylation modification of a compound, characterized in that, The reaction end product system includes: (1) S-adenosyl-methylcysteine; and / or, S-adenosyl-L-cysteine; and, (2) A second methyltransferase; the second methyltransferase is an oxygen methyltransferase, a carbon methyltransferase, a thiomethyltransferase, or a protein methyltransferase, such as an oxygen methyltransferase; preferably, the oxygen methyltransferase is ScOMT or NfOMT and, (3) Substrate; and / or, product; Preferably, the reaction end product system further includes: A first methyltransferase having an amino acid sequence as shown in SEQ ID NO: 2 or 3 and any one of the first methyltransferase mutants as described in claim 10 or 11; And / or, methyl donor; More preferably, the substrate is a flavonoid compound such as luteolin, scutellarin, myricetin, hesperidin, or kaempferol.

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