Oxymethyltransferase and application thereof in biosynthesis of isofraxidin and fraxidin

Abstract

The invention relates to the technical field of synthetic biology, in particular to oxygen methyl transferase and application thereof in biosynthesis of isofraxidin and fraxidin. According to the application, the core gene elements of the biosynthetic pathway of the isofraxidin, namely the oxygen methyl transferase genes SgOMT3 and Ese08G000662. T1, are identified, and the protein coded by the oxygen methyl transferase genes has the function of catalyzing fraxetin to generate the isofraxidin. Based on the oxymethyltransferase provided by the invention, synthesis of isofraxidin can be realized in bacteria, yeasts, algae and plant chassis in combination with a metabolic pathway of isofraxidin. The invention provides an artificial synthesis path of the isofraxidin, and provides a theoretical basis for realizing efficient, green and low-carbon production of the isofraxidin by utilizing biosynthesis.

Description

Technical Field

[0001] The invention relates to the technical field of synthetic biology, and in particular to an oxygen methyltransferase and application thereof in the biosynthesis of isoflurane and quinidine. Background Art

[0002] Sarcandra glabra (Thunb.) Nakai is an evergreen semi-shrub of the Sarcandra genus of the Orchidaceae family and is a traditional Chinese herbal medicine. It grows in the shade and wet places under the forest on hillsides and valleys, and avoids high temperature, dryness and strong light. The research on the chemical composition of Sarcandra glabra is relatively systematic and comprehensive. According to the existing research progress, the whole grass of Sarcandra glabra mainly contains compounds such as sesquiterpenes, flavonoids, organic acids, coumarins, and volatile oils. Modern pharmacological studies have shown that Sarcandra glabra has antibacterial, anti-inflammatory and analgesic, anti-ulcer, influenza virus inhibition, fracture healing, immunity enhancement, anti-tumor, and liver protection effects. At present, there are many coumarin compounds extracted from Sarcandra glabra, among which isoflurane is one of the main active ingredients of Sarcandra glabra, and is used as a quality control indicator for Sarcandra glabra medicinal materials and preparations in the Chinese Pharmacopoeia. At present, the resources of wild Sarcandra glabra are becoming less and less, and the demand of pharmaceutical companies for isoflurane, an important active ingredient extracted from Sarcandra glabra raw materials, is gradually increasing. At present, the metabolic pathway of isofraquinone has not been elucidated, and it is difficult to synthesize isofraquinone through biosynthesis. Through omics methods, the catalytic enzyme for synthesizing isofraquinone can be found, and then through synthetic biology methods, isofraquinone can be synthesized heterologously in chassis such as tobacco, yeast or algae. This provides an important theoretical basis and economic value for the protection of wild resources of Coral Grass and clinical medication, and helps to promote the diversity of sources of isofraquinone compounds.

[0003] The present invention functionally identifies an oxygen methyltransferase gene (OMT) of Sarcandra glabra through omics screening, heterologous expression, enzyme activity detection and other technologies. The OMT gene mainly catalyzes quercetin to produce isofraquinone and quercetin. The discovery of the gene is conducive to the later heterologous synthesis of isofraquinone through a synthetic biology chassis, thereby meeting the market demand for important active ingredients of Sarcandra glabra, and has huge market value. Summary of the invention

[0004] The purpose of the present invention is to provide an oxygen methyltransferase gene (OMT) gene SgOMT3 involved in the synthesis pathway of isofraquinone, the protein encoded by which can catalyze the methylation modification of quercetin at the C(7) or C(8) position to ultimately generate isofraquinone or quercetin.

[0005] The first aspect of the present invention provides an oxygen methyltransferase, wherein the amino acid sequence of the oxygen methyltransferase comprises or consists of the following sequence:

[0006] A1) the amino acid sequence shown in SEQ ID NO: 2; or

[0007] A2) an amino acid sequence having the same protein activity as that of SEQ ID NO: 2 by adding, deleting or replacing one or more amino acids; or

[0008] A3) the amino acid sequence shown in SEQ ID NO: 4; or

[0009] A4) An amino acid sequence having the same protein activity as that of SEQ ID NO: 4 by adding, deleting or replacing one or more amino acids.

[0010] The second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned oxygen methyltransferase.

[0011] Preferably, the sequence of the nucleic acid molecule is any one of B1)-B2):

[0012] B1) the nucleotide sequence shown in SEQ ID NO: 1; or

[0013] B2) The nucleotide sequence shown in SEQ ID NO:3.

[0014] The third aspect of the present invention provides a biomaterial, wherein the biomaterial is any one of C1)-C3):

[0015] C1) an expression cassette containing the above nucleic acid molecule;

[0016] C2) a recombinant vector containing the above nucleic acid molecule;

[0017] C3) A host cell containing at least one of the above nucleic acid molecule, the expression cassette described in C1), and the recombinant vector described in C2).

[0018] The fourth aspect of the present invention provides the use of any one of the above-mentioned oxygen methyltransferase, the above-mentioned nucleic acid molecule, and the above-mentioned biological material in the synthesis of isofraquinone and / or quinraquinone.

[0019] Preferably, the application uses fraxinusin as a substrate.

[0020] Preferably, the quercetin is methylated at position C(7) or C(8).

[0021] The fifth aspect of the present invention provides a method for producing the above-mentioned oxygen methyltransferase, wherein the above-mentioned nucleic acid molecule, or at least one of the expression cassette and recombinant vector in the above-mentioned biological material is transformed into a host cell, so that the host cell produces the above-mentioned oxygen methyltransferase.

[0022] The sixth aspect of the present invention provides a method for synthesizing isofraquinone and / or quinaquinone, using quinaquinone as a substrate, and under the action of the above-mentioned oxygen methyltransferase and / or the oxygen methyltransferase produced by the above-mentioned method, catalyzing the methylation modification of quinaquinone at the C(7) or C(8) position to synthesize isofraquinone and / or quinaquinone.

[0023] The seventh aspect of the present invention provides a method for synthesizing isofraquinone and / or quinaquinone, wherein the above-mentioned nucleic acid molecule or any one of the above-mentioned biological materials C1)-C2) is transformed into a host cell, so that the host cell produces oxygen methyltransferase; using quinaquinone as a substrate, under the action of the produced oxygen methyltransferase, catalyzes the methylation modification of quinaquinone at the C(7) or C(8) position to synthesize isofraquinone and / or quinaquinone.

[0024] The beneficial effects of the present invention are as follows: the key candidate gene for producing isofraquinone is successfully screened for the first time using multi-omics data, and the key point is to verify the candidate gene has the function of catalyzing the production of isofraquinone using an in vitro enzyme expression system. The complete isofraquinone metabolic pathway can be used to realize the synthesis of isofraquinone in bacteria, yeast, algae and plant chassis.

[0025] Combined with the artificial synthesis pathway of isoflurane provided in the present application, in-depth mining of functional element modules based on genomic big data, redesign and optimization and adaptation of the synthesis pathways and regulatory modules in algae, Saccharomyces cerevisiae or Nicotiana tabacum can break through the regulatory and efficiency limitations of natural biosynthesis, and provide theoretical and practical support for improving the product synthesis efficiency of isoflurane and quinidine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 To determine the content of isoflurane in different tissues of Corallis glabra.

[0027] Figure 2 This is a proposed synthetic pathway for isoflurane.

[0028] Figure 3 Pearson correlation analysis between isoflurane content and candidate OMT genes in Sarcandra glabra.

[0029] Figure 4 This is the total ion chromatogram of the SgOMT3 protein gene of Sarcandra glabra.

[0030] Figure 5 To determine the content of isoflurane in the root tissues of Acanthopanax senticosus and Sarcandra glabra.

[0031] Figure 6 This is the total ion chromatogram of the Ese08G000662.t1 protein gene in Acanthopanax senticosus. DETAILED DESCRIPTION

[0032] The present invention provides two oxygen methyltransferase genes (OMT) genes SgOMT3 and Ese08G000662.t1 involved in the synthesis pathway of isoflurane. The nucleotide sequence of the coding region of the gene SgOMT3 is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO: 2. The nucleic acid sequence of the gene Ese08G000662.t1 is shown in SEQ ID NO: 3, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO: 4.

[0033] In some embodiments of the present invention, the above-mentioned oxygen methyltransferase SgOMT3 can be derived from different species and / or different strains of the genus Sarcocoria.

[0034] In some embodiments of the present invention, the oxygen methyltransferase may also be derived from a gene with the same function as the methyltransferase SgOMT3, such as Ese08G000662.t1 in Acanthopanax senticosus. The Ese08G000662.t1 may be derived from different species and / or strains of Acanthopanax.

[0035] In some embodiments of the present invention, when the starting material is quercetin, under the catalysis of the oxygen methyltransferase provided by the present invention (as shown in SEQ ID NO:1, SEQ ID NO:3), quercetin is methylated at the C(7) or C(8) position to ultimately generate isoflurane or quercetin.

[0036] In another embodiment of the present invention, when the starting materials are phenylalanine, 6'-hydroxyferuloyl-CoA and scopoletin, which are precursors of quercetin, the oxygen methyltransferase provided by the present invention can be selected according to the specific synthesis pathway, combined with one or more of phenylalanine ammonia lyase (PAL), cinnamic acid-4-hydroxylase (C4H), cinnamic acid-3-hydroxylase (C3H), hydroxycinnamoyl-CoA-shikimate hydroxycinnamoyltransferase (HCT), feruloyl-CoA 6'-hydroxylase 1 (F6'H1), and scopoletin 8-hydroxylase (S8H) to achieve the synthesis of isoflurane or quercetin.

[0037] In another embodiment of the present invention, SgOMT3 or Ese08G000662.t1 is also introduced into the synthetic biology chassis by overexpression or knockout, thereby achieving heterologous synthesis of isoflurane.

[0038] Optionally, the synthetic biology chassis includes a microbial system and a plant system. The microbial system includes bacteria and / or fungi; the plant system includes but is not limited to Coral Grass, Acanthopanax Senticosus, and Nicotiana tabacum.

[0039] In some embodiments of the invention, the synthetic biology chassis is a microorganism.

[0040] In the present invention, the term "SgOMT3" may represent the Sarcandra glabra isoquinone oxygen methyltransferase gene, and the specific meaning may be determined in combination with the context.

[0041] Below in conjunction with embodiment, technical scheme of the present invention is clearly and completely described, obviously, described embodiment is only a part of embodiment of the present invention, rather than all embodiments. Embodiment is carried out according to conventional experimental conditions or according to the condition suggested by manufacturer's specification. Unless otherwise defined, all professional terms used in the present invention have the same meaning as those skilled in the art are generally understood. In addition, any method and material similar or identical to the described content can be used in the inventive method, based on the embodiment in the present invention, all other embodiments obtained by those skilled in the art under the premise of not paying creative work, all belong to the scope of protection of the present invention.

[0042] The experimental methods used in the following examples were carried out according to conventional methods or those recommended by the manufacturer unless otherwise specified. The materials, reagents and consumables used were all commercially available unless otherwise specified.

[0043] In some embodiments of the present invention, the Sarcandra glabra plants used are collected from Louyuan State-owned Forest Farm in Sanyuan District, Sanming City, Fujian Province. In other embodiments, other varieties of Sarcandra glabra plants may be selected as a substitute.

[0044] In some embodiments of the present invention, the plant total RNA was extracted using FastPure Plant Total RNAlsolation Kit (Polysaccharides & Polvphenolics-rich, Vazyme); the reverse transcription synthesis of cDNA used PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa); PCR amplification used KOD OneTM PCR Master Mix (ToYoBo), PCR Master Mix (With Dye) (Yeasen); Gel Extract Kit D2500 (Omega) was used for gel extraction; Plasmid Mini-Extraction Kit (DP103-03, TIANGEN) was used to extract plasmid DNA; HindⅢ (NEB) and KpnⅠ (NEB) were used to cut DNA with restriction endonucleases; pEASY-Basic Seamless Cloning and Assembly Kit (Quanshijin) was used for gene cloning and ligation; DL5000 DNA marker (TaKaRa) was used for DNA molecular weight calibration; DH5α and BL21 (DE3) (Shanghai Weidi) were used for gene cloning and vector construction; Zhejiang Shangya Biotechnology Co., Ltd. was commissioned to synthesize the primers required for realization; Zhejiang Shangya Biotechnology Co., Ltd. was commissioned to perform gene sequencing. In other embodiments, other suitable reagents and tools can also be selected as needed to carry out the above experiments.

[0045] Example 1. Establishment of Isofrapidin Detection System

[0046] To determine the content of isofraquinone in the leaves, stems, roots and flowers of Coral Grass, 0.1 g of crushed fresh sample was weighed for each different tissue, 5 ml of methanol was added to the four tissues for ultrasonic extraction for 1 hour, and then vacuum frozen and concentrated to 1 ml, and then filtered through a 0.22 μm filter membrane. The filtered samples were placed in a sample injection bottle for loading. LC-MS / MS was used to determine the content of isofraquinone. The results are shown in Figure 1 As shown, it was proved that the content of isoflurane in the roots of Sarcandra glabra was higher than that in the leaves, stems and flowers.

[0047] Example 2: Prediction of the synthesis pathway of isoflurane and mining of candidate OMT genes

[0048] The inventors have found that isofraxidin has the same skeleton as compounds such as scopolamine, scopolamine, and fraxidin in terms of structure. Therefore, the inventors speculate that the synthesis of isofraxidin should be that phenylalanine is first converted into 6'-hydroxyferuloyl-CoA by the action of enzymes such as phenylalanine ammonia lyase (PAL), cinnamic acid-4-hydroxylase (C4H), and cinnamic acid-3-hydroxylase (C3H), and then successively converted into fraxidin by the catalysis of feruloyl-CoA 6'-hydroxylase 1 (F6'H1) and scopolamine 8-hydroxylase (S8H), and finally converted into isofraxidin or fraxidin by the catalysis of a specific OMT gene ( Figure 2 ).

[0049] In order to verify the above speculation, the present invention uses the OMT gene set published by Chrysanthemum indicum as the target, and finally obtains 18 OMT genes of Coral Grass from the genome and transcriptome data of Coral Grass, which are divided into 2 clades, Clade1: 15 caffeic acid O-methyltransferase (COMT) families; Clade2: 3 caffeic acid coenzyme AO-methyltransferase (CCoAOMT) families. Pearson correlation analysis was performed on the transcriptome data of Coral Grass with the content of isofraxidin in the four tissues of Coral Grass (leaves, stems, roots and flower tissues) and 13 OMT genes (COMT family, except 2 OMT genes with an expression level of 0), and finally 4 candidate OMT genes were screened. Pearson analysis was performed on the above 13 OMT genes and the isofraxidin content in the four tissues, and only 4 OMT genes were positively correlated with isofraxidin ( Figure 3 ), which are SgOMT1, gOMT2, SgOMT3 and SgOMT4 respectively.

[0050] Example 3: Cloning of candidate genes and construction of heterologous expression vectors

[0051] Through the experimental verification of the four candidate OMT genes in Example 2, an oxygen methyltransferase gene with the function of catalyzing the production of isoflurane was found: SgOMT3. SgOMT3 contains 1065 nucleotides and encodes 354 amino acids.

[0052] Take an appropriate amount of fresh root tissue sample of Corallis glabra and grind it with liquid nitrogen. Use the Vazyme kit method to extract total RNA from Corallis glabra. Synthesize cDNA according to the instructions of TaKaRa's first-strand cDNA synthesis kit PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa). Based on the nucleotide sequence of the SgOMT3 gene (SEQ ID NO: 1), the synthetic primers of the SgOMT3 gene were designed, and the primers contained KpnⅠ and HindⅢ restriction site sequences. Using the cDNA of coral root as a template, the SgOMT3 gene was amplified by PCR and recombined with the linear pCold-TF vector digested with KpnⅠ and HindⅢ. The recombinant plasmid was obtained by using the pEASY-Basic Seamless Cloning and Assembly Kit. The homologous recombination product was transferred into DH5α competent cells, transformed and plated, and positive clones were screened by colony PCR. The strains identified as positive clones were sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing verification, and the plasmids of the positive strains with correct sequencing sequences were extracted. The positive recombinant plasmid was named pCold-SgOMT3.

[0053] The amplification primer sequences used for SgOMT3 are as follows:

[0054] SgOMT3-Forward: aggcatatggagctcggtaccATGGCCAAAACCACCAACG (the lowercase sequence is the vector sequence, and the uppercase sequence is the SgOMT3-specific primer sequence) (SEQ ID NO: 5).

[0055] SgOMT3-Reverse: agactgcaggtcgacaagcttTTATTTGTGGAACTCCATGACCC (the lowercase sequence is the vector sequence, and the uppercase sequence is the SgOMT3-specific primer sequence) (SEQ ID NO: 6).

[0056] Example 4: Heterologous expression of SgOMT3 protein in vitro enzyme activity

[0057] The positive plasmid obtained in Example 3 was transformed into the E. coli expression competent BL21 (DE3) strain, and after colony PCR screening, 50 μl of bacterial solution was inoculated into 5 ml of LB liquid containing ampicillin resistance (Amp), and cultured at 37°C overnight; 2 ml was taken from the 5 ml bacterial solution cultured overnight and inoculated into a medium containing 200 ml of liquid LB (containing Amp resistance). After culturing at 37°C for 3-4 hours, the OD600 value was measured to be about 0.8. After the induced culture medium was placed on ice and allowed to stand for 1 hour, 400 μl IPTG (0.5M) was added to a final concentration of 1 mM. After induction at 18°C ​​for 20 hours, the bacterial solution was transferred to a 500 mL large centrifuge tube, 8000 rpm, 4°C, 10 minutes, the supernatant was discarded, and the bacteria were collected. 15 ml of lysis solution (Lysis Buffer) and 200 μl of PMSF were added, vortexed and resuspended, and then ultrasonically broken. After the bacteria are broken, the supernatant is collected by centrifugation and filtered with a 0.45μm membrane. Add 600μL Ni-NTA filler to the supernatant, rotate and mix well, and incubate overnight at 4℃. The next day, use 40, 80, and 150μM Wash Buffer to wash the impurities, and finally add 5ml (250μM) Elusion Buffer to elute the target protein. After the eluted target protein is concentrated and desalted using a desalting concentration column, take a portion for detection and store the rest at -80℃ for use.

[0058] The enzyme activity system (200 μl) includes the following components: 10 μg purified protein, 0.5 mM quercetin, 0.4 M Tris-HCl (pH 7.5), 4 mM DTT, 2 mM MgCl2, 0.5 mM SAM, and the rest is made up with ddH2O. React at 37 °C for 1 h. After the enzyme activity reaction is completed, add 200 μl acetonitrile to stop the reaction, then add 800 μl ethyl acetate to vortex and shake, and stand in the dark for 30 min. Centrifuge at 12000 rpm for 10 min, draw the upper liquid into a new 1.5 ml centrifuge tube, freeze-concentrate in vacuum, add 200 μl chromatographic methanol to reconstitute, and detect on LC-MS (Thermo Scientific, Germany). Chromatographic column: Waters Acquity BEHC18 column (2.5 μm, 2.1 mm × 100 mm). Mobile phase: chromatographic methanol (phase A), ddH2O (phase B). Chromatographic program: 0min 90% B; 0–0.5min 90% B; 0.5–6.9min 5% B; 6.9–10min 90% B. The aspiration volume was 1 μl, the flow rate was 0.30 ml / min, and the column temperature was 40°C. pCold-TF was used as a control.

[0059] The in vitro enzyme expression system combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to analyze whether the protein encoded by the SgOMT3 gene has the function of catalyzing the production of isoflurane from fraxinusin. Figure 4 As shown, when the SgOMT3 gene is expressed, the production of isofraquinone or quinaquinone can be detected. That is, the SgOMT3 enzyme can catalyze the methylation modification of quinaquinone at the C(7) or C(8) position, and finally generate isofraquinone or quinaquinone.

[0060] Example 5: Determination of the content of isofraxidin in Acanthopanax senticosus and mining of OMT homologous genes

[0061] This example further determines the content of isofraquinone in the root of Acanthopanax senticosus and finds genes with the same function as the methyltransferase SgOMT3 based on the genome data of Acanthopanax senticosus (Eleutherococcussenticosus, Araliaceae). To determine the content of isofraquinone in the root tissue of Acanthopanax senticosus, 0.1 g of crushed fresh sample was weighed, 5 ml of methanol was added to the root tissue for ultrasonic extraction for 1 hour, and the filtered sample was placed in a sample injection bottle for loading, and the established isofraquinone LC-MS detection method of the present invention was used to detect the content of isofraquinone in the root tissue of Acanthopanax senticosus. The results showed that the content of isofraquinone in the root tissue of Corallaria sinensis was significantly higher than that in the root tissue of Acanthopanax senticosus ( Figure 5 ).

[0062] The present invention takes SgOMT3 of Coral Grass as a target, wherein a highly homologous gene, Ese08G000662.t1, is obtained from Acanthopanax Senticosus, wherein Ese08G000662.t1 contains 1098 nucleotides and encodes 365 amino acids.

[0063] Take an appropriate amount of fresh root tissue sample of Acanthopanax senticosus and grind it with liquid nitrogen. Use the Vazyme kit method to extract total RNA from Acanthopanax senticosus. Synthesize cDNA according to the instructions of TaKaRa's first-strand cDNA synthesis kit PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa). Based on the nucleotide sequence of the Ese08G000662.t1 gene (SEQ ID NO: 3), the synthetic primers of the Ese08G000662.t1 gene were designed, and the primers contained KpnⅠ and HindⅢ restriction site sequences. Using the cDNA of Acanthopanax senticosus root as a template, the Ese08G000662.t1 gene was amplified by PCR and recombined with the linear pCold-TF vector double-digested with KpnⅠ and HindⅢ. The recombinant plasmid was obtained by using the pEASY-Basic Seamless Cloning and Assembly Kit kit. The homologous recombination product was transferred into DH5α competent cells, transformed and plated, and positive clones were screened by colony PCR. The strains determined as positive clones were sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing verification, and the plasmids of the positive strains with correct sequencing sequences were extracted. The positive recombinant plasmid was named pCold-Ese08G000662.t1.

[0064] The amplification primer sequences used for Ese08G000662.t1 are as follows:

[0065] Ese08G000662.t1-Forward: aggcatatggagctcggtaccATGGATTCAAGGGCCGAGA (the lowercase sequence is the vector sequence, and the uppercase sequence is the Ese08G000662.t1 specific primer sequence) (SEQ ID NO: 7).

[0066] Ese08G000662.t1-Reverse: agactgcaggtcgacaagcttCTATTTAGAAAATTCCATGACCCAAG (the lowercase sequence is the vector sequence, and the uppercase sequence is the Ese08G000662.t1 specific primer sequence) (SEQ ID NO: 8).

[0067] Example 6: Heterologous expression of Ese08G000662.t1 protein in vitro enzyme activity

[0068] The positive plasmid obtained in Example 5 was transformed into the competent E. coli for expression, and other specific experimental operations were basically the same as those in Example 4.

[0069] The in vitro enzyme expression system combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to analyze whether the protein encoded by the Ese08G000662.t1 gene has the function of catalyzing the production of isoflurane from fraxinusin. Figure 6 As shown, the protein encoded by the Ese08G000662.t1 gene has the ability to catalyze the production of isofraquinone and also produces a byproduct, quinaquinone. That is, the protein encoded by the Ese08G000662.t1 gene can catalyze the methylation modification of quinaquinone at the C(7) or C(8) position, and finally produce isofraquinone or quinaquinone.

[0070] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. An oxygen methyltransferase, characterized in that The amino acid sequence of the oxygen methyltransferase comprises or consists of the following sequence: A1) the amino acid sequence shown in SEQ ID NO: 2; or A2) an amino acid sequence having the same protein activity as that of SEQ ID NO: 2 by adding, deleting or replacing one or more amino acids; or A3) the amino acid sequence shown in SEQ ID NO: 4; or A4) An amino acid sequence having the same protein activity as that of SEQ ID NO: 4 by adding, deleting or replacing one or more amino acids.

2. A nucleic acid molecule encoding the oxygen methyltransferase according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The sequence of the nucleic acid molecule includes or consists of the following sequence: B1) the nucleotide sequence shown in SEQ ID NO: 1; or B2) The nucleotide sequence shown in SEQ ID NO:

3.

4. A biomaterial, characterized in that: The biological material is any one of C1)-C3): C1) an expression cassette containing the nucleic acid molecule according to claim 2 or 3; C2) a recombinant vector containing the nucleic acid molecule according to claim 2 or 3; C3) A host cell containing at least one of the nucleic acid molecule according to claim 2 or 3, the expression cassette according to C1), and the recombinant vector according to C2).

5. Use of any one of the oxygen methyltransferase according to claim 1, the nucleic acid molecule according to claim 2 or 3, and the biomaterial according to claim 4 in the synthesis of isofraquinone and / or quinraquinone.

6. The use according to claim 5, characterized in that: The application uses fraxetin as a substrate.

7. The use according to claim 6, characterized in that: The quercetin is methylated at the C(7) or C(8) position.

8. A method for producing the oxygen methyltransferase according to claim 1, characterized in that: The nucleic acid molecule according to claim 2 or 3, or at least one of the expression cassette and the recombinant vector in the biological material according to claim 4, is used to transform a host cell, so that the host cell produces the oxygen methyltransferase.

9. A method for synthesizing isofraquinone and / or quinraquinone, characterized in that: With quercetin as a substrate, under the action of the oxygen methyltransferase described in claim 1 and / or the oxygen methyltransferase produced by the method described in claim 8, quercetin is catalyzed to undergo methylation modification at the C(7) or C(8) position to synthesize isofraquinone and / or quinraquinone.

10. A method for synthesizing isofraquinone and / or quinraquinone, characterized in that: The nucleic acid molecule described in claim 2 or 3, or any one of the biological materials C1)-C2) described in claim 4 is transformed into a host cell, so that the host cell produces oxygen methyltransferase; using quercetin as a substrate, under the action of the produced oxygen methyltransferase, catalyzes the methylation modification of quercetin at the C(7) or C(8) position to synthesize isofraquinone and / or quercetin.

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