Dracaena cambogia flavone compound o-methyltransferase gene and application

Abstract

The application belongs to the technical field of genetic engineering and enzyme engineering, and discloses a dragon blood tree flavone compound O-methyltransferase gene and application thereof. Three functionally differentiated O-methyltransferase genes DcOMT1, DcOMT2 and DcOMT3 are cloned from dragon blood tree, and the coded proteins can catalyze O-methylation reaction of flavonoids. The three enzymes show different catalytic modes for dracorhodin D: DcOMT1 preferentially catalyzes 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone to generate dracorhodin B and further catalyzes dracorhodin B; DcOMT2 catalyzes dracorhodin B; and DcOMT3 catalyzes 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone. The functions of the three enzymes are complementary, and can be used alone or in combination to realize precise synthesis of multiple different methylated flavones from a single substrate.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to the O-methyltransferase (OMT) gene of flavonoid compounds isolated and identified from dragon blood tree, the protein encoded by it, and the application of these proteins in catalyzing the O-methylation reaction of flavonoid compounds. Background Technology

[0002] Methylation (O-methylation) of flavonoids is a key pathway in nature that enriches their structural diversity and biological activity. Methylated flavonoids typically exhibit greater lipid solubility, better membrane permeability, higher metabolic stability, and unique pharmacological activities (i.e., the "magic methyl effect"). For example, 3'-O-methylcatechin and 5'-O-methylquercetin have shown superior biological activity compared to their parent nuclei in studies. O-methyltransferases are the core catalysts in this biosynthetic step.

[0003] Currently, although various plant methylated flavonoid metabolites (OMTs) have been reported, they generally suffer from limited catalytic function. For example, most identified OMTs exhibit high catalytic activity only for one or a class of specific substrates (such as hydroxyl groups at specific positions on the A or B rings of flavonoids), or show poor regioselectivity, and lack the ability to perform stepwise or selective methylation of different phenolic hydroxyl sites on the same substrate. This limited diversity severely restricts the efficiency of producing multiple high-value methylated flavonoid products or mixtures using a single enzyme catalyst. Therefore, identifying OMT gene combinations with different regioselectivities, different methylation levels (monomethylation / dimethylation) regulatory capabilities, and complementary functions is crucial for constructing an efficient toolkit or microbial cell factory for the biosynthesis of methylated flavonoids.

[0004] The red resin formed when a dragon's blood tree is injured is the main source of dragon's blood resin. Dragon's blood resin is known as a "holy medicine for promoting blood circulation," possessing the effects of promoting blood circulation, removing blood stasis, relieving pain, stopping bleeding, and promoting tissue regeneration. Flavonoids are the core active substances carrying these effects. Statistics show that of the 170 flavonoids isolated and identified from dragon's blood trees, 78.2% were O-methylated, suggesting that it may contain abundant and functionally differentiated OMT gene resources. However, there are currently no publicly available reports on the systematic isolation, identification, and combined application of multiple functionally complementary flavonoid-specific OMTs capable of site-specific and degree-differentiated targeting of the phenolic hydroxyl groups on the B-ring of flavonoids from a single plant source. Summary of the Invention

[0005] This invention aims to overcome the limitations of single OMT functions in existing technologies by providing a set (three types) of O-methyltransferase genes derived from the same *Dracaena cochinchinensis* species. These genes possess novel sequences and exhibit efficient and complementary regioselectivity for different flavonoid substrates or different B-ring phenolic hydroxyl sites on the same substrate, as well as differential regulatory capabilities on the degree of methylation (monomethylation / dimethylation). The invention also includes the proteins encoded by these genes and the applications of this protein combination. This combination enables the efficient and specific synthesis of various methylated flavonoids, particularly achieving precise, stepwise, or complete methylation modification of the phenolic hydroxyl groups at the 2'- and / or 4'- positions of the flavonoid B-ring.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the application of an O-methyltransferase in catalyzing the O-methylation reaction of flavonoids, wherein the O-methyltransferase is selected from proteins having the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.6;

[0008] When the flavonoid compound is dracoside D:

[0009] (1) The protein shown in SEQ ID NO.2 catalyzes the formation of 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone from dracoside D, and further catalyzes the formation of dracoside B;

[0010] (2) The protein shown in SEQ ID NO.4 catalyzes the formation of dracospermoid D from dracospermoid B;

[0011] (3) The protein shown in SEQ ID NO.6 catalyzes the formation of 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone from dragon's blood D.

[0012] Further, the protein shown in SEQ ID NO.2 is encoded by a gene having the nucleotide sequence shown in SEQ ID NO.1; the protein shown in SEQ ID NO.4 is encoded by a gene having the nucleotide sequence shown in SEQ ID NO.3; and the protein shown in SEQ ID NO.6 is encoded by a gene having the nucleotide sequence shown in SEQ ID NO.5.

[0013] In a second aspect, the present invention also provides a recombinant expression vector comprising the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.5.

[0014] Thirdly, the present invention also provides a host cell comprising the above-mentioned recombinant expression vector.

[0015] Fourthly, the present invention also provides a flavonoid methylation reaction kit, comprising the above-mentioned O-methyltransferase, reaction buffer and methyl donor S-adenosylmethionine.

[0016] Fifthly, the present invention also provides a method for in vitro catalytic O-methylation of flavonoid compounds, comprising the following steps:

[0017] (a) Provide the above-mentioned O-methyltransferase or kit;

[0018] (b) The O-methyltransferase is mixed with a flavonoid substrate and a methyl donor, S-adenosylmethionine, to carry out an enzymatic reaction;

[0019] (c) Collect the reaction products to obtain methylated flavonoid compounds.

[0020] Furthermore, when the flavonoid substrate is dracosanol D, the proteins shown in SEQ ID NO.2 and SEQ ID NO.6 are added stepwise for combined catalysis, thereby improving the synthesis efficiency of dracosanol B; the combined catalysis specifically involves:

[0021] The first step involves adding the protein shown in SEQ ID NO.6 to catalyze the formation of 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone from dragon's blood D.

[0022] The second step involves adding the protein shown in SEQ ID NO.2 to catalyze the formation of dragon's blood B from the 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone.

[0023] Furthermore, when the protein shown in SEQ ID NO.2 is used for catalysis, the flavonoid substrate is dracoside D, isoliquiritin, or naringenin.

[0024] The beneficial effects of this invention are:

[0025] (1) The three O-methyltransferases provided by this invention exhibit clear functional differentiation, especially showing fine differential regulation of the methylation site and degree of phenolic hydroxyl groups on the B ring of flavonoids. They can be used as modular tools to flexibly select or combine according to the target product, so as to achieve precise synthesis from a single substrate to a variety of different methylation products.

[0026] (2) By using the three O-methyltransferases provided by the present invention alone or in combination, the efficiency and flexibility of synthesizing different methylated flavonoids can be greatly improved, which greatly enhances the flexibility and efficiency of enzymatic synthesis of dragon's blood active ingredients.

[0027] (3) The three OMT genes (DcOMT1, DcOMT2, DcOMT3) provided by this invention can all express soluble active proteins efficiently in conventional Escherichia coli expression systems, which is convenient for large-scale preparation and application.

[0028] (4) The DcOMT1 provided by the present invention not only has the ability to stepwise methylate D of dragon's blood, but also can efficiently catalyze the 4'-position methylation of the B ring of various flavonoids such as isoliquiritin and naringenin, showing good substrate broad spectrum and site specificity, and can be extended to the enzymatic modification of active ingredients from licorice and citrus. Attached Figure Description

[0029] Figure 1 This is an electrophoresis image of PCR amplification of three Dragon's Blood OMT genes (DcOMT1 / 2 / 3) in Example 1 of the present invention; where M: DNA Marker; 1 / 2 / 3: PCR amplification bands of DcOMT1, DcOMT2, and DcOMT3 (approximately 1000-1100bp).

[0030] Figure 2 This is an SDS-PAGE analysis of the crude enzyme extraction supernatant and precipitate of recombinant DcOMT1, DcOMT2, and DcOMT3 proteins in Example 2 of the present invention. Lanes 1-3: crude enzyme supernatant of DcOMT1, DcOMT2, and DcOMT3, respectively; Lanes 4-6: crude enzyme precipitate of DcOMT1, DcOMT2, and DcOMT3, respectively.

[0031] Figure 3 This is an SDS-PAGE analysis image of the recombinant DcOMT1, DcOMT2, and DcOMT3 proteins purified by Ni-NTA affinity in Example 2 of this invention. Lane M: Protein molecular weight standard (Protein Marker); Lanes 1-3: Purified proteins of DcOMT1, DcOMT2, and DcOMT3, respectively (target band size approximately 40-41 kDa).

[0032] Figure 4 This is an SDS-PAGE analysis of recombinant DcOMT1 and DcOMT2 proteins after imidazole gradient elution and concentration in Example 2 of this invention. Lanes 1, 3, and 5: gradient elution fractions of DcOMT1 at 20 mM, 40 mM, and 250 mM imidazole concentrations, respectively; Lane 7: target protein band after DcOMT1 concentration; Lanes 2, 4, and 6: gradient elution fractions of DcOMT2 at 20 mM, 40 mM, and 250 mM imidazole concentrations, respectively; Lane 8: target protein band after DcOMT2 concentration.

[0033] Figure 5This is an SDS-PAGE analysis image of the recombinant DcOMT3 protein after imidazole gradient elution and concentration in Example 2 of the present invention. Lanes 1-3: gradient elution fractions of DcOMT3 at imidazole concentrations of 20 mM, 40 mM, and 250 mM, respectively; Lane 4: target protein band after DcOMT3 concentration.

[0034] Figure 6 This is the LC-MS total ion chromatogram of the products from the reaction of DcOMT1, DcOMT2, and DcOMT3 catalyzing dracosanol D in Example 3 of the present invention. Wherein, 1: DcOMT1 / 2 / 3 purified protein was added to the reaction substrate dracosanol D; 2: DcOMT1 / 2 / 3 purified protein was added to the reaction product 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone; 3: DcOMT1 / 2 / 3 purified protein was added to the reaction product dracosanol B.

[0035] Figure 7 The image shows the ESI-TOF-MS mass spectrum (negative ion mode detection) of the products generated from dragon's blood D catalyzed by DcOMT1 in Example 3 of this invention, displaying the [MH] corresponding to each product. - Quasi-molecular ion peak.

[0036] Figure 8 The image shows the ESI-TOF-MS mass spectrum (negative ion mode detection) of the products generated from dragon's blood D catalyzed by DcOMT2 in Example 3 of this invention, displaying the [MH] corresponding to each product. - Quasi-molecular ion peak.

[0037] Figure 9 The image shows the ESI-TOF-MS mass spectrum (negative ion mode detection) of the products generated from dragon's blood D catalyzed by DcOMT3 in Example 3 of this invention, displaying the [MH] corresponding to each product. - Quasi-molecular ion peak.

[0038] Figure 10 This is a quantitative comparison chart of the peak areas of the products obtained after DcOMT1, DcOMT2, and DcOMT3 catalyze the reaction of dragon's blood D in Example 3 of the present invention.

[0039] Figure 11 The LC-MS product spectra of DcOMT1 catalyzing the reactions of isoliquiritigenin and naringenin, respectively.

[0040] Figure 12 The synthetic route for the synthesis of dracospermoid D to dracospermoid B by the combined catalysis of DcOMT1 and DcOMT3 is described. Detailed Implementation

[0041] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0042] Example 1: Cloning of OMT genes from three types of dragon blood trees

[0043] Three highly expressed OMT candidate genes with significant sequence differences were screened from the *Dracaena cochinchinensis* transcriptome data. Specific primers were designed for RT-PCR amplification, and after cloning and sequencing, three full-length coding sequences were obtained, named DcOMT1 (SEQ ID NO.1, encoding the protein shown in SEQ ID NO.2), DcOMT2 (SEQ ID NO.3, encoding the protein shown in SEQ ID NO.4), and DcOMT3 (SEQ ID NO.5, encoding the protein shown in SEQ ID NO.6). Sequence alignment showed that the amino acid sequence similarity among the three was approximately 65-75%, belonging to the same family but with significant differentiation.

[0044] 1. Primer design

[0045] Based on the BamHI and XhoI restriction sites of the pET-28a(+) vector, specific primers with homologous arms were designed (Table 1) to amplify the CDS sequences of the DcOMT1 (SEQ ID NO.1), DcOMT2 (SEQ ID NO.3), and DcOMT3 (SEQ ID NO.5) genes, respectively.

[0046] Table 1

[0047]

[0048] 2. Total RNA extraction and cDNA synthesis from Dragon Blood Tree

[0049] Cut an appropriate amount of fresh dragon's blood leaf tissue and place it in a sterilized and pre-cooled mortar. Add liquid nitrogen and grind rapidly and thoroughly. Weigh approximately 100 mg of powder into a 1.5 mL RNase-free centrifuge tube, add 1 mL of Trizol reagent, vortex to mix, and incubate at room temperature for 5-10 min to allow for complete lysis of the nucleoproteins. Add 0.2 mL of chloroform, vortex for 30 s, incubate at room temperature for 5 min, and then centrifuge at 4 ℃ and 12,000 r / min for 11 min. Transfer the uppermost aqueous phase to a new RNase-free centrifuge tube, add an equal volume of isopropanol solution, vortex to mix, and incubate at room temperature for 15 min. Centrifuge again at 4 ℃ and 12,000 r / min for 11 min, and discard the supernatant. Wash the precipitate with 700 μL of 75% ethanol, centrifuge at 4 ℃ and 12,000 r / min for 3 min, discard the supernatant, and dry at room temperature for 5-10 min (do not heat to avoid excessive drying which would make the RNA difficult to dissolve). Add 30-50 μL of RNase-free ddH2O to fully dissolve the RNA, determine the concentration, and store at -80 ℃ or use for subsequent experiments.

[0050] The extracted RNA was reverse transcribed into cDNA using the Evo M-MLV Reverse Transcription Kit II (Accurate Biotechnology). The specific steps are as follows:

[0051] (1) Take out the extracted dragon blood tree RNA from the -80 ℃ freezer, thaw it on ice and then measure the RNA concentration.

[0052] (2) The amount of RNA used was calculated based on 1 μg of RNA. Genomic DNA contamination was removed using gDNA Clean Reagent. After mixing the reaction system, the mixture was briefly centrifuged and placed in a PCR instrument at 42 °C for 2 min. After removal, the mixture was immediately placed on ice.

[0053] (3) The above DNA-depleted reaction solution was further reverse transcribed into cDNA. The reverse transcription reaction system was prepared according to the kit instructions, mixed thoroughly, and briefly centrifuged before being placed in a PCR instrument. Reaction conditions: 37 ℃, 15 min; 85 ℃, 5 s. After the reaction, the mixture was stored at -20 ℃ for later use as a template for subsequent PCR amplification.

[0054] 3. Target gene amplification and recovery

[0055] Using cDNA as a template, the target gene fragment was obtained by PCR amplification using the specific primers designed above (Table 1). The PCR reaction system is shown in Table 2, and the PCR amplification procedure is shown in Table 3. The amplification products were verified by 1% agarose gel electrophoresis. Figure 1 The target fragment was recovered using a gel recovery kit, and after the concentration was determined, it was stored at -20 ℃ for later use.

[0056] Table 2 PCR reaction system

[0057]

[0058] Table 3 PCR amplification program

[0059]

[0060] 4. Carrier Construction and Transformation

[0061] The pET-28a(+) vector was linearized by double digestion with BamHI and XhoI. After gel recovery, it was mixed with the target fragment at a ratio of 2:1 (target gene fragment: linearized pET-28a(+) vector). 2× CE Mix was added, and ligation was carried out at 50℃ for 1 h.

[0062] Transform DH5α competent cells with the ligation product: Thaw 50 μL of competent cells on ice, add 10 μL of ligation product, incubate on ice for 10 min, heat shock at 42℃ for 50 s, incubate on ice for 2 min; add 500 μL of antibiotic-free LB medium, and recover at 37℃ and 200 rpm for 30 min; then centrifuge to collect the cells, resuspend them and spread them on LB plates containing 50 μg / mL kanamycin, and incubate upside down at 37℃ overnight.

[0063] 5. Identification and sequencing of positive clones

[0064] Single colonies were picked and inoculated into LB medium containing 50 μg / mL kanamycin. After incubation at 37°C for 4 h in a shaker, colony PCR was performed for verification. The colony PCR reaction system is shown in Table 4, and the colony PCR amplification program is shown in Table 5. After electrophoresis, positive bacterial cultures were selected and sent to a sequencing company for Sanger sequencing. The correctly sequenced DcOMT1, DcOMT2, and DcOMT3 recombinant plasmids and their corresponding glycerol bacteria were stored at -80°C for later use.

[0065] Table 4 Colony PCR Reaction System

[0066]

[0067] Table 5 Colony PCR Amplification Procedure

[0068]

[0069] Example 2: Expression, purification and identification of three recombinant DcOMT proteins

[0070] The genes for DcOMT1 (SEQ ID NO.1), DcOMT2 (SEQ ID NO.3), and DcOMT3 (SEQ ID NO.5) were constructed into the pET-28a(+) vector and transformed into *E. coli* BL21(DE3) for induced expression. The DcOMT1, DcOMT2, and DcOMT3 proteins were purified using the same Ni-NTA affinity chromatography strategy to obtain high-purity DcOMT1, DcOMT2, and DcOMT3 proteins. The molecular weights of all three proteins were between 39 and 42 kDa.

[0071] 1. Plasmid transformation and induced expression

[0072] The correctly sequenced DcOMT1, DcOMT2, and DcOMT3 recombinant plasmids were transformed into *E. coli* BL21(DE3) competent cells, respectively. Transformation procedure: 50 μL of competent cells were thawed on ice, 2–5 μL of recombinant plasmid were added, and the cells were incubated on ice for 30 min, followed by heat shock at 42°C for 60 s, and then incubated on ice for 2 min. 600 μL of antibiotic-free LB medium was added, and the cells were incubated at 37°C and 200 rpm for 45 min to 1 h. 100 μL of the bacterial culture was plated on LB agar plates containing 50 μg / mL kanamycin and incubated upside down overnight at 37°C.

[0073] Single colonies were picked and inoculated into 5 mL of LB medium containing kanamycin and cultured overnight at 37°C and 200 rpm (approximately 12 h). Then, 1% of the colonies were transferred to 200 mL of LB medium containing kanamycin and cultured at 37°C with shaking for 5-6 h. OD values ​​were then measured using a microplate reader. 600 When the concentration is 0.6-0.8, add IPTG to the bacterial culture until the final concentration is 0.3 mM (e.g., add 120 μL of 0.5 M IPTG stock solution to 200 mL of bacterial culture), and then incubate at 16℃ and 150 rpm for 16-18 h.

[0074] 2. Collection and disruption of bacterial cells

[0075] After induction, the bacterial culture was transferred to a 50 mL centrifuge tube and centrifuged at 8000 rpm for 5-10 min at 4°C to collect the bacterial pellet, which was then stored at -20°C. The bacterial pellet was resuspended in 10 mL Binding Buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl) by pipetting and then transferred to a 1.5 mL EP tube. The pellet was then sonicated on ice at the following parameters: power 35%, on for 3 seconds, off for 3 seconds, for a total of 20 min. The lysate was centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was collected for later use.

[0076] 3. Ni-NTA affinity chromatography purification

[0077] Add the supernatant to the Ni-NTA affinity column in multiple fractions, adjusting the flow rate to 2-3 seconds per drop. First, wash for 3 column volumes (2 mL Elution Buffer + 48 mL Binding Buffer) with 20 mM imidazole elution buffer, then wash for 3 column volumes (4 mL Elution Buffer + 46 mL Binding Buffer) with 40 mM imidazole elution buffer to remove non-specifically bound proteins. Finally, wash for 3 column volumes (25 mL Elution Buffer + 25 mL Binding Buffer) with 250 mM imidazole elution buffer, collecting the target protein eluent. Pour the 250 mM imidazole elution buffer into a 30 kDa ultrafiltration tube, centrifuge at 3500 × g for 5 min at 4 °C, add Binding Buffer to dilute and wash twice, and concentrate by multiple centrifugations to approximately 500 μL.

[0078] 4. SDS-PAGE identification and protein concentration determination

[0079] The purified protein sample was mixed with 5× protein loading buffer at a 1:1 volume ratio, denatured in a 95℃ metal bath for 5 min, centrifuged, and then loaded for SDS-PAGE electrophoresis (10% separating gel). Electrophoresis conditions: first, electrophoresis at a constant voltage of 100 V for 20 min, then at a constant voltage of 120 V until bromophenol blue reached the bottom of the gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue for 3 h, and then destained with destaining solution until the bands were clear. Figure 2-5 The results showed that all three recombinant proteins exhibited a clear target band at 39-42 kDa, consistent with the theoretical molecular weight.

[0080] Protein concentrations were determined using the BCA method: 18 μL of 0.9% NaCl, 200 μL of Coomassie Brilliant Blue reagent, and 2 μL of purified protein sample were added to a 96-well plate. After mixing, the absorbance was measured at 595 nm using a microplate reader. The concentrations of DcOMT1 protein were 1.132 mg / mL, DcOMT2 was 1.248 mg / mL, and DcOMT3 was 1.227 mg / mL. Based on the standard curve y = 0.2234x + 0.7318, the required volume for a quantitative reaction of 50 μg of enzyme was calculated. 32.6 μL of DcOMT1 protein, 25.3 μL of DcOMT2 protein, and 26.4 μL of DcOMT3 protein were required.

[0081] 5. Protein purification

[0082] Take the remaining volume of purified protein, add 1 / 7 volume of 80% glycerol, mix well, then flash freeze in liquid nitrogen and store at -80℃.

[0083] Example 3: In vitro enzyme activity detection of dracoside D by three DcOMTs

[0084] 1. In vitro enzymatic reaction system

[0085] A 100 μL in vitro reaction system was established using dragon's blood D as a substrate. The reaction system is shown in Table 6. The blank control group was prepared by replacing the purified protein with an equal volume of fragmentation buffer. Each group was divided into three replicates, and the reaction was carried out at 37℃ in a shaker for 12 h.

[0086] Table 6 In vitro enzymatic reaction system

[0087]

[0088] 2. Reaction Termination and Sample Preparation

[0089] After the reaction was complete, 200 μL of ethyl acetate was immediately added to terminate the reaction. The mixture was thoroughly vortexed and centrifuged at 8000 rpm for 10 min. The upper organic phase was carefully transferred to a new EP tube. The extraction was repeated twice. The combined organic phases were dried under nitrogen or naturally in a clean bench and stored at -20℃ until analysis. Before detection, 200 μL of chromatographic grade methanol was added to the dried sample for redissolution, and the mixture was sonicated for 20-30 min. After filtration through a 0.22 μm needle filter, the solution was transferred to the inner tube of the sample vial for LC-MS analysis.

[0090] 3. LC-MS detection conditions

[0091] Chromatographic conditions: C18 reversed-phase column; mobile phase A: 0.1% formic acid and deionized water; mobile phase B: acetonitrile; gradient elution program: 0-2 min, 22%-45% B; 2-6 min, 45% B; 6-7 min, 45%-95% B; 7-8 min, 95%-22% B; 8-11 min, 22% B; flow rate: 0.3 mL / min; column temperature: 35℃; sample chamber temperature: 4℃; injection volume: 3 μL.

[0092] Mass spectrometry conditions: A high-energy electrospray ionization source (HESI source) was used for scanning in negative ion mode; the capillary temperature was 350℃, the spray voltage was 2.8 kV, and the S-lens level was 35 kV; both the sheath gas and the auxiliary gas were high-purity nitrogen (purity >99.99%), the sheath gas flow rate was 35 arb, and the auxiliary gas flow rate was 10 arb; parallel reaction monitoring (PRM) mode was used for scanning.

[0093] 4. Catalytic Results

[0094] HPLC-MS analysis results ( Figure 6-10 The results showed that the three recombinant proteins exhibited significant differences in their catalytic activity and product distribution for dragon's blood D. The peak areas and relative conversion rates are shown in Table 7.

[0095] Table 7. LC-MS results of three DcOMT-catalyzed dracosperm D.

[0096]

[0097] Specifically, DcOMT1 catalyzes the formation of dracosperm D primarily into 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone at m / z 301, and can further catalyze the formation of a small amount of dracosperm B at m / z 315; DcOMT2 catalyzes the formation of dracosperm D into dracosperm B at m / z 315; and DcOMT3 catalyzes the formation of dracosperm D primarily into 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone at m / z 301.

[0098] Example 4: In vitro enzymatic catalysis of naringenin and isoliquiritigenin by DcOMT1

[0099] 1. Experimental materials

[0100] Substrates: Isoliquiritigenin (purity ≥98%), Naringenin (purity ≥98%), Enzyme: DcOMT1 protein purified in Example 2 (1.132 mg / mL), Other reagents are the same as in Example 3.

[0101] 2. In vitro enzymatic reaction system

[0102] A 100 μL in vitro reaction system was established using isoliquiritigenin and naringenin as substrates. The reaction system is shown in Table 8. The blank control group was prepared by replacing the purified protein with an equal volume of fragmentation buffer. Each group was divided into three replicates, and the reaction was carried out at 37℃ in a shaker for 12 h.

[0103] Table 8 In vitro enzymatic reaction system

[0104]

[0105] 3. Reaction Termination and Sample Preparation

[0106] Same as in Example 3. After the reaction was complete, 200 μL of ethyl acetate was added for extraction, dried under nitrogen, and then redissolved in 200 μL of chromatographic grade methanol. The mixture was then filtered through a 0.22 μm filter membrane before injection.

[0107] 4. LC-MS detection conditions

[0108] The HPLC-MS conditions were the same as in Example 3.

[0109] 4. Catalytic Results

[0110] HPLC-MS analysis results ( Figure 11The results showed that DcOMT1 exhibited highly efficient monomethylation activity at the 4'-position of the B ring for both isoliquiritigenin and naringenin, and did not further catalyze the formation of dimethylated products (the 5,7-hydroxyl groups on the A ring were not methylated), indicating that it has regioselectivity for the 4'-position of the B ring.

[0111] Example 5: The combination of DcOMT1 and DcOMT3 can improve the conversion rate of dracospermin D to dracospermin B.

[0112] 1. Experimental Objective

[0113] The stepwise combination catalysis of DcOMT3 and DcOMT1 was investigated to improve the efficiency of the conversion of dracosanol D to dracosanol B, and to verify the synergistic effect of the two enzymes in the synthesis of methylated flavonoids.

[0114] 2. Experimental Materials

[0115] Substrate: Dragon's blood D (purity ≥98%); Enzymes: DcOMT1 protein (1.132 mg / mL, 50 μg requires 32.6 μL) and DcOMT3 protein (1.227 mg / mL, 50 μg requires 26.4 μL) purified in Example 2; Methyl donor SAM and other reagents are the same as in Example 3.

[0116] 3. In vitro enzymatic reaction system

[0117] A stepwise enzyme dosing strategy was adopted. Figure 12 Establish the following reaction system:

[0118] (1) First step reaction: A 100 μL reaction system was established using dracosanol D as a substrate. 0.5 μL of dracosanol D, 2 μL of SAM, and 50 μg (26.4 μL) of purified DcOMT3 protein were added to the system, and the volume was brought up to 100 μL with 50 mM Tris-HCl (pH 8.0). The blank control was replaced with an equal volume of fragmentation buffer instead of DcOMT3 protein. Each group was divided into 3 replicates, and the reaction was carried out precisely at 37℃ in a shaker for 6 h.

[0119] (2) Second step reaction: After the first step reaction is completed, add 50 μg (32.6 μL) of purified DcOMT1 protein directly to the above reaction system, add 2 μL of SAM, and adjust the total volume to 200 μL with 50 mM Tris-HCl (pH 8.0). After mixing, continue the reaction in a shaker at 37℃ for 6 h.

[0120] Single enzyme control group: Drocoderm D was catalyzed by equal amounts of DcOMT1 or DcOMT3 protein alone, with a total reaction time of 12 h. The reaction system and conditions were the same as above.

[0121] 4. Reaction Termination and Sample Preparation

[0122] After the second step of the reaction is completed, immediately add 400 μL of ethyl acetate to terminate the reaction, vortex thoroughly, centrifuge at 8000 rpm for 10 min, and carefully aspirate the upper organic phase; repeat the extraction twice. Combine the organic phases, dry them under nitrogen in a clean bench, and store at -20℃ until analysis. Before detection, add 200 μL of chromatographic grade methanol to the dried sample for redissolution, sonicate for 20-30 min, filter through a 0.22 μm needle filter membrane, and then inject the sample.

[0123] Note: When the reaction of the single enzyme control group was terminated, 200 μL of ethyl acetate was added for extraction, and the rest of the treatment was the same.

[0124] 5. LC-MS detection conditions

[0125] Same as Example 3.

[0126] 6. Catalytic Results

[0127] The HPLC-MS analysis results are shown in Table 9. The stepwise combination catalysis of DcOMT3 and DcOMT1 can significantly improve the yield of dragon's blood B.

[0128] Table 9. Comparison of the efficiency of different catalytic strategies in the synthesis of dragon's blood B

[0129]

[0130] Specifically, when DcOMT1 single enzyme catalyzes dracosanol D for 12 h, the relative conversion rate of dracosanol B (m / z 315) is only 1.82%; when DcOMT3 single enzyme catalyzes dracosanol D for 12 h, the intermediate product 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone (m / z 301) is mainly generated, with a relative conversion rate of 22.99%, and almost no dracosanol B is generated. However, by adopting a stepwise combination strategy of first catalyzing with DcOMT3 for 6 h, and then continuing catalysis with DcOMT1 for another 6 h, the relative conversion rate of dracosanol B (m / z 315) is increased to approximately 10.49%.

[0131] The above results indicate that DcOMT3 exhibits strong selective monomethylation ability for the 2'-phenolic hydroxyl group of dracosanol D, efficiently generating the intermediate 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone; DcOMT1 possesses further 4'-methylation activity for this intermediate, converting it into dracosanol B. The stepwise combination of the two enzymes effectively overcomes the low efficiency of direct dimethylation of dracosanol D catalyzed by DcOMT1 alone. The "monomethylation-remethylation" cascade strategy significantly improves the synthesis efficiency of dracosanol B, fully demonstrating the synergistic application value of the OMT enzyme combination provided in this invention for the precise synthesis of methylated flavonoids.

[0132] The above embodiments fully demonstrate the functional differentiation and combined application value of the three OMT genes and their encoded proteins provided by the present invention. The present invention is not limited to the specific embodiments described above.

Claims

1. The application of an O-methyltransferase in catalyzing the O-methylation reaction of flavonoids, characterized in that, The O-methyltransferase is selected from proteins having the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.6; When the flavonoid compound is dracoside D: The protein shown in SEQ ID NO.2 catalyzes the formation of 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone from dragon's blood D, and further catalyzes the formation of dragon's blood B; The protein shown in SEQ ID NO.4 catalyzes the formation of dracospermin D from dracospermin B. The protein shown in SEQ ID NO.6 catalyzes the formation of 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone from dragon's blood D.

2. The application according to claim 1, characterized in that: (1) The protein shown in SEQ ID NO.2 is encoded by a gene having the nucleotide sequence shown in SEQ ID NO.1; (2) The protein shown in SEQ ID NO.4 is encoded by a gene having the nucleotide sequence shown in SEQ ID NO.3; (3) The protein shown in SEQ ID NO.6 is encoded by a gene having the nucleotide sequence shown in SEQ ID NO.

5.

3. A recombinant expression vector, characterized in that, It comprises the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.5 as described in claim 2.

4. A host cell, characterized in that, It includes the recombinant expression vector of claim 3.

5. A flavonoid methylation reaction kit, characterized in that, It comprises the O-methyltransferase of claim 1, a reaction buffer, and the methyl donor S-adenosylmethionine.

6. A method for the in vitro catalytic O-methylation of flavonoid compounds, characterized in that, Includes the following steps: (a) Provide the O-methyltransferase of claim 1 or the kit of claim 5; (b) The O-methyltransferase is mixed with a flavonoid substrate and a methyl donor, S-adenosylmethionine, to carry out an enzymatic reaction; (c) Collect the reaction products to obtain methylated flavonoid compounds.

7. The method according to claim 6, characterized in that, When the flavonoid substrate is dracosanol D, the proteins shown in SEQ ID NO.2 and SEQ ID NO.6 are added stepwise for combined catalysis, thereby improving the synthesis efficiency of dracosanol B; the combined catalysis specifically involves: The first step involves adding the protein shown in SEQ ID NO.6 to catalyze the formation of 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone from dragon's blood D. The second step involves adding the protein shown in SEQ ID NO.2 to catalyze the formation of dragon's blood B from the 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone.

8. The method according to claim 6, characterized in that, When the protein shown in SEQ ID NO.2 is used for catalysis, the flavonoid substrate is dracoside D, isoliquiritin, or naringenin.

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