An oxygen methyltransferase and its application in biocatalytic synthesis of O-methylated products

Abstract

The present invention provides an oxygen methyltransferase and its application in the biocatalytic synthesis of O-methylated products. The applicant screened and obtained oxygen methyltransferases AgOMT1 and AgOMT2 from the transcriptome data of roots, leaves and petioles of celery. Four different furanocoumarin substrates (zanthoxylum toxicol, bergamotol, 5-hydroxyzanthoxylum toxicol and 8-bergamotol) can be catalyzed by AgOMT1 and generate corresponding O-methylated products. Oxygen methyltransferase AgOMT2 can catalyze bergamotol to generate bergamotol, and the catalytic reaction rate is high, which fills the gap in the biosynthetic pathway of furanocoumarin series compounds and provides an optional enzymatic element for the methylation modification of similar compounds.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to an oxygen methyltransferase and an application thereof in biocatalytic synthesis of O-methylated products. Background Art

[0002] Methylation is a ubiquitous reaction that occurs in a variety of organisms, including bacteria, fungi, plants, and mammals. Methylated small and macromolecules undergo significant changes in physical properties, including structural stability, protein affinity, hydrophilicity, and solubility. Methylation can also improve the biological activity of some small molecule compounds. For example, oxygen methylation of plant flavonoids changes their solubility and intracellular partitioning, increasing their antibacterial activity. In animals, oxygen methylation plays an important role in the biosynthesis of catecholamines and melatonin and the detoxification of reactive hydroxyls.

[0003] Coumarin is a class of active natural products derived from the secondary metabolic pathways of plants or microorganisms. It is widely distributed in nature, especially in plant groups. In nature, furanocoumarins are widely distributed and have been isolated and identified in many higher plants, especially in citrus species of Rutaceae (such as citrus, sweet orange, lemon, grapefruit and bergamot), fig of Moraceae, psoralea corylifolia of Leguminosae, and parsley, celery and coriander of Apiaceae. Furocoumarins and pyrocoumarins have significant biological activities and diverse structural variations. They are active ingredients of many traditional medicines and important secondary metabolites in many plants and microorganisms. Analyzing the biosynthetic pathways of furanocoumarins and pyrocoumarins can not only help us deeply study and understand the complex chemical reaction processes in organisms, reveal how organisms synthesize complex organic molecules through specific metabolic pathways, but also lay the foundation for the construction of efficient biosynthetic systems of such compounds in the future. Oxymethyltransferase in its biosynthetic pathway is an important synthetic step, and there is no complete report on it worldwide.

[0004] Therefore, it is necessary to develop more oxygen-methyltransferases with high catalytic efficiency that can participate in catalyzing the multi-step oxygen-methylation reactions in the biosynthesis of furanocoumarin isoanisole. Summary of the invention

[0005] The invention aims to provide an oxygen methyltransferase and its application in biocatalytic synthesis of O-methylated products, which participates in catalyzing multi-step oxygen methylation reactions in the biosynthesis of furanocoumarin isoanisole lactone.

[0006] The present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, an oxygen methyltransferase is provided, wherein the oxygen methyltransferase comprises one of the following:

[0008] Oxygen methyltransferase AgOMT1, the amino acid sequence of which is shown in SEQ ID NO.1;

[0009] Oxygen methyltransferase AgOMT2, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] Furthermore, the oxygen methyltransferase also includes one of the following:

[0011] 1) An amino acid sequence having the same function as the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 obtained by replacing and / or deleting and / or adding one or more amino acid residues;

[0012] 2) having a fusion amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.1 or SEQ ID NO.2;

[0013] 3) An amino acid sequence having 95% or more homology with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 and having the same function.

[0014] In the second aspect of the present invention, the use of the oxygen methyltransferase in biocatalysis is provided, wherein the oxygen methyltransferase AgOMT1 and AgOMT2 biocatalyze bergamotol to produce bergamotinoside lactone.

[0015] Furthermore, the application also includes: the oxygen methyltransferase AgOMT1 catalyzes Zanthoxylum toxicans to produce Zanthoxylum toxin, catalyzes 5-hydroxy Zanthoxylum toxin to produce bergamotolide, and catalyzes 8-bergamotolide to produce isoanisole lactone.

[0016] In the third aspect of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector is capable of expressing the oxygen methyltransferase.

[0017] The nucleic acid molecule encoding the oxygen methyltransferase in the recombinant expression vector includes one of the following:

[0018] 1) having a deoxyribonucleotide sequence as shown in SEQ ID NO.3 or SEQ ID NO.4;

[0019] 2) having a deoxyribonucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2;

[0020] 3) a deoxyribonucleotide sequence encoding a gene having oxygen methyltransferase activity obtained by substituting and / or deleting and / or adding one or more nucleotides to the deoxyribonucleic acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4;

[0021] 4) A deoxyribonucleotide sequence having a homology of 95% or more (e.g., 96%, 97%, 98%, 99%, 100%) with the deoxyribonucleic acid sequence defined as SEQ ID NO.3 or SEQ ID NO.4 and encoding an oxygen-methyltransferase.

[0022] In the fourth aspect of the present invention, a recombinant bacterium or an engineered cell line comprising the recombinant expression vector is provided.

[0023] In the fifth aspect of the present invention, provided is the use of the recombinant expression vector or the recombinant bacteria or engineered cell line in biocatalysis. It is characterized in that the recombinant expression vector includes one or more of an Escherichia coli expression vector, a yeast expression vector, a Subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector; the host cell includes an Escherichia coli host cell (such as Escherichia coli BL21, Escherichia coli DH5α, etc.), a yeast host cell (such as Saccharomyces cerevisiae, Pichia pastoris, etc.), a Subtilis host cell (such as Bacillus subtilis R25, etc.), a lactic acid bacteria host cell (such as Lacticacid bacteria COCC101, etc.), an actinomycete host cell (such as Streptomyces spp., etc.), a filamentous fungus host cell (such as Trichoderma viride, etc.), an insect cell (such as Bombyx mori, etc.), and a mammalian cell (such as Chinese hamster ovary cell CHO, baby hamster kidney cell BHK, etc.).

[0024] In a sixth aspect of the present invention, a method for biosynthesis of an O-methylated product is provided, wherein the O-methylated product comprises one of zanthoxylum toxin, bergamotolactone, and isoanisole lactone, and the method comprises one of the following:

[0025] Using bergamotol as a substrate, the oxygen methyltransferase AgOMT1 or AgOMT2 or its enzyme lysate is used to catalyze the synthesis of bergamotol lactone;

[0026] Using Zanthoxylum bungeanum as substrate, the oxygen methyltransferase AgOMT1 or its enzyme lysis solution is used to catalyze the synthesis of Zanthoxylum bungeanum toxin;

[0027] Using 5-hydroxy zanthoxylum bungeanum toxin as a substrate, the oxygen methyltransferase AgOMT1 or its enzyme lysis solution is used to catalyze the synthesis of bergamotinoside lactone;

[0028] Taking 8-bergamot lactone as a substrate, the oxygen methyltransferase AgOMT1 or its enzyme lysis solution is used to catalyze the synthesis of isoanisole lactone.

[0029] Furthermore, the oxygen methyltransferase AgOMT1 or AgOMT2 can be obtained by constructing a vector for expression and purification.

[0030] In the seventh aspect of the present invention, a fermentation production method of an O-methylated product is provided, wherein the O-methylated product includes one of zanthoxylum toxin, bergamot glycoside lactone, and isoanisole lactone, and the method includes the following steps: fermenting and culturing the recombinant bacteria or engineered host cell line, adding a substrate, and obtaining the compound after fermentation.

[0031] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0032] 1. The present invention provides an oxygen methyltransferase and its application in biocatalysis. The applicant screened the oxygen methyltransferase from the transcriptome data of the roots, leaves and petioles of Celery. Four different furanocoumarin substrates (zanthoxylum phenol, bergamotol, 5-hydroxyzanthoxylum phenol and 8-bergamotol) can be catalyzed by AgOMT1 to generate corresponding O-methylated products. The oxygen methyltransferase AgOMT2 can catalyze bergamotol to generate bergamotol, and the catalytic reaction rate is high;

[0033] 2. The oxygen methyltransferase provided by the present invention uses engineered Escherichia coli for protein expression, and the addition of substrate can efficiently catalyze the generation of corresponding O-methylated products. The entire reaction process has the advantages of simple operation, high safety factor, short production cycle, high efficiency and environmental friendliness.

[0034] 3. In the present invention, the analysis and discovery of coumarin oxygen methyltransferase with high catalytic efficiency fills the gap in the biosynthetic pathway of furanocoumarin series compounds and provides an alternative enzymatic element for the methylation modification of similar compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0036] Figure 1It is the recombinant plasmid pET-28a(+)-AgOMT1.

[0037] Figure 2 This is the gel image of protein purification of AgOMT1 and AgOMT2. Figure 2 The left side of the figure shows the results of small-scale expression, and the right side shows the protein detection of each step in the purification process. The theoretical size of the AgOMT1 protein (including the tag) in Figure A is 43.91 kDa, and the theoretical size of the AgOMT2 protein (including the tag) in Figure B is 43.95 kDa.

[0038] Figure 3 Activity test and enzyme kinetic analysis of AgOMT1 and AgOMT2. A. AgOMT1 uses Zanthoxylum phenol as substrate to generate oxygen-methylated product Zanthoxylum toxicum. B. AgOMT1 and AgOMT2 use bergamotol as substrate to generate oxygen-methylated product bergamotol. C. AgOMT1 uses 5-hydroxy Zanthoxylum toxicum as substrate to generate oxygen-methylated product isoanisole. D. AgOMT1 uses 8-hydroxybergamotol as substrate to generate oxygen-methylated product isoanisole.

[0039] Figure 4 This is the mass spectrometry analysis of the methylation reaction of Zanthoxylum bungeanum toxicity catalyzed by AgOMT1. The left side shows the molecular ion peak of the extracted target compound, with an error value of <10ppm, and the right side shows the secondary mass spectrum.

[0040] Figure 5 Mass spectrometric analysis of the methylation reaction of bergamotol catalyzed by AgOMT1 and AgOMT2.

[0041] Figure 6 Mass spectrometric analysis of the methylation reaction of 5-hydroxyxanthoxanthin and 8-hydroxybergamotolactone catalyzed by AgOMT1.

[0042] Figure 7 This is a test of the catalytic activity of AgOMT1 and AgOMT2 towards coumarin compounds.

[0043] Figure 8 This is a test of the catalytic activity of AgOMT1 towards flavonoids. DETAILED DESCRIPTION

[0044] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0045] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0046] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0047] The following is a detailed description of an oxygen methyltransferase of the present application and its application in biocatalysis in combination with examples and experimental data.

[0048] Plasmid pET-28a (+) was purchased from Novagen. Competent E. coli TOP10 and BL21 (pGro7) were purchased from Beijing Qingke Biotechnology. Restriction endonucleases were purchased from New England Biolabs. Plasmid extraction and gel recovery kits were purchased from OMEGA. Reagents used in common culture media were purchased from Oxford, Amresco and Sinopharm Chemical Reagent Co., Ltd. All antibiotics used were purchased from Sigma-Aldrich.

[0049] The formula of LB medium is as follows: Tryptone: 10 g / L, Yeast extract: 5 g / L, NaCl: 10 g / L.

[0050] The experimental reagents not specifically described in the present invention are all conventional reagents in the art and can be prepared according to conventional methods in the art or purchased commercially.

[0051] Example 1. Screening of O-methyltransferases in Celery and Discovery of O-methyltransferases AgOMT1 and AgOMT2

[0052] Trinity was used to perform reference splicing of the transcriptome data of roots, leaves and petioles of Celery, and quantitative analysis was completed based on RSEM. Sequences with high expression levels were screened out and annotated with Pfam using Hmmer. Gene sequences with annotation results of O-methyltransferase were selected and clustered for expression analysis together with the upstream identified AgUDT1 gene and AgCOC1 gene. This work was completed using TBtools. The full-length sequences of O-methyltransferase genes clustered with AgUDT1 and AgCOC1 were extracted, and preliminary functional sorting was performed using the public databases NCBI and uniprot.

[0053] Nucleotide sequence of AgOMT1 (SEQ ID NO.3)

[0054]

[0055] Amino acid sequence of O-methyltransferase AgOMT1 (SEQ ID NO.1)

[0056] MAEITKSHGVNGNDEIAQAQVDIWRFVFGFTETAAVKCAIELGIPDILENQADPMTLSQLSSALSCSSTALFRIMRFLMNRGIFKEKITKEGSMGYVQTPLSRLLRKDGDNSLTAMLLWQNSSILIDPWHYLSARVLDDKASAFVCAHGKSLWEIAAEDPEQRRLIDDAMACDTRTTV GALVDGCPEVFDGLSSVVNVGGGNGTALRVLIERCPWIRGINFDLNEVVSVAPESEGIEHVGGDMFKSVPKANAAFLKWILHDWSDGECIQILKKCREAISEYGTAGKVIIVEAVIEVNGGDKLKDVGLMLDMLMLAQTNKGKERTAEEWAFILREAGFTRHTIKSFQSALSVIEAYPY

[0057] Nucleotide sequence of AgOMT2 (SEQ ID NO.4)

[0058]

[0059] Amino acid sequence of AgOMT2 (SEQ ID NO.2)

[0060] MAETKTSPSQDEEACLLAIQLATSTVLPMILKSAIELDLLNTITKAGPGNYLSPSDLASKLSMSNPDAPVMLDRILKVLATYKVLDCKPSELSNGHVEWLYCWTPVCKFLSNNEDGASIAPLLLVHQDKVPMKSWYYLTDAVRDGGTAFNKAYGMSIFDCASQDPMFNKVFNVSMRGHS TITMKKILETYNGFKGLKSIVDVGGGTGATLNMIISKYPTIKGINFDLPHVVGDAPSHPGVEHVGGNMFESVPKGDAIFLKWIFHSWSDEDCLRILKKCCEALEDNKKVIVAEFIYPEVPGGSDSGTKSVVHLDAIMLAYVPGGKERTEKEYKSLATRAGFKSFSKVCCAFGTWIMEFSK

[0061] Example 2: Construction of pET-28a(+)-AgOMT1 and pET-28a(+)-AgOMT2 recombinant plasmids

[0062] We optimized the codons of AgOMT1 and AgOMT2 by gene synthesis and constructed them on the pET-28a(+) expression vector. For details, see Figure 1 .

[0063] Taking AgOMT1 as an example, we fused His tags to both the N-terminus and the C-terminus of the gene so that the protein it expressed could have a good affinity with the nickel column. The specific construction method is as follows:

[0064] 1. Synthesize and obtain nucleic acid molecules with nucleotide sequences of AgOMT1 and AgOMT2 as shown in SEQ ID NO.1 and SEQ ID NO.3;

[0065] 2. The nucleic acid molecules were cloned into the vector pET-28a(+), and the restriction sites were BamHI at the N-terminus and XhoⅠ at the C-terminus, respectively, to obtain the recombinant plasmids pET-28a(+)-AgOMT1 and pET-28a(+)-AgOMT2.

[0066] Example 3. Expression and purification of AgOMT1 and AgOMT2 proteins

[0067] 1. Transform the recombinant plasmid into E. coli expression competent cells

[0068] ① Take out the BL21 (pGro7) competent cells from the -80℃ refrigerator, insert them into ice, and add the recombinant plasmids pET-28a(+)-AgOMT1 and pET-28a(+)-AgOMT2 respectively after the bacterial block melts. Gently tap the bottom of the centrifuge tube to evenly mix the plasmid and competent cells, and let it stand on ice for 25 minutes.

[0069] ② Heat shock in 42℃ metal bath for 90s, quickly put back into ice and let stand for 2min.

[0070] ③ Transfer the competent cells to the clean bench, add 700 μL of antibiotic-free LB medium to the centrifuge tube, and culture at 37°C, 220 rpm, with shaking for 90 minutes to revive the cells.

[0071] ④ Centrifuge the bacterial solution at 12000rpm for 1min, take 100μL of supernatant, and use the pipette tip to blow and aspirate to redisperse the bacterial clumps. Spread the bacterial solution on a plate of LA medium containing Kan, and incubate it upside down in a constant temperature incubator at 37℃ for 12h.

[0072] 2. Inducible expression of AgOMTs in Escherichia coli

[0073] ① Pick a single clone colony. In the clean bench, take 3 shaking tubes and add 6mL LB medium and 6μL Kan to each. Use a sterilized toothpick to pick a single colony and inoculate it into the LB medium. Incubate at 37℃, 220rpm, and shake for about 6h.

[0074] ② Transfer. Take two sterilized 2L conical flasks, add 900mL LB medium, 900μL Kan, add L-arabinose at a ratio of 1mg / mL to each flask in a clean bench, and finally add 9mL bacterial solution. Incubate at 37℃, 220rpm, and shake for about 3h.

[0075] ③ Induction. In a clean bench, take 4 mL of bacterial solution into a shaking tube without adding inducer. Add IPTG inducer with a final concentration of 0.1 mM to the remaining bacterial solution. Induce expression for 18 hours at 18°C ​​and 180 rpm.

[0076] ④ Collect the bacteria. Centrifuge at 4°C, 10,000 rpm for 45 min, discard the culture medium supernatant, and retain the bacteria. Use a desktop centrifuge to collect the uninduced bacterial solution in the shaking tube, centrifuge at 12,000 rpm for 1 min.

[0077] 3. Protein purification of AgOMTs

[0078] (1) Nickel column affinity chromatography

[0079] ① Collect the induced bacteria, add about 3 times the volume of the bacteria into Binding Buffer, and resuspend on a mixer.

[0080] ②Put the resuspended bacterial solution on ice and use an ultrasonic cell disruptor at 60W for 30min. Ultrasonicate for 2s each and cool for 5s.

[0081] ③ The broken bacterial liquid was transferred into a centrifuge tube and centrifuged at 4°C, 15,000 rpm for 60 minutes in a floor centrifuge.

[0082] ④ After centrifugation, the supernatant was filtered through a 0.45 μm filter membrane and placed on ice.

[0083] ⑤ Use a pre-packed nickel column and a peristaltic pump for protein affinity chromatography. Before loading, wash the nickel column with 10 volumes of Milli-Q water and then equilibrate it with 5 volumes of Binding Buffer.

[0084] ⑥ Add sample. The supernatant after passing through the membrane is passed through the nickel column twice to load the target protein.

[0085] ⑦ Elute the impurities. Use 25mM imidazole eluent, 50mM imidazole eluent and 100mM imidazole eluent to wash the impurities in sequence until the Brandford protein indicator solution hardly changes color. Samples of each gradient eluent are retained for testing.

[0086] ⑧ Elute the target protein. Use 200mM imidazole eluent and 300mM imidazole eluent to elute the target protein. Collect the eluted proteins in 5mL centrifuge tubes and number them until the color changes to a lighter color when detected by Brandford protein indicator solution.

[0087] ⑨ The protein eluate of each gradient was detected by SDS gel electrophoresis. The sample containing high-purity target protein was collected, concentrated to a small volume with a protein ultrafiltration tube, and then added with imidazole-free Store Buffer, repeatedly diluted and concentrated until the imidazole concentration was negligible.

[0088] ⑩ The concentration of the concentrated protein was detected using an ultra-micro spectrophotometer to calculate the total amount of protein.

[0089] (2) Gel chromatography (molecular sieve)

[0090] ①AKTA purifier, flow rate set to 1 mL / min, Store Buffer balanced for 2 column volumes.

[0091] ② The protein concentrate purified by affinity chromatography is injected into the sample loading end and separated using the molecular sieve principle.

[0092] ③ Collect the protein eluates at each time gradient, detect them by SDS gel electrophoresis, and concentrate the high-purity part to a small volume using an ultrafiltration tube.

[0093] ④Measure the protein concentration, divide into packages, freeze in liquid nitrogen and store in a -80℃ refrigerator.

[0094] 4. SDS-PAGE was performed on AgOMT1 and AgOMT2 proteins. The results were as follows: Figure 2 shown.

[0095] Depend on Figure 2 It can be seen that compared with the uninduced group, new bands appeared in the whole bacterial protein and supernatant protein under IPTG induction, and their size was about 43KDa, which was consistent with the theoretical calculated values ​​of AgOMT1 and AgOMT2. A pre-loaded nickel column was used for affinity purification, and a large amount of target protein was obtained under the elution conditions of 200mM imidazole and 300mM imidazole. The purer part was collected and concentrated, and the impurities were further removed by molecular sieve. After the purification, the recombinant protein was concentrated and the concentration was tested, and it was divided into tubes and stored at -80℃.

[0096] Example 4: In vitro activity test and kinetic analysis

[0097] 1. In vitro enzyme activity test of AgOMTs

[0098] In vitro enzyme catalysis experiments were performed according to the following reaction system to test the catalytic activity of the AgOMT1 and AgOMT2 proteins obtained in Example 3 on various substrates. The group without enzyme was used as a negative control, and other conditions were the same.

[0099] During the activity test, the reaction temperature was set at 28°C, the reaction time was set at 3h, and the mixture was incubated at 300rpm in a constant temperature shaker. After the reaction was completed, an equal amount of methanol was added to quench the mixture, and the mixture was centrifuged at 13,300rpm for 10min. The supernatant was filtered through a 0.22μm filter membrane and placed in a sample vial for analysis.

[0100] Table 1-AgOMTs recombinase reaction system (total reaction system 150 μL)

[0101] Components Concentration / dosage Sodium phosphate buffer (pH 8.0) 100mM SAM (methyl donor) 1mM Substrate 100μM Enzymes About 5 μg

[0102] 2. Liquid chromatography analysis: HPLC analysis and LC-MS identification of products

[0103] (1) HPLC-UV analysis:

[0104] The HPLC-UV analysis was performed according to the following elution procedure: flow rate 1 mL / min; injection volume 10 μL; detection wavelengths 268 nm and 310 nm.

[0105] Table 2 Elution program 1

[0106] Elution time (min) Phase A (0.1% formic acid-water) Phase B (Methanol) 0 90％ 10％ 10 40％ 60％ 20 10％ 90％ 25 10％ 90％ 25.01 90％ 10％ 30 90％ 10％ 0 90％ 10％

[0107] (2) High performance liquid chromatography-mass spectrometry analysis:

[0108] The HPLC-MS analysis was performed according to the following elution program: flow rate 0.3 mL / min; injection volume 2 μL; ion scanning range: m / z 100-1000; detection mode: positive ion.

[0109] Table 3 Elution program 2

[0110]

[0111]

[0112] 3. In vitro activity test and kinetic analysis of AgOMTs

[0113] Preliminary analysis showed that both AgOMT1 and AgOMT2 are SAM-dependent O-methyltransferases. We used four linear furanocoumarins, zanthoxylum phenol, bergamotol, 5-hydroxyzanthoxylum phenol, and 8-bergamotol as substrates and S-adenosylmethionine (SAM) as a methyl donor to test the activities of AgOMT1 and AgOMT2.

[0114] The results showed that compared with the control group, AgOMT1 could catalyze the O-methylation of zanthoxylum phenol, bergamotol, 5-hydroxyzanthoxylum toxin and 8-bergamotol to generate zanthoxylum toxin, bergamotol and isoanisole. Figure 3 Within the specified reaction time, the conversion rates of the four reactions were close to 100%. The generated products were compared with the standard under UV and mass spectrometry detection conditions. The mass spectrometry results showed that Figure 4 , Figure 5 and Figure 6 AgOMT2 can only catalyze the conversion of bergamotol to bergamotolide, with a reaction conversion rate close to 100%, but the enzyme cannot complete the O-methylation of other similar substrates, see Figure 3 B. To explore the catalytic efficiency of AgOMT1 and AgOMT2 on substrates, we conducted enzyme reaction kinetic analysis and conducted a preliminary study on their catalytic reaction rates.

[0115] The results showed that AgOMT1 exhibited a high catalytic reaction rate for three substrates: zanthoxylum toxicogenin, 5-hydroxyzanthoxylum toxicogenin and 8-hydroxybergamot lactone.

[0116] When using Zanthoxylum bungeanum as substrate, the maximum reaction rate of AgOMT1 was about 0.346 mM / s, the Km value was about 198 μM, and the Kcat value was about 0.474 s -1 , Kcat / Km value is about 2.4mM -1 s -1 ,See Figure 3 A;

[0117] When 5-hydroxyzanthoxylum bungeanum was used as the substrate, the maximum reaction rate of AgOMT1 was about 0.152 mM / s, the Km value was about 82.3 μM, and the Kcat value was about 0.208 s -1 , Kcat / Km value is about 2.5mM -1 s -1 ,See Figure 3 C;

[0118] When 8-hydroxybergamot lactone was used as the substrate, the maximum reaction rate of AgOMT1 was about 0.175 mM / s, the Km value was about 122 μM, and the Kcat value was about 0.240 s -1 , Kcat / Km value is about 2.0mM -1 s -1 ,See Figure 3 D.

[0119] In contrast, the catalytic reaction rate of AgOMT1 on bergamot alcohol was lower, with a maximum reaction rate of about 0.0156 mM / s, a Km value of about 120 μM, and a Kcat value of about 0.0129 min -1 , Kcat / Km value is about 0.11mM -1 min -1 ,See Figure 3 B.

[0120] The catalytic performance of AgOMT2 with the same catalytic function in this reaction is better than that of AgOMT1, with a maximum reaction rate of about 0.285 mM / s, a Km value of about 478 μM, and a Kcat value of about 0.234 min -1 , Kcat / Km value is about 0.49mM -1 min -1 ,See Figure 3 B.

[0121] Although our activity test results showed that the four different furanocoumarin substrates could be catalyzed by AgOMT1 to generate the corresponding O-methylated products, the catalytic reaction rate of bergamotol was much lower than that of other substrates.

[0122] AgOMT2 specifically catalyzes this reaction, does not accept other similar substrates, and has better catalytic performance than AgOMT1. In general, the activities of AgOMT1 and AgOMT2 complement each other and together constitute an efficient biocatalyst for furanocoumarins in celery.

[0123] Example 5. Substrate properties of AgOMT1 and AgOMT2

[0124] 1. To explore the substrate characteristics of AgOMT1 and AgOMT2, we used a variety of phenolic compounds for testing. We first selected coumarin and its analogs as substrates and tested whether new products were generated under ultraviolet conditions. Figure 7 .

[0125] Depend on Figure 7 It can be seen that AgOMT1 can accept DMS as a substrate and generate new products, but the conversion efficiency is low. When using cypermethrin, umbelliferone and p-hydroxycinnamic acid as substrates, no obvious new peaks were observed in the reaction group under UV detection compared with the control group. AgOMT2 does not accept the above four types of substrates, and no product peaks were observed in the reaction group.

[0126] AgOMT1 has a certain catalytic activity for DMS, but does not show obvious substrate acceptance for cypermethrin, umbelliferone and p-hydroxycinnamic acid. AgOMT2 has no obvious activity for the above substrates.

[0127] 2. Based on the clustering of AgOMT1 in the phylogenetic tree, we selected a variety of flavonoids and N-acetyl-5-hydroxytryptamine as substrates for testing, see Figure 8 .

[0128] Depend on Figure 8 It can be seen that AgOMT1 has good catalytic activity for flavonoid compounds kaempferol and quercetin. Compared with the enzyme-free group, new product peaks were detected in both reaction groups under UV conditions. However, for the dihydroflavonoid compounds naringenin and N-acetyl-5-hydroxytryptamine, AgOMT1 had no catalytic effect, and there was no difference in the test results between the reaction group and the enzyme-free group. AgOMT1 has a catalytic effect on kaempferol and quercetin, but has no activity on the dihydroflavonoids naringenin and N-acetyl-5-hydroxytryptamine.

[0129] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0130] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0131] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0132] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An oxygen methyltransferase, characterized in that The oxygen methyltransferase comprises one of the following: Oxygen methyltransferase AgOMT1, the amino acid sequence of which is shown in SEQ ID NO.1; Oxygen methyltransferase AgOMT2, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. An oxygen methyltransferase according to claim 1, characterized in that The oxygen methyltransferase also includes one of the following: 1) An amino acid sequence having the same function as the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 obtained by replacing and / or deleting and / or adding one or more amino acid residues; 2) having a fusion amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.1 or SEQ ID NO.2; 3) An amino acid sequence having 95% or more homology with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 and having the same function.

3. Use of the oxygen methyltransferase according to any one of claims 1 to 2 in biocatalysis, characterized in that: The application includes: the oxygen methyltransferase AgOMT1 or AgOMT2 biocatalyzes bergamotol to generate bergamotinoside lactone.

4. The use according to claim 3, characterized in that: The application also includes: the oxygen methyltransferase AgOMT1 catalyzes Zanthoxylum toxicans to generate Zanthoxylum toxin, catalyzes 5-hydroxy Zanthoxylum toxin to generate bergamotolide, and catalyzes 8-bergamotolide to generate isoanisole lactone.

5. A recombinant expression vector, characterized in that: The recombinant expression vector is capable of expressing the oxygen methyltransferase according to claim 1.

6. The recombinant expression vector according to claim 5, characterized in that The nucleic acid molecule encoding the oxygen methyltransferase in the recombinant expression vector includes one of the following: 1) having a deoxyribonucleotide sequence as shown in SEQ ID NO.3 or SEQ ID NO.4; 2) having a deoxyribonucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2; 3) a deoxyribonucleotide sequence encoding a gene having oxygen methyltransferase activity obtained by substituting and / or deleting and / or adding one or more nucleotides to the deoxyribonucleic acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4; 4) A deoxyribonucleotide sequence having a homology of 95% or more with the deoxyribonucleic acid sequence defined in SEQ ID NO.3 or SEQ ID NO.4 and encoding an oxygen methyltransferase.

7. A recombinant bacterium or engineered host cell line comprising the recombinant expression vector according to claim 5 or 6, characterized in that: The recombinant expression vector includes one or more of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector; the host cell includes one of an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell, or a mammalian cell.

8. Use of the recombinant expression vector according to claim 5 or 6 or the recombinant bacteria or engineered cell line according to claim 7 in biocatalysis.

9. A method for biosynthesis of an O-methylated product, characterized in that: The O-methylated product includes one of Zanthoxylum bungeanum toxin, bergamotolactone, and isoanisole lactone, and the method includes one of the following: Using bergamotol as a substrate, the oxygen methyltransferase AgOMT1 or AgOMT2 or its enzyme lysate as claimed in claim 1 is used to catalyze the synthesis of bergamotol lactone; Using Zanthoxylum bungeanum as a substrate, the oxygen methyltransferase AgOMT1 or its enzyme lysate described in claim 1 is used to catalyze the synthesis of Zanthoxylum bungeanum toxin; Using 5-hydroxy Zanthoxylum bungeanum toxin as a substrate, the oxygen methyltransferase AgOMT1 or its enzyme lysate described in claim 1 is used to catalyze the synthesis of bergamotinoside lactone; Taking 8-bergamot lactone as a substrate, the oxygen methyltransferase AgOMT1 or its enzyme lysate described in claim 1 is used to catalyze the synthesis of isoanisole lactone.

10. A fermentation production method for an O-methylated product, characterized in that: The O-methylated product includes one of Zanthoxylum bungeanum toxin, bergamot glycoside lactone, and isoanisole lactone. The method includes the following steps: fermenting and culturing the recombinant bacteria or engineered host cell line according to claim 7, adding a substrate, and obtaining the compound after fermentation.

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