An oxygen methyltransferase and its application in the biosynthesis of isoflurane and quinidine

Abstract

This invention relates to the field of synthetic biology, specifically to an oxygen methyltransferase and its application in the biosynthesis of isozyridine and aziridine. This application identifies the core gene elements of the isozyridine biosynthetic pathway—the oxygen methyltransferase genes SgOMT3 and Ese08G000662.t1—which encode proteins capable of catalyzing the conversion of fraxinus to isozyridine. Based on the oxygen methyltransferase of this application, combined with the isozyridine metabolic pathway, isozyridine can be synthesized in bacteria, yeast, algae, and plant substrates. This application provides an artificial synthetic pathway for isozyridine, offering a theoretical basis for the efficient, green, and low-carbon production of isozyridine through biosynthesis.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, specifically to an oxymethyltransferase and its application in the biosynthesis of isozypicridine and isozypicridine. Background Technology

[0002] Sarcandra glabra (Thunb.) Nakai, a traditional Chinese herbal medicine, is an evergreen semi-shrub belonging to the genus Sarcandra in the family Chloranthus. It grows in shady and damp places on hillsides and in valleys, and dislikes high temperatures, dryness, and direct sunlight. Research on the chemical composition of Sarcandra glabra is relatively systematic and comprehensive. Current research indicates that the whole plant mainly contains sesquiterpenes, flavonoids, organic acids, coumarins, and volatile oils. Modern pharmacological studies have demonstrated that Sarcandra glabra possesses antibacterial, anti-inflammatory, analgesic, anti-ulcer, influenza virus-inhibiting, fracture-healing-promoting, immunity-enhancing, anti-tumor, and hepatoprotective effects. Currently, several coumarin compounds have been extracted from Sarcandra glabra, among which isopyridine is one of the main active ingredients and is used as a quality control indicator for Sarcandra glabra medicinal materials and preparations in the Chinese Pharmacopoeia. Currently, wild Sarcandra glabra resources are becoming increasingly scarce, while pharmaceutical companies are increasingly demanding isopyridine, an important active ingredient extracted from Sarcandra glabra raw materials. The metabolic pathway of isozonidine remains unexplored, making its biosynthesis challenging. Identifying the catalytic enzymes involved in isozonidine synthesis using omics approaches, and then heterologously synthesizing isozonidine in substrates such as tobacco, yeast, or algae through synthetic biology methods, would provide a crucial theoretical foundation and economic value for the conservation of wild coral reef resources and their clinical application, contributing to the diversification of isozonidine compound sources.

[0003] This invention identifies the function of an oxygen methyltransferase (OMT) gene of *Sargassum fusiforme* through omics screening, heterologous expression, and enzyme activity detection. The OMT gene primarily catalyzes the production of isozymine and isozymine from fraxinus. Discovering this gene facilitates the later heterologous synthesis of isozymine via synthetic biology, thereby meeting market demand for important active ingredients of *Sargassum fusiforme* and possessing significant market value. Summary of the Invention

[0004] The purpose of this invention is to provide an oxygen methyltransferase (OMT) gene SgOMT3 that participates in the isozyptin synthesis pathway. The protein encoded by this gene can catalyze the methylation modification of fraxin at the C(7) or C(8) position, ultimately generating isozyptin or fraxin.

[0005] A first aspect of the present invention provides an oxymethyltransferase, the amino acid sequence of which comprises or is composed of the following sequences:

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

[0007] A2) An amino acid sequence in which one or more amino acids are added, deleted, or substituted, and which has the same protein activity, as shown in SEQ ID NO:2; or

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

[0009] A4) An amino acid sequence in which one or more amino acids are added, deleted, or replaced in the amino acid sequence shown in SEQ ID NO:4, and which has the same protein activity.

[0010] A second aspect of the invention provides a nucleic acid molecule encoding the aforementioned oxygen methyltransferase.

[0011] Preferably, the sequence of the above 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] A third aspect of the present invention provides a biomaterial, said biomaterial being any one of C1)–C3):

[0015] C1) An expression cassette containing the above-mentioned nucleic acid molecules;

[0016] C2) A recombinant vector containing the above-mentioned nucleic acid molecules;

[0017] C3) A host cell containing at least one of the above-mentioned nucleic acid molecules, 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-described oxymethyltransferase, the above-described nucleic acid molecule, or the above-described biological material in the synthesis of isozopridine and / or isozopridine.

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

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

[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 above-mentioned expression cassette or recombinant vector in the biological material, is transformed into a host cell to induce the host cell to produce the oxygen methyltransferase.

[0022] The sixth aspect of the present invention provides a method for synthesizing isozypicoride and / or aziridine, using fraxin as a substrate, and catalyzing fraxin at the C(7) or C(8) position under the action of the above-mentioned oxymethyltransferase and / or the oxymethyltransferase generated by the above method to synthesize isozypicoride and / or aziridine.

[0023] The seventh aspect of the present invention provides a method for synthesizing isozypicoride and / or aziridine, wherein any one of the above-mentioned nucleic acid molecules and the above-mentioned biological materials (C1)–C2) is transformed into a host cell to induce the host cell to produce an oxymethyltransferase; using fraxinerin as a substrate, under the action of the generated oxymethyltransferase, fraxinerin is catalyzed to undergo methylation modification at the C(7) or C(8) position to synthesize isozypicoride and / or aziridine.

[0024] The beneficial effects of this invention are as follows: For the first time, key candidate genes for the production of isozincide were successfully screened using multi-omics data. The key point is that the candidate genes were verified to have the function of catalyzing the production of isozincide using an in vitro enzyme expression system. Furthermore, isozincide can be synthesized in bacteria, yeast, algae, and plant substrates using the complete isozincide metabolic pathway.

[0025] Based on the artificial synthesis pathway of isozincide provided in this application, and through in-depth mining of functional element modules using genomic big data, the synthetic pathways and regulatory modules in algae, Saccharomyces cerevisiae, or Tobacco monoculture can be redesigned and optimized. This can overcome the regulatory and efficiency limitations of natural biosynthesis and provide theoretical and practical support for improving the product synthesis efficiency of isozincide and isozincide. Attached Figure Description

[0026] Figure 1 The content of isozymine terpineol in different tissues of *Sarcandra glabra* was determined.

[0027] Figure 2 This is a hypothesized synthetic route for isozymine.

[0028] Figure 3 Pearson correlation analysis was performed to determine the content of isozymine and the candidate OMT gene of *Sargassum fusiforme*.

[0029] Figure 4 The total ion chromatogram of the SgOMT3 protein gene of Coral sedge.

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

[0031] Figure 6 The total ion chromatogram of the Ese08G000662.t1 protein gene from Acanthopanax senticosus. Detailed Implementation

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

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

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

[0035] In some embodiments of the present invention, when the starting material is fraxin, under the catalytic action of the oxymethyltransferase provided by the present invention (such as SEQ ID NO:1, SEQ ID NO:3), fraxin is methylated at the C(7) or C(8) position to finally generate isozyridine or isozyridine.

[0036] In another embodiment of the present invention, when the starting materials are phenylalanine, 6'-hydroxyferuloyl-CoA, and scopolamine, the oxymethyltransferase provided by the present invention can be selected according to the specific synthetic pathway and 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 scopolamine 8-hydroxylase (S8H) to achieve the synthesis of isozyridine or isozyridine.

[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 isozymine.

[0038] Optionally, the synthetic biology chassis includes microbial systems and plant systems. The microbial systems include bacteria and / or fungi; the plant systems include, but are not limited to, *Sarcandra glabra*, *Eleutherococcus senticosus*, and *Nicotiana scabra*.

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

[0040] In this invention, the term "SgOMT3" can refer to the *Sophora alopecuroides* isozymine pyridoxine methyltransferase gene, and its specific meaning can be determined by referring to the context.

[0041] The technical solution of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All embodiments were carried out under conventional experimental conditions or conditions recommended in the manufacturer's instructions. Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. Furthermore, any methods and materials similar to or identical to those described can be used in the methods of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples shall be performed according to conventional methods or the manufacturer's recommendations. Unless otherwise specified, all materials, reagents, and consumables used are commercially available.

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

[0044] In some embodiments of the present invention, total plant RNA extraction was performed using the FastPure Plant Total RNA Isolation Kit (Polysaccharides & Polvphenolics-rich, Vazyme); cDNA synthesis was performed using the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa); and PCR amplification was performed using the KOD One™ PCR Master Mix (ToYoBo). PCR Master Mix (With Dye) (Yeasen); Gel Extraction was performed using a Gel Extract Kit D2500 (Omega); Plasmid DNA extraction was performed using a Plasmid Mini-Prep Kit (DP103-03, TIANGEN); DNA was digested with restriction endonucleases HindIII (NEB) and KpnI (NEB); Gene cloning and ligation were performed using the pEASY-Basic Seamless Cloning and Assembly Kit (Taiwan); DNA molecular weight determination was performed using the DL5000 DNA marker (TaKaRa); Gene cloning and vector construction were performed using DH5α and BL21 (DE3) (Shanghai Weidi); Primers required for the assay were synthesized by Zhejiang Shangya Biotechnology Co., Ltd.; Gene sequencing was performed by Zhejiang Shangya Biotechnology Co., Ltd. In other embodiments, other suitable reagents and tools may be selected as needed to perform the above experiments.

[0045] Example 1: Establishment of an isozine dermatidine detection system

[0046] To determine the content of isopyridine in the leaves, stems, roots, and flowers of *Sarcandra glabra*, 0.1 g of pulverized fresh sample was weighed from each tissue. 5 ml of methanol was added, and the four tissues were ultrasonically extracted for 1 h. The extract was then concentrated to 1 ml under vacuum and filtered through a 0.22 μm filter membrane. The filtered sample was then loaded into a sample vial. The isopyridine content was determined by LC-MS / MS. The results are as follows: Figure 1 As shown, the content of isozymine in the roots of *Sarcandra glabra* is higher than that in the leaves, stems, and flowers.

[0047] Example 2: Speculation of the isozypterin synthesis pathway and discovery of candidate OMT genes

[0048] The inventors discovered that isofraxidin shares a structural skeleton with compounds such as fraxidin, scopolamine, and fraxidin. Therefore, the inventors hypothesize that the synthesis of isofraxidin involves the following steps: phenylalanine is first synthesized into 6'-hydroxyferuloic acid coenzyme A via the action of enzymes such as phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (C4H), and cinnamic acid-3-hydroxylase (C3H). Subsequently, fraxidin is generated by the catalysis of feruloyl-CoA 6'-hydroxylase 1 (F6'H1) and scopolamine 8-hydroxylase (S8H). Finally, fraxidin is catalyzed by a specific OMT gene to produce isofraxidin. Figure 2 ).

[0049] To verify the above hypothesis, this invention used the OMT gene set published by *Chrysanthemum indicum* as a target, and ultimately obtained 18 *Sarcandra glabra* OMT genes from the genomic and transcriptomic data of *Sarcandra glabra*. These genes were divided into two clades: clade 1: 15 caffeic acid O-methyltransferase (COMT) family genes; clade 2: 3 caffeic acid coenzyme AO-methyltransferase (CCoAOMT) family genes. Pearson correlation analysis was performed on the isozyridine content in four tissues (leaf, stem, root, and flower tissues) of *Sarcandra glabra* and the transcriptomic data of the 13 OMT genes (COMT family, excluding two OMT genes with expression levels of 0), ultimately screening out four candidate OMT genes. Pearson analysis was then performed on the isozyridine content in the four tissues of these 13 OMT genes, and only four OMT genes showed a positive correlation with isozyridine. Figure 3 The four denoted as SgOMT1, gOMT2, SgOMT3 and SgOMT4 are respectively.

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

[0051] Through experimental verification of four candidate OMT genes in Example 2, one OMT gene with the function of catalyzing the production of isozymine: SgOMT3 was found. SgOMT3 contains 1065 nucleotides, encoding 354 amino acids.

[0052] Fresh root tissue samples of *Sarcandra glabra* were collected and ground with liquid nitrogen. Total RNA was extracted from *Sarcandra glabra* using the Vazyme kit. cDNA was synthesized according to the instructions of the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa). Primers for the synthesis of the SgOMT3 gene were designed based on the nucleotide sequence (SEQ ID NO:1), and the primers contained KpnⅠ and HindⅢ restriction enzyme sites. Using cDNA from *Sarcandra glabra* roots as a template, the SgOMT3 gene was amplified by PCR and recombined with a linear pCold-TF vector digested with KpnI and HindIII. The recombinant plasmid was obtained using the pEASY-Basic Seamless Cloning and Assembly Kit. The homologous recombination product was transferred into DH5α competent cells, transformed, 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 plasmids of positive strains with correct sequencing sequences were extracted. The positive recombinant plasmid was named pCold-SgOMT3.

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

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

[0055] SgOMT3-Reverse: agactgcaggtcgacaagcttTTATTTGTGGAACTCCATGACCC (lowercase sequence is vector sequence, uppercase sequence is 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 *E. coli* competent cells BL21(DE3). After colony PCR screening, 50 μl of bacterial culture was inoculated into 5 ml of LB broth containing ampicillin resistance (Amp) and cultured overnight at 37°C. 2 ml of the overnight culture was then inoculated into 200 ml of liquid LB broth (containing Amp resistance). After culturing at 37°C for 3-4 h, the OD600 value was approximately 0.8. The induction medium was placed on ice and allowed to stand for 1 h, then 400 μl of IPTG (0.5 M) was added to a final concentration of 1 mM. After induction at 18°C ​​for 20 h, the bacterial culture was transferred to a 500 mL centrifuge tube and centrifuged at 8000 rpm, 4°C, for 10 min. The supernatant was discarded, and the bacterial cells were collected. 15 ml of lysis buffer and 200 μl of PMSF were added, the cells were vortexed and resuspended, and then sonicated. After bacterial cell lysis, the supernatant was collected by centrifugation and filtered through a 0.45 μm membrane. 600 μL of Ni-NTA packing material was added to the supernatant, and the mixture was incubated overnight at 4°C with rotational mixing. The following day, contaminating proteins were washed with 40, 80, and 150 μM Wash Buffer, respectively. Finally, 5 ml (250 μM) Elusion Buffer was added to elute the target protein. The eluted target protein was concentrated and desalted using a desalting column. A portion was used for analysis, and the remainder was stored at -80°C for later use.

[0058] The enzyme activation system (200 μl) consisted of the following components: 10 μg purified protein, 0.5 mM fraxetin, 0.4 M Tris-HCl (pH 7.5), 4 mM DTT, 2 mM MgCl2, 0.5 mM SAM, with the remainder made up with ddH2O. The reaction was carried out at 37 °C for 1 h. After the enzyme activation reaction was complete, 200 μl of acetonitrile was added to stop the reaction, followed by 800 μl of ethyl acetate. The mixture was vortexed and allowed to stand in the dark for 30 min. The mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was transferred to a new 1.5 ml centrifuge tube. After vacuum freeze concentration, 200 μl of chromatographic methanol was added to reconstitute the solution. The solution was then analyzed by 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: 0 min 90% B; 0–0.5 min 90% B; 0.5–6.9 min 5% B; 6.9–10 min 90% B. Aspirate volume 1 μl, flow rate 0.30 mL / min, column temperature 40 °C. pCold-TF was used as a control.

[0059] The presence of the protein encoded by the SgOMT3 gene in the in vitro enzyme expression system, combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS), was analyzed to determine whether it catalyzes the conversion of fraxetin to isozytin. Results are as follows: Figure 4 As shown, when the SgOMT3 gene is expressed, the production of isozypicillin or aziridine can be detected. That is, the SgOMT3 enzyme can catalyze the methylation modification of fraxin at the C(7) or C(8) position, ultimately generating isozypicillin or aziridine.

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

[0061] This embodiment further determined the content of isozypicillin in the roots of Eleutherococcus senticosus and identified a gene with the same function as the methyltransferase SgOMT3 based on the genomic data of Eleutherococcus senticosus (Araliaceae). To determine the content of isozypicillin in the root tissue of Eleutherococcus senticosus, 0.1 g of crushed fresh sample was weighed, and 5 ml of methanol was added for ultrasonic extraction for 1 h. After vacuum freezing and concentration to 1 ml, the sample was filtered through a 0.22 μm filter membrane. The filtered sample was then loaded into a sample vial. The content of isozypicillin in the root tissue of Eleutherococcus senticosus was detected using the established LC-MS detection method of this invention. The results showed that the content of isozypicillin in the root tissue of *Sarcandra glabra* was significantly higher than that in the root tissue of Eleutherococcus senticosus. Figure 5 ).

[0062] This invention targets SgOMT3 of *Sarcandra glabra*, and obtains a highly homologous gene from *Eleutherococcus senticosus*: Ese08G000662.t1. Ese08G000662.t1 contains 1098 nucleotides, encoding 365 amino acids.

[0063] Fresh samples of Acanthopanax senticosus root tissue were taken and ground with liquid nitrogen. Total RNA was extracted from Acanthopanax senticosus using the Vazyme kit. cDNA was synthesized according to the instructions of the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa). Primers for the synthesis of the Ese08G000662.t1 gene were designed based on the nucleotide sequence (SEQ ID NO:3) of the Ese08G000662.t1 gene, and the primers contained KpnⅠ and HindⅢ restriction enzyme sites. Using cDNA from Acanthopanax senticosus root as a template, the Ese08G000662.t1 gene was amplified by PCR and recombined with a linear pCold-TF vector digested with KpnⅠ and HindⅢ. The recombinant plasmid was obtained using the pEASY-Basic Seamless Cloning and Assembly Kit. The homologous recombination product was transformed into DH5α competent cells, transformed, 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 plasmids of positive strains with correct sequencing sequences were extracted. The positive recombinant plasmid was named pCold-Ese08G000662.t1.

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

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

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

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

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

[0069] The study analyzed whether the protein encoded by the Ese08G000662.t1 gene catalyzes the conversion of fraxetin to isozymine using an in vitro enzyme expression system combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS). The results are as follows: Figure 6 As shown, the protein encoded by the Ese08G000662.t1 gene has the ability to catalyze the production of isozinidine, and also produces the byproduct aziridine. That is, the protein encoded by the Ese08G000662.t1 gene can catalyze the methylation modification of fraxin at the C(7) or C(8) position, ultimately producing isozinidine or aziridine.

[0070] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. An oxygen methyltransferase, characterized in that, The amino acid sequence of the oxymethyltransferase is the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence shown in SEQ ID NO:

4.

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

3. The nucleic acid molecule according to claim 2, characterized in that, The sequence of the nucleic acid molecule is the nucleotide sequence shown in SEQ ID NO:1 or the nucleotide sequence shown in SEQ ID NO:

3.

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

5. The use of any one of the following in the synthesis of isozyptin and / or isozyptin: the oxymethyltransferase of claim 1, the nucleic acid molecule of claim 2 or 3, or the biomaterial of claim 4, characterized in that, The application uses fraxin as a substrate.

6. The application according to claim 5, characterized in that, The fraxin is methylated at the C(7) or C(8) position.

7. A method for producing the oxymethyltransferase of claim 1, characterized in that, The host cell is transformed with the nucleic acid molecule of claim 2 or 3, or at least one of the expression cassette or recombinant vector in the biological material of claim 4, so that the host cell produces the oxygen methyltransferase.

8. A method for synthesizing isopyridine and / or aziridine, characterized in that, Using fraxin as a substrate, under the action of the oxymethyltransferase described in claim 1 and / or the oxymethyltransferase generated by the method described in claim 7, fraxin is catalyzed to undergo methylation modification at the C(7) or C(8) position to synthesize isozyridine and / or aziridine.

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