Application of dendrobium methyltransferase in synthesis of flavonoid compounds for regulating gastrointestinal activity

Abstract

The invention discloses application of Dendrobium huoshanense DhOMT8 gene coded methyltransferase in biosynthesis of a flavonoid compound for regulating gastrointestinal activity, and belongs to the technical field of biology. The nucleotide sequence of the DhOMT8 gene encodes a nucleotide sequence of an amino acid sequence as shown in SEQ ID NO. 2. The methylated flavonoid compound is obtained by a biological synthesis method for carrying out enzyme catalysis by using the DhOMT8 protein in vitro, and a new method is provided for synthesizing the flavonoid compound for regulating gastrointestinal activity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of dendrobium methyltransferase in synthesizing flavonoid compounds for regulating gastrointestinal activity. Background Art

[0002] Dendrobium has a long history of medicinal use. The Shennong's Herbal Classic lists it as a top-grade product, recording that it "treats injuries, removes numbness, relieves qi, replenishes the five internal organs, strengthens yin, and thickens the stomach and intestines after long-term use". After more than 2,000 years of development, there are more than 30 species of Dendrobium for medicinal use in my country, including Dendrobium candidum, Dendrobium huoshanense, Dendrobium nobile, Dendrobium chrysotoxum, Dendrobium tassel, and Dendrobium dentata. Dendrobium "benefits the stomach and produces fluid, nourishes yin and clears heat", and its "benefits the stomach" effect is definite, and it is currently a commonly used "gastrointestinal medicine" in clinical practice. Ancient books often mention its effects of "thickening the stomach and intestines", "calming down the stomach qi", "clearing stomach heat", "strengthening the spleen and appetizing", and many other related pharmacological effects have been reported (Yan Meiqiu, Chen Suhong, Lv Guiyuan. Research and application progress on the pharmacological effects of Dendrobium "thickening the stomach and intestines" [J]. Chinese Herbal Medicine, 2016, 47(21): 3918-3924.). Methoxytransferases of flavonoids participate in the biosynthesis and metabolism of flavonoids in plants. These enzymes can catalyze the conversion of the hydroxyl group (-OH) on the flavonoid skeleton to the methoxy group (-OCH 3 ) substitution reaction to produce methoxylated flavonoid derivatives. Methoxytransferases introduce methyl groups at the hydroxyl position of flavonoids, changing their chemical structure and thus affecting their biological activity. Methylated flavonoids are generally more lipid-soluble and more easily pass through cell membranes, increasing their bioavailability and stability. Some methylated flavonoids have the effect of regulating gastrointestinal activity (Huang Xiaoyan, Zou Menglong, Chen Yalu, et al. Analysis of the mechanism of action of Astragalus in the treatment of ulcerative colitis based on network pharmacology and molecular docking [J]. New Chinese Medicines and Clinical Pharmacology, 2021, 32(6): 815-824.). In addition, methylated flavonoids also exhibit enhanced antioxidant and anti-inflammatory effects. Therefore, the study of flavonoid methoxytransferases helps to gain a deeper understanding of the biosynthetic mechanism of these compounds and provide a theoretical basis and potential biotechnology means for the development of new drugs.

[0003] Studies have shown that the chemical composition of Huoshan Dendrobium contains a rich class of methylated flavonoids, which have the ability to regulate gastrointestinal activity. Its methyltransferase can catalyze the methylation of the oxygen atoms of the hydroxyl groups in flavonoid compounds. Methylated flavonoid compounds mainly require chemical synthesis and enzyme synthesis. The chemical synthesis method has harsh reaction conditions, involves organic reagents, has a low yield and is prone to produce by-products that are harmful to the environment and human body. It is not the best choice for synthesizing methylated flavonoids. In comparison, the enzyme synthesis method is more suitable. So far, no reports on the methyltransferase of Huoshan Dendrobium have been found. Therefore, it is of great significance to identify and develop enzymes with the function of synthesizing methylated flavonoid compounds and apply them in the synthesis of flavonoid compounds that regulate gastrointestinal activity. Summary of the invention

[0004] The present invention mainly aims at the above technical problems and provides a methyltransferase gene for catalyzing the production of flavonoid compounds regulating gastrointestinal activity, as well as a method for biosynthesizing flavonoid compounds regulating gastrointestinal activity, so as to solve the shortcomings of existing chemical synthesis of flavonoid compounds regulating gastrointestinal activity.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] In one aspect, the present invention provides a methyltransferase, whose amino acid sequence comprises the sequence shown in SEQ ID NO.2.

[0007] In another aspect, the present invention provides a methyltransferase gene, the sequence of which is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2,

[0008] Or based on the principle of complementary pairing, the methyltransferase gene provided by the present invention can be a sequence that is fully complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2.

[0009] In a preferred embodiment, the nucleotide sequence of the methyltransferase gene comprises a nucleotide sequence as shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO. 1.

[0010] It is well known to those skilled in the art that a gene sequence may also contain introns, promoters and various regulatory elements, and thus the nucleotide sequence of the above-mentioned methyltransferase gene may also contain introns, promoters and various regulatory elements.

[0011] On the other hand, the present invention provides an application of a methyltransferase gene in the synthesis of flavonoid compounds that regulate gastrointestinal activity, wherein the nucleic acid sequence of the methyltransferase gene is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2, or a sequence that is fully complementary to a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0012] In a preferred embodiment, the methyltransferase gene sequence is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2, or a sequence that is completely complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2.

[0013] In a preferred embodiment, the nucleotide sequence of the methyltransferase gene is as shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO. 1.

[0014] On the other hand, the present invention provides the use of the aforementioned methyltransferase in the synthesis of flavonoid compounds regulating gastrointestinal activity.

[0015] In another aspect, the present invention provides a method for synthesizing a flavonoid compound that regulates gastrointestinal activity, the method comprising the following steps:

[0016] 1) obtaining the aforementioned methyltransferase;

[0017] 2) Using the methyltransferase in step 1) to catalyze the synthesis of flavonoid compounds that regulate gastrointestinal activity in an enzyme activity reaction system.

[0018] In a preferred embodiment, the methyltransferase is obtained by prokaryotic expression.

[0019] In a preferred embodiment, the methyltransferase is obtained by biosynthesis.

[0020] In a preferred embodiment, the enzyme activity reaction system contains the above-mentioned methyltransferase, SAM, a substrate for synthesizing flavonoid compounds regulating gastrointestinal activity, and a buffer.

[0021] In another aspect, the present invention provides a method for synthesizing a flavonoid compound having gastrointestinal activity, characterized in that the method comprises the following steps:

[0022] 1) obtaining a methyltransferase comprising the amino acid sequence shown in SEQ ID NO.2;

[0023] 2) Using the above methyltransferase to catalyze the synthesis of flavonoid compounds that regulate gastrointestinal activity in an enzyme activity reaction system.

[0024] In a preferred embodiment, the methyltransferase is obtained by prokaryotic expression.

[0025] In a preferred embodiment, the methyltransferase is obtained by chemical synthesis.

[0026] In a preferred embodiment, the enzyme activity reaction system contains the above-mentioned methyltransferase, SAM, a substrate for synthesizing flavonoid compounds regulating gastrointestinal activity, and a buffer.

[0027] In a preferred embodiment, the enzyme activity reaction system also contains divalent cations.

[0028] In a preferred embodiment, the reaction temperature of the enzyme activity reaction system is 4-70°C.

[0029] In a preferred embodiment, the pH of the enzyme activity reaction system is 7.0-10.0.

[0030] In a preferred embodiment, the substrate for synthesizing the flavonoid compound regulating gastrointestinal activity is isorhamnetin.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] 1) Compared with the prior art, the present invention utilizes an enzyme-catalyzed biosynthesis method to obtain flavonoid compounds that regulate gastrointestinal activity, which has the advantages of simpler steps, less pollution, and more uniform products compared with chemical synthesis methods.

[0033] 2) The present invention provides a new methyltransferase having a wider suitable reaction temperature range (4-70°C).

[0034] 3) The methyltransferase in the present invention is not metal ion dependent.

[0035] 4) The methyltransferase of the present invention reaches a high reaction rate in a short time, and can completely convert the substrate into the product to obtain a pure product. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0037] Figure 1 The results of cloned gene fragments. A: Gene cloning, M is a 2000 bp DNA marker, and lane 1 is DhOMT8.

[0038] Figure 2The SDS-PAGE gel electrophoresis diagram of pET-28a(+)-DhOMT8 fusion protein. The left side is the protein marker (15-180kDa), and the right side is the target protein (1, 2, and 3 are empty load, DhOMT8 crude enzyme, and DhOMT8 purified protein, respectively).

[0039] Figure 3 Results of the reaction between DhOMT8 and substrates: A: liquid phase diagram of the reaction between DhOMT8 and quercetin; B: liquid phase diagram of the standard; C: MS peak diagram of the catalytic product.

[0040] Figure 4 These are the results of enzymatic kinetic parameter detection of DhOMT8 catalyzing the conversion of quercetin to isorhamnetin. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0043] Example 1

[0044] Cloning of target methyltransferase gene

[0045] 1. Total RNA extraction kit was used to extract total RNA from Dendrobium huoshanense. The experimental operation was carried out according to the instruction manual of IIQ RT SuperMix reverse transcription kit to obtain the ideal cDNA. The full-length fragment of DhOMT8 gene was amplified by polymerase chain reaction (PCR) using this as a template. The specific primers were designed according to the transcriptome data of this experiment, see Table 1:

[0046] Table 1 Primers sequence

[0047]

[0048] The cDNA of Dendrobium huoshanense extracted in the above experiment was used as a PCR template, and the target fragment was amplified using primers DhOMT8-F and DhOMT8-R. The polymerase used in PCR was Nanjing Novozyme 2×Phanta Max Master Mix. According to Table 10, 25 μL of PCR reaction system was prepared.

[0049] Table 2 25μL PCR reaction system for amplifying target gene (25μL)

[0050]

[0051]

[0052] Place the prepared PCR reaction system in a PCR instrument and amplify the target gene according to the PCR reaction program in 11.

[0053] Table 3 PCR reaction procedures for amplified genes

[0054]

[0055] After the reaction was completed, the product size was detected by 1% agarose gel electrophoresis. The electrophoresis diagram is shown in Figure A.

[0056] 2. The amplified product was connected to the T vector (pET-28a, Nanjing Novozymes Biotech Co., Ltd.) and transformed into Escherichia coli DH5α. A single colony was selected and cultured in LB medium and verified by bacterial liquid PCR. The bacterial liquid PCR reaction system was: 12.5 μL Green Taq Mix (P131, Nanjing Novozymes Biotech Co., Ltd.), 1 μL of upstream and downstream primers, 2 μL template (bacterial liquid), dd H 2 O to 25 μL. The samples with the correct bacterial liquid PCR results were sent for sequencing.

[0057] The nucleotide sequence obtained by sequencing is shown in SEQ ID NO.1. The gene sequence contains 1077 nucleotides and encodes 358 amino acids (shown in SEQ ID NO.2).

[0058] >SEQ ID NO.1

[0059] ATGGGCAGTTACAATGCCACCGCCGATGGAAGTATCGACGACGAGGCCGCCTGCCTCTA

[0060] CGCGTTGCAGCTCGTCAGCTTCTCCATCCTCCCCCATGACCCTCAAGGCCGCCATCGAACT

[0061] CAAACTCCTCGAAACCATCTCCCTCGCCGGCCCCGGCGCTCAACTCTCTCCGTCAGAAC

[0062] TTGCCTCTCGCCTCCCCTTCATCACAAACCCCCAAGCCCCCGTCATGCTCGACCGCATCC

[0063] TCCGCCTTCTGGCCAGCTACTCCATCCTCACTTGCTCTATCTCCCCATCCGGCGAGCGCC

[0064] GCTATGGCGCCGCACCGGTTTGTAAATTCCTCACGCCCAATTCCGATGGAGTTTCTATGG

[0065] CTCCGCTTGCTCTAATGAATCAGGATAAGGTGTTGATGGAGAGCTGGTACTATCTAAAGG

[0066] ATGCGGTGATCGAAGGCGGTATTCCGTTCAATAAGGCGTATGGGATGACGGCGTTTGAGT

[0067] ACCATGGAACGGATCCAAGGTTCAATAAGGTGTTTAACGATGGGATGTCAGGCCATTCA

[0068] ACTATCATTACGAATAAATTGTTGGAGATTTATGAGGGTTTTGATGGGTTGGGTTCGCTGG

[0069] TGGATGTGGGAGGCGGAGTGGGGGCGACGTTGGGAAAGATTACGGCCAAGTATACCGG

[0070] GATTAGAGGAATTAATTTTGATCTCCCGCATGTAATCTCCGAGGCTCCTCCTCTTCCAGGA

[0071] GTGGAGCATGTGGGCGGAGATATGTTCGAGAGTGTTCCCACCGCTGATGCCATTTTCATG

[0072] AAGTGGATTCTTCATGATTGGAGCGACGATCATTGCCTCAAATTGTTGAAGAATTGTTGG

[0073] AAGGCATTGCCTGAAAATGGGAAAGTGATTGTGGCCGAGTGCATTCTTCCGGTGGAGCC

[0074] CGAACAAACTGTGGCAGCAAAAGGTGTCTTTCATGTCGACCTCATCATGTTGGCTCATA

[0075] ACCCTGGAGGAAAAGAGAGAACGGAGAATGAATTTAAATCATTGGCAAAGGAGGCTGG

[0076] CTTCTCTGATTTCAAATCCATCTACATATTTTCAGGCTGTTGGGTTATGGAATTCATCAAGT

[0077] AG

[0078] >SEQ ID NO.2

[0079] MGSYNATADGSIDDEAACLYALQLVSFSILPMTLKAAIELKLLETISLAGPGAQLSPSELASR

[0080] LPFITNPQAPVMLDRILRLLASYSILTCSISPSGERRYGAAPVCKFLTPNSDGVSMAPLALMN

[0081] QDKVLMESWYYLKDAVIEGGIPFNKAYGMTAFEYHGTDPRFNKVFNDGMSGHSTIITNKLL

[0082] EIYEGFDGLGSLVDVGGGVGATLGKITAKYTGIRGINFDLPHVISEAPPLPGVEHVGGDMFE

[0083] SVPTADAIFMKWILHDWSDDHCLKLLKNCWKALPENGKVIVAECILPVEPEQTVAAKGVF

[0084] HVDLIMLAHNPGGKERTENEFKSLAKEAGFSDFKSIYIFSGCWVMEFIK

[0085] Example 2 Expression and detection of target protein

[0086] Expand the target bacterial solution with correct sequencing and extract the recombinant plasmid pET-28a-DhOMT8. Transform the recombinant plasmid into E. coli BL21 (DE3) competent cells, plate, and select clones to LB medium, culture at 37°C, 200rpm, and obtain the mother solution for 16 hours. Inoculate the mother solution into LB medium containing 50μg / mL KaN at a ratio of 1:100 and culture at 37°C until OD 600 =0.6-0.8 (with sterile LB medium as control), add IPTG with a final concentration of 0.5 mM, and finally culture at 16°C, 200 rpm for 18-20 h.

[0087] Collect the induced expression bacterial solution by centrifugation, resuspend the bacteria in pre-cooled Tris-HCl (50mM) buffer at pH = 9.0, centrifuge, collect the bacteria again, and repeat once to remove the residual culture medium. Finally, add 15mL of the above Tris-HCl buffer per gram of wet bacteria. The obtained bacterial suspension can be frozen in a -20°C refrigerator overnight, and then low-temperature crushed on an ultrasonic cell crusher with a power of 150W, working for 3s, and resting for 3s. When the bacterial suspension is clear, the crushing can be stopped. Finally, the crushed liquid is centrifuged at 4°C and 14000rpm for 4min. The supernatant is the crude enzyme solution, which is stored in a -20°C refrigerator.

[0088] According to the His-Tag tag on the fusion protein, the target protein was purified by nickel affinity chromatography, and the target protein was eluted with imidazole solutions of different concentrations. Finally, the molecular weight and purity of the target protein were detected by 10% SDS-PAGE electrophoresis. Figure 2 As shown, the target protein appeared around 43 kDa (1, 2, and 3 were used for empty load, crude enzyme, and purified protein, respectively), indicating that the protein was successfully expressed in the supernatant and the purified protein could be used for subsequent experiments.

[0089] Example 3 In vitro enzyme activity detection

[0090] 1. Enzyme activity reaction system: 10 μg pure enzyme, 0.5 mM quercetin, 5 mM SAM methyl donor, 14 mM β-mercaptoethanol, dilute to 200 μL with 50 mM, pH = 9.0 Tris-HCl buffer. After the reaction solution was incubated at 45°C for 45 minutes, double volume of pre-cooled methanol was added, vortexed and mixed, and centrifuged at 4°C, 14000 rpm for 4 minutes. The supernatant was filtered and detected by high performance liquid chromatography.

[0091] 2. HPLC conditions

[0092] Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile

[0093] Elution gradient: 0-5min: 5%-20% B; 5-8min: 20%-22% B; 8-17min: 22%-25% B; 17-23min: 25%-35% B; 23-25min: 35%-50% B; 25-32min: 50%-95% B; 32-37min: 95% B; 37-38min: 95%-5% B; 38-42min: 5% B.

[0094] 3. LC-MS conditions:

[0095] The detection mode was positive ion mode, capillary voltage was 3500 V, sheath gas temperature was 280 °C, and flow rate was 11 L min -1 , drying gas temperature 320℃, flow rate 10L·min -1 , nozzle voltage 155V, flow rate 0.250mL·min -1 .

[0096] 4. Test results:

[0097] Results Figure 3 When DhOMT8 used quercetin as substrate, a product peak was observed in the reaction. By comparing with the standard control and based on the retention time and mass-to-charge ratio m / z, the peak was determined to be the product isorhamnetin.

[0098] Example 4

[0099] Enzyme kinetic parameters detection

[0100] Using quercetin as substrate and SAM as methyl donor, the effects of pH, temperature, metal ions and reaction time on the catalytic activity were investigated.

[0101] 1. Temperature: Quercetin was used as the methyl acceptor and SAM as the methyl donor to investigate the changes in enzyme activity at different reaction temperatures (4°C, 30°C, 40°C, 50°C, 60°C and 70°C). The reaction system contained 10μg of pure protein, 0.5mM substrate, 5mM SAM, 14mM β-mercaptoethanol, and was fixed to 200μL with 50mM, pH=9.0 Tris-HCl buffer. The reaction solution was reacted at each temperature for 45min. After the reaction, twice the volume of pre-cooled methanol was immediately added to the reaction system to terminate the reaction. After centrifugation, the supernatant was filtered and tested by HPLC. Three parallel tests were set for each group of reactions. The conversion rate of the substrate was estimated based on the peak area ratio in the chromatogram.

[0102] 2. pH: Investigate different pH buffers of 50mM: 7.0-8.0, Na 2 HPO4 -NaH 2 PO 4 Buffer; 8.0-9.0, Tris-HCl Buffer; 9.0-10.0, Na 2 CO 3 -NaHCO 3 Effect of Buffer on Enzyme Activity. The reaction system is: 14mM β-mercaptoethanol, 5mM SAM, 10μg protein, 0.5mM compound quercetin in a 200μL reaction system, supplemented with the corresponding buffer to the specified volume. The reaction solution was placed at 45°C for 40min, and immediately added with twice the volume of pre-cooled methanol after the reaction. After centrifugation, the supernatant was filtered and tested by HPLC. To ensure the reliability of the data, three replicate samples were set up for each group of reactions, and the final conversion rate was calculated by the peak area of ​​the chromatogram.

[0103] 3. Metal ions: The acceptor molecule of the reaction solution is selected as quercetin, and the donor is selected as SAM. Different divalent metal ions are investigated: Co 2+ 、Zn 2+ , Ba 2+ , Ca 2+ Mg 2+ The effect of EDTA on enzyme activity. A 200 μL reaction system contains 14 mM β-mercaptoethanol, 5 mM SAM donor, 10 μg protein, 0.5 mM substrate, 5 mM divalent metal ions, and 50 mM Tris-HCl buffer at pH = 9.0 is added to the specified volume. The reaction solution is placed at 30°C for 4 minutes. After the reaction, two volumes of pre-cooled methanol are immediately added and centrifuged at 14000 rpm and 4°C for 4 minutes. The supernatant is filtered and tested by HPLC. Three replicate samples are set up for each reaction. The final conversion rate is calculated by the peak area of ​​the chromatogram.

[0104] 4. Reaction time: 200 μL Tris-HCl (50 mM, pH = 9.0) reaction system contains 14 mM β-mercaptoethanol, 5 mM SAM, 10 μg purified protein, 0.5 mM substrate quercetin, and reacts for 3 min, 5 min, 8 min, 10 min, 20 min, 30 min, 40 min, 60 min in a 50 ° C water bath. After the reaction, quickly add two times the volume of pre-cooled methanol to each reaction solution to terminate the reaction, and centrifuge at 14000 rpm and 4 ° C for 4 min. Take the supernatant and filter it for detection on HPLC, and calculate the conversion rate of the substrate. To ensure the accuracy of the data, three parallel tests were set up for each group, and the substrate conversion rate was calculated according to the ratio of the peak area in the HPLC spectrum.

[0105] 6. Test results:

[0106] Enzyme kinetic studies have shown that ( Figure 4 ), DhOMT8 has the highest enzyme activity at 50℃, and has a good conversion rate in the range of 4~70℃. The optimum pH of the reaction solution is 9.0, and it is suitable for reactions in the pH range of 7.0~9.0, and its catalytic activity in Tris-HCl buffer is higher than that of Na 2 CO 3 -NaHCO 3 The enzyme is not dependent on metal ions. The relative conversion efficiency can reach more than 70% in 10 minutes and 100% in 30 minutes.

[0107] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a methyltransferase in the synthesis of flavonoid compounds for regulating gastrointestinal activity, characterized in that: The amino acid sequence of the methyltransferase is shown in SEQ ID NO.2, and the flavonoid compound regulating gastrointestinal activity is isorhamnetin.

2. Use of a methyltransferase gene in the synthesis of flavonoid compounds regulating gastrointestinal activity, characterized in that The methyltransferase gene sequence is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2, or a sequence fully complementary to a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2, and the flavonoid compound regulating gastrointestinal activity is isorhamnetin.

3. A method for synthesizing a flavonoid compound that regulates gastrointestinal activity, wherein the flavonoid compound that regulates gastrointestinal activity is isorhamnetin, and the method comprises the following steps: 1) obtaining a methyltransferase having an amino acid sequence as shown in SEQ ID NO.2; 2) Using the methyltransferase in step 1) to catalyze the synthesis of flavonoid compounds that regulate gastrointestinal activity in an enzyme activity reaction system.

4. The method according to claim 3, characterized in that In step 1), the methyltransferase is obtained by prokaryotic expression or by chemical synthesis.

5. The method for synthesizing a flavonoid compound for regulating gastrointestinal activity according to claim 3, characterized in that: The enzyme activity reaction system in step 2) contains the methyltransferase described in step 1), S-adenosylmethionine (SAM) and a substrate for synthesizing flavonoid compounds regulating gastrointestinal activity.

6. The method according to claim 5, characterized in that The enzyme activity reaction system in step 2) also contains divalent cations.

7. The method according to claim 5, characterized in that The reaction temperature of the enzyme activity reaction system in step 2) is 4-70°C.

8. The method for synthesizing a flavonoid compound for regulating gastrointestinal activity according to claim 5, characterized in that: The pH of the enzyme activity reaction system in step 2 is 7.0-10.0.

Images