CN116426495A - Flavonoid-O-methyltransferase and its Application in the Synthesis of Iswogonin and Scutellaria flavonoids - Google Patents

Abstract

The invention provides a flavone-O-methyltransferase which comprises one or more of a flavone-8-methyltransferase with an amino acid sequence shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5 and a flavone-7-methyltransferase with an amino acid sequence shown as SEQ ID No.6 and SEQ ID No.7, and provides a nucleotide sequence for encoding the amino acid sequence, a recombinant vector containing a polynucleotide sequence for encoding the amino acid, a recombinant host cell and application thereof. The flavone-O-methyltransferase disclosed by the invention can be used for synthesizing flavone such as wogonin, iso-wogonin, mosoroflavone and the like, can catalyze the synthesis and methylation reaction of flavonoid substances with similar structures, and lays a foundation for producing methylated flavonoid compounds by an industrial large-scale fermentation production or enzyme conversion method.

Description

Flavone-O-methyltransferase and Its Function in the Synthesis of Iswogonin and Scutellaria flavonoids Applications

This application is a Chinese patent application 202010117745.1 submitted on February 25, 2020. The title of the invention is "Flavone-O-Methyltransferase and Its Application in the Synthesis of Wogonin, Iswogonin, and Saccharin" divisional application.

technical field

The present invention relates to the field of biotechnology, specifically, the present invention relates to five flavone-8-methyltransferases, two flavone-7-methyltransferases and their functions in flavones such as wogonin, isowogonin and scutellaria The application in the synthesis of flavones and the like also relates to a method for using these enzymes to biosynthesize methylated flavones.

Background technique

Wogonin (5,7-dihydroxy-8-methoxyflavone), isowogonin (5,8-dihydroxy-7-methoxyflavone), scutellaria flavone (5-hydroxy-7, 8-dimethoxyflavone) (the structures of the three flavones are shown below) are the active ingredients of traditional Chinese herbal medicines Scutellaria baicalensis and Scutellaria barbata, which have strong inhibitory effects on key targets of cancer. The methylation at the 8-position and 7-position is the main structural feature of the above compounds. In the biosynthesis process of plant natural products, the process of methylation is catalyzed by methyltransferase (methyltransferase) dependent on S-adenosyl-L-methionine, which has a strong Substrate and catalytic site specificity.

In 2013, Anna Berim and David R. Gang discovered two enzymes that can catalyze the 8-position methylation of flavonoids in Ocimum basilicum L., namely ObF8OMT-1 and ObPFOMT-1, both of which can Using pilosin as a substrate to synthesize kerophyllin, and realized the functional expression of both in Saccharomyces cerevisiae in 2018. Wogonin in Scutellaria baicalensis is synthesized by 8-hydroxylation of chrysin followed by 8-methylation, and the 8-methyltransferase in Scutellaria baicalensis was identified in 2019.

Methylated flavonoids such as wogonin, isowogonin, and scutellaria flavonoids have been extracted from Scutellaria barbata, a plant of the Labiatae family, but there is no 8-position methylated flavonoid from Scutellaria barbata. Therefore, there is an urgent need to screen the corresponding F8OMT and F7OMT and develop a biological method to synthesize wogonin, isowogonin and Su The method of the flavonoids of chrysanthemum.

Contents of the invention

In order to solve the problems in the prior art, the present invention provides a flavone-O-methyltransferase, which comprises: flavone-O-methyltransferase having the amino acid sequence shown in SEQ ID NO.1, 2, 3, 4 or 5 8-methyltransferase, and/or flavone-7-methyltransferase having the amino acid sequence shown in SEQ ID NO.6 or 7.

The present invention also provides a polynucleotide encoding the flavone-O-methyltransferase, the nucleotide sequence of which is shown in SEQ ID NO.8, 9, 10, 11, 12, 13 or 14. Wherein the nucleotide sequences of SEQ ID NO.8, 9, 10, 11 and 12 respectively encode flavone-8-methyltransferases having amino acid sequences as shown in SEQ ID NO.1, 2, 3, 4 or 5, and the nuclear The nucleotide sequences of SEQ ID NO.13 and 14 encode flavone-7-methyltransferases having the amino acid sequence shown in SEQ ID NO.6 or 7, respectively.

The present invention also provides a recombinant vector comprising the above-mentioned polynucleotide encoding the flavone-O-methyltransferase.

According to the present invention, the recombinant vector includes bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, animal cell virus, retrovirus or other vectors well known in the art. Vectors suitable for the present invention include, but are not limited to: shuttle plasmid vectors; T7 promoter-based expression vectors expressed in bacteria, such as pET-28a; vectors expressed in yeast, such as YEp series vectors; The broken MSXND expression vector expressed in cells, etc. Any vector can be used to construct a recombinant expression vector as long as it can replicate and exist stably in the host cell. Plasmid vectors are preferred, such as shuttle plasmid vectors.

The present invention also provides a recombinant host cell, which comprises one, two or more recombinant vectors comprising the above-mentioned polynucleotide encoding the flavone-O-methyltransferase.

According to the present invention, the host cells may be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferable examples include Escherichia coli, yeast and the like.

According to an embodiment of the present invention, the recombinant host cell is transformed into one, two or more recombinant vectors comprising polynucleotides encoding flavone-8-methyltransferase, and/or one, Two or more recombinant vectors comprising polynucleotides encoding flavone-7-methyltransferase.

According to the embodiment of the present invention, the recombinant host cell can also express other required enzymes, such as flavone hydroxylase (such as flavone 8-hydroxylase (F8H)), P450 reductase (CPR) and the like.

The present invention also provides the application of the flavone-O-methyltransferase, which is used in the synthesis of 8-O-methylflavone, 7-O-methylflavone or 7,8-dimethoxyflavone compound . Preferably, it can be used in the synthesis of wogonin, isowogonin or scutellarin.

The present invention also provides a method for synthesizing 8-O-methylflavone, which comprises the following steps: using a flavone-8-methyltransferase having an amino acid sequence as shown in SEQID NO.1, 2, 3, 4 or 5 Catalyzed steps.

Preferably, the present invention provides a method for synthesizing wogonin, which comprises the following steps: using a flavone-8-methyltransferase having an amino acid sequence as shown in SEQID NO.1, 2, 3, 4 or 5 to catalyze the removal of The step of wogonin; or include the following steps: using flavone-8-methyltransferase and flavone-8-hydroxylase catalyzed by the amino acid sequence shown in SEQ ID NO.1, 2, 3, 4 or 5 Chrysin steps.

According to an embodiment of the present invention, the flavone-8-methyltransferase and the flavone-8-hydroxylase can be in the form of the enzyme itself, or can be transformed into a polynucleotide containing the enzyme encoding the enzyme A recombinant host cell form of a recombinant vector.

According to the method of synthetic 8-O-methylflavone of the present invention, it may further comprise the steps:

Step (1): constructing a recombinant vector comprising a polynucleotide encoding a flavone-8-methyltransferase having an amino acid sequence as shown in SEQ ID NO.1, 2, 3, 4 or 5;

Step (2): transferring one, two or more recombinant vectors obtained in step (1) into host cells;

Step (3): fermenting the recombinant host cell obtained in step (2) together with the substrate to produce 8-O-methylflavone.

Preferably, the 8-O-methyl flavone is wogonin or scutellaria flavone.

Preferably, the substrate is norwogonin or chrysin.

According to an embodiment of the present invention, the recombinant host cell can also express other required enzymes, such as flavone-7-O-methyltransferase, flavone hydroxylase (such as flavone 8-hydroxylase (F8H)), P450 Reductase (CPR), etc.

The present invention also provides a method for synthesizing 7-O-methylflavone, which comprises the following steps: using a flavone-7-methyltransferase having the amino acid sequence shown in SEQID NO.6 or 7 to catalyze.

Preferably, the present invention provides a method for synthesizing isowogonin, which comprises the following steps: using a flavone-7-methyltransferase having an amino acid sequence as shown in SEQ ID NO.6 or 7 to catalyze nor-wogonin or include the following steps: using flavone-8-hydroxylase and flavone-7-methyltransferase having the amino acid sequence shown in SEQ ID NO.6 or 7 to catalyze the step of chrysin.

Preferably, the present invention also provides a method for synthesizing the flavonoids of thalassicum flavonoids, which includes the following steps: using flavone-8-transmethylation with the amino acid sequence shown in SEQ ID NO.1, 2, 3, 4 or 5 Enzymes and flavone-7-methyltransferases with the amino acid sequence shown in SEQ ID NO.6 or 7 catalyze the step of demethyl wogonin; The flavone-8-methyltransferase with the amino acid sequence shown in ID NO.1, 2, 3, 4 or 5 and the flavone-7-methyltransferase with the amino acid sequence shown in SEQ ID NO.6 or 7 catalyze poplar prime steps.

According to the present invention, the flavone-7-methyltransferase and the flavone-8-hydroxylase may be in the form of the enzyme itself, or may be transformed into a recombinant vector comprising a polynucleotide encoding the enzyme. host cell form.

According to the method of synthetic 7-O-methylflavone of the present invention, it may further comprise the steps:

Step (1): constructing a recombinant vector comprising a polynucleotide encoding a flavone-7-methyltransferase having an amino acid sequence as shown in SEQ ID NO.6 or 7;

Step (2): transferring one, two or more recombinant vectors obtained in step (1) into host cells;

Step (3): fermenting the recombinant host cell obtained in step (2) together with the substrate to produce 7-O-methylflavone.

Preferably, the 7-O-methyl flavone is isowogonin or scutellaria flavone.

Preferably, the substrate is norwogonin or chrysin.

According to an embodiment of the present invention, the recombinant host cell can also express other required enzymes, such as flavone-8-O-methyltransferase, flavone hydroxylase (such as flavone 8-hydroxylase (F8H)), P450 Reductase (CPR), etc.

It should be understood that, within the scope of the present invention, as long as the conventional knowledge in the field is not violated, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other, thereby forming New or preferred technical solutions. Due to space limitations, we will not repeat them here.

Beneficial effect

Flavone-8-methyltransferase (F8OMT) and flavone-7-methyltransferase (F7OMT) of the present invention have good catalytic activity of synthesizing O-methylated flavones, and they can be used for flavones such as wogonin, The synthesis of isowogonin and scutellaria flavonoids, etc., can catalyze the synthesis and methylation of flavonoids with similar structures containing 7-O-methyl and/or 8-O-methyl substituents. Industrial large-scale fermentation production or enzymatic conversion methods have laid the foundation for the production of methylated flavonoids.

Description of drawings

Figure 1 is a schematic diagram of the plasmid map of the recombinant plasmid Y33-FOMT.

Fig. 2 is a schematic diagram of the plasmid map of the recombinant plasmid Y22-FOMT.

Fig. 3 is an evolutionary analysis diagram of flavone-O-methyltransferase of Scutellaria barbata.

Fig. 4 is an HPLC-MS analysis chart of flavone-O-methyltransferase derived from Scutellaria barbatae catalyzing the 8-position methylation of norwogonin to synthesize wogonin and the 7-position methylation to generate isowogonin.

Fig. 5 is an HPLC-MS analysis graph of SbarF7OMT derived from Scutellaria barbata and SbaiF7OMT derived from Scutellaria baicalensis catalyzing the 7-position methylation of norwogonin to generate isowogonin.

Fig. 6 is an HPLC analysis chart of the synthesis of wogonin and isowogonin by catalyzing dechrysin as a substrate by the Saccharomyces cerevisiae engineering strain.

Fig. 7 is the HPLC-MS analysis diagram of SbarF8OMT and SbarF7OMT catalyzed the 8-position and 7-position methylation reaction of norwogonin to synthesize the flavonoid of scutellarin.

Fig. 8 is the ¹ H and ¹³ C NMR spectra of isowogonin synthesized by biological method.

Fig. 9 is a schematic diagram of generating methylated flavonoids from norwogonin as a substrate under the action of flavone-O-methyltransferase.

Fig. 10 is a schematic diagram of generating methylated flavonoids from chrysin as a substrate under the action of flavone-O-methyltransferase.

Detailed ways

The specific implementation manners of the present invention will be further described below in conjunction with the drawings and examples. The following examples are only used to illustrate the technical solution of the present invention more clearly, but not to limit the protection scope of the present invention.

Unless otherwise specified, the gene sequences used in the present invention were synthesized by Nanjing GenScript Biotechnology Co., Ltd., and the materials used in the examples are all commercially available products. The experimental methods used in the following examples are conventional methods unless otherwise specified.

Example 1 Obtaining of Encoding Nucleotide and Construction of Vector

According to the nucleotide sequence information provided by the present invention (SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No .14), performing gene synthesis. The shuttle plasmid YCPlac33-PE was used as the vector, and the exogenous sequence was inserted between the restriction sites SalI and XbaI, and the recombinant vector was constructed by using the Gibson assembly method (Figure 1). In addition, for SEQ ID No. 14, the shuttle plasmid YCPlac22-PE was used as the carrier, and the foreign sequence was inserted between the restriction site SalI and XbaI, and the recombinant vector was constructed by using the Gibson assembly method (Figure 2).

Example 2 Construction of Wogonin-producing Saccharomyces cerevisiae Host Bacteria

2.1 Production of wogonin using nor-wogonin as substrate

Utilize conventional lithium acetate conversion method to comprise the recombinant vector of nucleotide sequence SEQ ID No.8 or SEQ ID No.9 or SEQ ID No.10 or SEQ ID No.11 or SEQ ID No.12 described in embodiment 1 Transform into Saccharomyces cerevisiae host strain W303-1B, pick the clones that can grow on uracil-deficient complete (CM) medium, and use 1 mM norwogonin as the substrate to ferment and produce wogonin.

2.2 Production of wogonin with chrysin as substrate

The recombinant vector comprising the nucleotide sequence SEQ ID No.12 described in Example 1 was transformed into a Saccharomyces cerevisiae host containing flavone-8-hydroxylase (F8H) and P450 reductase (CPR) using a conventional lithium acetate transformation method Bacteria, pick the clones that can grow on tryptophan and uracil-deficient CM medium, and use 1 mM chrysin as the substrate to ferment and produce wogonin.

Example 3 Construction of Saccharomyces cerevisiae host bacteria producing iso-wogonin

3.1 Production of iso-wogonin using nor-wogonin as substrate

The recombinant vector comprising nucleotide sequence SEQ ID No.13 or SEQ ID No.14 described in Example 1 is transformed into Saccharomyces cerevisiae host bacterium W303-1B by conventional lithium acetate transformation method. The clones grown on the deficient CM medium were fermented with norwogonin at a concentration of 1 mM to produce isowogonin.

3.2 Production of isowogonin with chrysin as substrate

The recombinant vector comprising the nucleotide sequence SEQ ID No.14 described in Example 1 was transformed into a Saccharomyces cerevisiae host containing flavone-8-hydroxylase (F8H) and P450 reductase (CPR) using a conventional lithium acetate transformation method Bacteria, pick the clones that can grow on tryptophan and uracil-deficient CM medium, and use 1 mM chrysin as the substrate to ferment and produce wogonin.

Example 4 The construction of Saccharomyces cerevisiae host bacteria producing thalassemia flavonoids

The YCPlac33-PE recombinant vector comprising the nucleotide sequence SEQ ID No.12 described in Example 1 and the YCPlac22-PE recombinant vector comprising the nucleotide sequence SEQ ID No.14 were co-transformed into In the host strain of Saccharomyces cerevisiae W303-1B, the clones that can grow on the CM medium deficient in uracil and tryptophan were picked, and the 1mM norscutellarein was used as the substrate to ferment and produce the flavonoid of scutellaria.

Example 5 Fermentative production of wogonin, iso-wogonin, and scutellaria flavonoids

5.1 Saccharomyces cerevisiae engineered strain seed liquid culture

Select CM medium (purchased from Suo Laibao Biotechnology Co., Ltd.) (formula: YNB w/o AA (0.67%), glucose (2g/L), Dropout powder (0.083%), wherein the Dropout powder contains (mg/ L): threonine 150, tyrosine 30, valine 150, lysine 30, glutamic acid 100, serine 150, aspartic acid 100, methionine 20, phenylalanine 50, iso Leucine 30, arginine 20; other nutrients (mg/L): adenine 50, uracil 50, histidine 100, leucine 100, tryptophan 100 (amino acid)), liquid medium to adjust pH 5.6, the solid medium was added with 1.5% agar powder (purchased from Suleibao Biotechnology Co., Ltd.), and the pH was adjusted to 6.5. Single clones were picked from the plates and transferred to 24-well plates containing 4 mL of sterilized medium, and the Saccharomyces cerevisiae engineered strains were cultured overnight at 30°C and 200 rpm.

5.2 Fermentation production

The seed solution was inoculated into a sterilized 24-well plate at an inoculation ratio of 1:50, and the corresponding substrate was added with a final concentration of 1 mM, and fermented at 30°C and 800 rpm for 4 days.

The HPLC-MS identification of embodiment 6 reaction product

6.1 After the fermentation, take 900 μL sample, add an equal volume of anhydrous methanol, and use an ultrasonic cleaner to perform ultrasonication for 30 minutes. Centrifuge at 12000rpm for 10min, and the supernatant is analyzed by high performance liquid phase.

6.2 HPLC, LC-MS detection conditions

HPLC analysis:

Instrument: Shimadzu HPLC 1200

Chromatographic column: Kinetex H15-168747 (4.6×250mm), ultraviolet detector, detection wavelength 290nm.

Mobile phase: A phase is 0.1% formic acid; B phase is acetonitrile; C phase is methanol

Starting concentration: A: 73% B: 22% C: 5%.

Flow rate: 1mL/min

Column temperature: 30°C

Detector: PDA detector

Gradient elution program: (concentration is the percentage of phase B)

MS analysis:

Mass spectrometer: Bruker-micrOTOF-II:

ESI ion source, positive ion mode

Nucleoplasmic ratio (m/z): 50-1000

Nitrogen flow rate: 6.0 L/min

Temperature: 180°C

Atomizer pressure: 1bar

Probe voltage: 14.5KV.

6.3 Functional identification of flavonoid-O-methyltransferase derived from Scutellaria barbata

According to the analysis of Scutellaria barbata transcriptome sequencing results, 12 flavone-O-methyltransferase candidate sequences were obtained (Figure 3), and Saccharomyces cerevisiae strains expressing flavone-O-methyltransferase were fermented with norwogonin as substrate , the results of HPLC detection showed that SbarFOMT-1, SbarFOMT-6, SbarFOMT-7, SbarFOMT-11, and SbarFOMT-12 could catalyze nor-wogonin to generate new products, and the peak time was 28.20min, which was the same as that of standard wogonin Consistent with that (Figure 4A), mass spectrometry results showed (Figure 4B) that the molecular weight of the new product was m/z, 285[M+H] ⁺ , which was consistent with the molecular weight of the standard wogonin, and the substance was determined to be wogonin. Among them, SbarFOMT-12 had the best catalytic activity, and SbarFOMT-12 was named SbarF7OMT. SbarFOMT-4 generates a new substance with a peak time of 27.50min (Figure 4A). Mass spectrometry results show that the molecular weight of the new product is m/z, 285[M+H] ⁺ (Figure 4C). It is speculated that this substance is wogonin The isomers of the isomers, determined by nuclear magnetic structure analysis, are consistent with the chemically synthesized iso-wogonin structure reported in the previous patent CN104926768A, confirming that the newly synthesized compound is iso-wogonin (Fig. 8), the SbarFOMT -4 named SbarF7OMT.

6.4 Functional identification of flavone-O-methyltransferase derived from Scutellaria baicalensis

The SbarF7OMT homologous sequence Sbai154288 derived from Scutellaria baicalensis was screened, and the Saccharomyces cerevisiae strain expressing Sbai154288 was fermented with norwogonin as a substrate. The results of HPLC detection showed that Sbai154288 could generate a new product from norwogonin, and the peak time was 27.50min is consistent with the standard product of wogonin (Figure 5A), mass spectrometry results show (Figure 5B) that the molecular weight of the new product is (m/z, 285[M+H]+), which is consistent with the molecular weight of the standard product of wogonin Consistent, determine that the substance is wogonin. Designated Sbai154288 as SbaiF7OMT.

6.5 Production of wogonin and isowogonin using chrysin as substrate

With 1mM chrysin as substrate, the fermentation strains W303-F8H-SbarF7OMT and W303-F8H-SbaiF7OMT were detected by HPLC. The results showed that both of them could catalyze chrysin to generate isowogonin at 27.50 min (Fig. 6A). The strain W303-F8H-SbarF8OMT was fermented with 1 mM chrysin as the substrate, and the product was detected by HPLC. Since the peak elution time of the substrate chrysin was not much different from that of wogonin, the mass spectrometry was used to extract the molecular weight [M+H] ⁺ 285 to analyze the results. The mass spectrum was extracted, and it was found that the strain W303-F8H-SbarF8OMT could catalyze chrysin to generate new products, and the peak time was consistent with that of the standard wogonin (Figure 6B), and the molecular weight was consistent (Figure 6C, Figure 6D), and the new product was determined For wogonin.

6.6 Using norscutellarein as the substrate to produce the flavonoids

Using 1mM norwogonin as a substrate, the strain W303-SbarF8OMT&SbarF7OMT was fermented, and the product was detected by HPLC. The results showed that chrysin was catalyzed to generate a new product at 33.55 min, which was consistent with the peak time of the standard product of chrysanthemum flavone (Fig. 7A). The results of mass spectrometry showed that the molecular weight of the new product was (m/z, 299[M+H] ⁺ ) (Fig. 7B), which was consistent with the molecular weight of the standard product of thalassicum flavonoids, and it was determined that the substance was thalassemia flavonoids.

Embodiment 7 NMR (nuclear magnetic resonance) identification fermentation product chemical structure

7.1 Inoculate the glycerol bacteria into a glass test tube containing 4 mL of sterilized uracil-deficient CM medium, and cultivate the engineering strain of Saccharomyces cerevisiae overnight at 30°C and 200 rpm. The seed solution was inoculated into sterilized 2L uracil-deficient CM medium at an inoculation ratio of 1:50, added with norwogonin at a final concentration of 0.5 mM, and fermented at 30°C and 200 rpm for 4 days. After the fermentation, centrifuge at 5000rpm for 10min, and extract the supernatant with an equal volume of ethyl acetate; extract the cell pellet with 100mL of methanol, and sonicate for 30min, spin-evaporate the methanol, and extract the remaining water with an equal volume of ethyl acetate; combine All the ethyl acetate was evaporated to dryness with a rotary evaporator; the extraction was repeated twice; the inner wall of the rotary evaporator was washed with 20 mL of methanol, centrifuged, and the concentrated product was separated and purified using a preparative liquid phase.

7.2 Preparative liquid phase separation conditions

HPLC analysis:

Instrument: Agilent1260 liquid chromatograph

Chromatographic column: Ultimate XB-C18 (21.2×250mm), ultraviolet detector, detection wavelength 290nm.

Mobile phase: phase A is ultrapure water; phase B is methanol

Starting concentration: A: 40% B: 60%

Flow rate: 10mL/min

Column temperature: 30°C

Detector: MWD detector

Gradient elution program: (concentration is the percentage of phase B)

7.3 Separation and purification of isowogonin

The fermentation product concentrated in Example 7.1 was separated by the preparative liquid phase, and the injection volume was 900 μL/time. The test results of the preparative liquid phase analysis showed that the peak eluting time of norwogonin was 23-26 minutes, and that of isowogonin The peak time is 36-38 minutes, open the collector to collect the samples of 36-38 minutes, repeat 20 times. The obtained liquid was concentrated and evaporated to dryness, and 500 uL deuterated DMSO was added to dissolve it, and 600 MHZ NMR detection was performed.

7.4 NMR (Nuclear Magnetic Resonance) Identification of the Chemical Structure of the New Fermentation Product Iswogonin

1H and 13C NMR spectra were acquired on a Bruker Avance III spectrometer at 600 MHz in DMSO-d6. Chemical shifts (δ) are expressed in ppm and J represents the coupling constant. The NMR results of isolated and purified isowogonin are shown in Figure 81H-NMR (600MHz, DMSO-d6): δ12.34 (1H, s), 8.14 (2H, d), 7.60 (3H, m), 6.97(1H,s),6.58(1H,s),3.91(3H,s);13C-NMR(600MHz,DMSO-d6):δ182.57,163.39,153.09,154.54,144.55,132.11,130.87,129.12,126.58, 126.36, 104.64, 104.078, 35.84, 56.39. It is consistent with the structure identification results of chemical synthesis reported previously.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (12)

1. A flavone-O-methyltransferase has the amino acid sequence shown in SEQ ID No.6 or 7.

2. A polynucleotide encoding the flavone-O-methyltransferase of claim 1, having the nucleotide sequence set forth in SEQ ID No.13 or 14.

3. A recombinant vector comprising the polynucleotide encoding a flavone-O-methyltransferase of claim 2.

4. A recombinant host cell comprising one, two or more recombinant vectors of claim 3;

preferably, the recombinant host cell further comprises one, two or more recombinant vectors encoding polynucleotides of the flavone-8-methyltransferase of the amino acid sequence shown as SEQ ID NO.1, 2, 3, 4 or 5.

5. The recombinant host cell of claim 4 wherein;

the recombinant host cell may also express other desired enzymes;

preferably, the recombinant host cell may also express Huang Tongqiang synthase and/or P450 reductase;

more preferably, the recombinant host cell may also express a flavone 8-hydroxylase (F8H) and/or a P450 reductase CPR.

6. Use of a flavone-O-methyltransferase according to claim 1 in the synthesis of 7-O-methylflavone or 7, 8-dimethoxy flavone compounds;

preferably, the 7-O-methyl flavone or 7, 8-dimethoxy flavone compound is iso-wogonin or threshepherd's-purse flavone.

7. A method for synthesizing 7-O-methyl flavone, comprising the steps of: a step of catalyzing with flavone-7-methyltransferase with the amino acid sequence shown as SEQ ID NO.6 or 7;

preferably, the flavone-7-methyltransferase is in the form of an enzyme per se or in the form of a recombinant host cell according to claim 4 or 5;

more preferably, the 7-O-methyl flavone is isowogonin or threo-limonin.

8. The method of claim 7, comprising the steps of:

step (1): constructing a recombinant vector comprising a polynucleotide encoding a flavone-7-methyltransferase having an amino acid sequence as set forth in SEQ ID No.6 or 7;

step (2): transferring one, two or more of the recombinant vectors obtained in step (1) into a host cell;

step (3): fermenting the recombinant host cell obtained in step (2) with a substrate to produce 7-O-methylflavone;

preferably, the substrate is norwogonin or chrysin.

9. A method of synthesizing a 7, 8-dimethoxy flavone compound, comprising the steps of: a step of catalyzing with a flavone-7-methyltransferase having an amino acid sequence as shown in SEQ ID No.6 or 7, and a step of catalyzing with a flavone-8-methyltransferase having an amino acid sequence as shown in SEQ ID No.1, 2, 3, 4 or 5;

preferably, the flavone-7-methyltransferase, flavone-8-methyltransferase is in the form of an enzyme per se or in the form of a recombinant host cell according to claim 4 or 5;

more preferably, the 7, 8-dimethoxy flavone compound is threo-limonin.

10. The method of claim 9, comprising the steps of:

step (1): constructing a recombinant vector comprising a polynucleotide encoding a flavone-7-methyltransferase having an amino acid sequence as set forth in SEQ ID No.6 or 7 and a polynucleotide encoding a flavone-8-methyltransferase having an amino acid sequence as set forth in SEQ ID No.1, 2, 3, 4 or 5;

step (2): transferring one, two or more of the recombinant vectors obtained in step (1) into a host cell;

step (3): fermenting the recombinant host cell obtained in step (2) with a substrate to produce 7, 8-dimethoxy flavone;

preferably, the substrate is norwogonin or chrysin.

11. A method for synthesizing isowogonin, comprising the steps of:

catalyzing the norwogonin by adopting flavone-7-methyltransferase with an amino acid sequence shown as SEQ ID No.6 or 7;

or comprises the following steps: catalyzing chrysin by using flavone-8-hydroxylase and flavone-7-methyltransferase with an amino acid sequence shown as SEQ ID NO.6 or 7;

preferably, the flavone-7-methyltransferase is in the form of an enzyme per se or in the form of a recombinant host cell according to claim 4 or 5;

more preferably, the flavone-8-hydroxylase is in the form of the enzyme itself or in the form of a host cell comprising a recombinant vector encoding a polynucleotide of the flavone-8-hydroxylase.

12. A method for synthesizing mosa flavone, which comprises the following steps:

a step of catalyzing norwogonin by using a flavone-8-methyltransferase having an amino acid sequence as shown in SEQ ID No.1, 2, 3, 4 or 5 and a flavone-7-methyltransferase having an amino acid sequence as shown in SEQ ID No.6 or 7;

or comprises the following steps: a step of catalyzing chrysin by using flavone-8-hydroxylase, flavone-8-methyltransferase with an amino acid sequence shown as SEQ ID NO.1, 2, 3, 4 or 5 and flavone-7-methyltransferase with an amino acid sequence shown as SEQ ID NO.6 or 7;

preferably, the flavone-8-methyltransferase, flavone-7-methyltransferase is in the form of an enzyme per se or in the form of a recombinant host cell according to claim 4 or 5;

more preferably, the flavone-8-hydroxylase is in the form of the enzyme itself or in the form of a host cell comprising a recombinant vector encoding a polynucleotide of the flavone-8-hydroxylase.

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