Biflavone oxymethyltransferase as well as coding gene and application thereof

Abstract

The invention discloses biflavone oxygen-methyltransferase as well as a coding gene and application thereof. The biflavone oxygen-methyltransferase has an amino acid sequence as shown in any one of SEQ ID NO: 1-5, or the amino acid sequence of the biflavone oxygen-methyltransferase and the amino acid sequence as shown in any one of SEQ ID NO: 1-5 have at least 95% of sequence identity. The biflavone oxymethyltransferase provides a new technical path for large-scale production of amentotaxus biflavone compounds, has important application value, not only provides an important gene element for biosynthesis of amentotaxus biflavone compounds with high pharmacological activity, such as bilobaliflavone, ginkgetin and isoginkgetin, but also provides an important gene element for biosynthesis of amentotaxus biflavone compounds with high pharmacological activity, such as bilobalitin, ginkgetin and isoginkgetin. The method has reference significance in widening production tools and application of biflavone compounds.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to biflavonoid oxymethyltransferase, its encoding gene, and its applications. Background Technology

[0002] Neurodegenerative diseases are a major global health challenge. Among numerous treatment strategies, natural biflavonoids have demonstrated unique advantages in the prevention and treatment of neurodegenerative diseases due to their multi-target regulatory effects. Specifically, preparations containing amentoflavone derivatives, such as Ginkgo biloba extract EGb 761, have been clinically proven to have significant cognitive function improvement and neuroprotective effects.

[0003] Representative active molecules among the apigenin derivatives include apigenin, ginkgolide, ginkgol, and isoginkol. Apigenin is a core structure formed by the intermolecular coupling of two apigenin monomers at the C3'-C8" positions. Ginkgolide is a monomethoxy derivative of apigenin with an oxymethylated hydroxyl group at the C4' position; ginkgol is a dimethoxy derivative formed by further methylation of the hydroxyl group at the C7 position; isoginkol has a similar structure to ginkgol, except that the second methoxy group is located at the 4"' position instead of the C7 position. These compounds exhibit significant inhibitory activity against β-amyloid (Aβ40) aggregation (IC50: 5-10 μM). However, the content of these compounds in natural plants is extremely low (isolation rate <0.001%), severely limiting their widespread clinical application. Besides their significant neurological activity, these compounds are also used in pharmacological and clinical research due to their wide range of cardiovascular disease applications.

[0004] Currently, the chemical synthesis of these compounds generally requires multi-step reactions under high temperature and strong oxidizing agent conditions, resulting in low yields, high costs, and heavy environmental burdens. Biosynthesis technology can achieve efficient and selective synthesis of biflavonoids under mild conditions, offering significant advantages for industrial applications. However, despite the discovery of biflavonoids nearly a century ago, the key enzymes in their biosynthetic pathway have remained unidentified, greatly hindering the development of related applications. Summary of the Invention

[0005] To address the aforementioned shortcomings in the prior art, this invention provides a biflavonoid oxymethyltransferase, its encoding gene, and its applications.

[0006] Specifically, the present invention provides a biflavonoid oxymethyltransferase having an amino acid sequence as shown in any one of SEQ ID NO:1-5 or having an amino acid sequence sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence as shown in any one of SEQ ID NO:1-5.

[0007] The present invention also provides an isolated nucleic acid that encodes the biflavonoid oxymethyltransferase described in this invention.

[0008] In some specific implementations, the nucleic acid comprises a nucleotide sequence as shown in any one of SEQ ID NO:6-10.

[0009] The present invention also provides a recombinant expression vector comprising the nucleic acid described herein.

[0010] In some specific implementations, the backbone of the recombinant expression vector is pET28a.

[0011] The present invention also provides a transformant comprising the nucleic acid described in the present invention, or the recombinant expression vector described in the present invention.

[0012] In some embodiments, the host cell used in the construction of the transformant is Escherichia coli.

[0013] In some specific implementations, the Escherichia coli is Escherichia coli BL21(DE3).

[0014] The present invention also provides a method for preparing biflavonoid oxymethyltransferase, which includes culturing the transformant described in the present invention to obtain a fermentation product.

[0015] In some embodiments, the culture medium used for the culture is LB liquid medium.

[0016] In some specific embodiments, the method further includes the step of purifying the fermentation product to obtain the biflavonoid oxymethyltransferase.

[0017] The present invention also provides an enzyme combination comprising at least two of the biflavonoid oxymethyltransferases described in the present invention.

[0018] This invention also provides the application of the biflavonoid oxymethyltransferase, the nucleic acid, the recombinant expression vector, the transformant, or the enzyme combination described herein in the preparation of Taxus chinensis biflavonoid compounds;

[0019] The cypress biflavonoids include ginkgo biflavonoids, ginkgolide, isoginkgolide, yew biflavonoids, podocarpus flavonoid A, and 4',7”-oxymethylcypress biflavonoids.

[0020] In some specific embodiments, the preparation of the ginkgo biflavonoid uses cypermethrin as a substrate, and the biflavonoid oxymethyltransferase used has an amino acid sequence as shown in SEQ ID NO:1 or 2, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or 2; the preparation of the ginkgolide uses ginkgo biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has an amino acid sequence as shown in SEQ ID NO:3, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3; the preparation of the isoginkgolide uses ginkgo biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has an amino acid sequence as shown in SEQ ID NO:4 or 5, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3. The amino acid sequence shown in NO:4 or 5 has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; the preparation of the erythroflavonoid uses erythroflavonoid as a substrate, and the oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:3 or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3; the preparation of the podocarpus flavonoid A uses erythroflavonoid as a substrate, and the oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:4 or 5 or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or 5; the preparation of the 4',7"-oxymethyl erythroflavonoid uses ginkgo biloba as a substrate, and the oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:1 or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in NO:1 has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0021] This invention also provides a method for preparing paclitaxel flavonoids, the method comprising:

[0022] The biflavonoid compounds of Taxus chinensis were prepared by contacting and reacting the biflavonoid oxymethyltransferase described in this invention with a substrate.

[0023] The cypress biflavonoids include ginkgo biflavonoids, ginkgolide, isoginkgolide, yew biflavonoids, podocarpus flavonoid A, and 4',7”-oxymethylcypress biflavonoids.

[0024] In some specific embodiments, the preparation of the ginkgo biflavonoids uses cypermethrin as a substrate, and the biflavonoid oxymethyltransferase used has an amino acid sequence as shown in SEQ ID NO:1 or 2, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or 2.

[0025] In some specific embodiments, the preparation of the ginkgo biloba extract uses biflavonoids as a substrate, and the biflavonoid oxymethyltransferase used has an amino acid sequence as shown in SEQ ID NO:3 or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3.

[0026] In some specific embodiments, the preparation of the isoginkgo biloba extract uses biflavonoids as a substrate, and the biflavonoid oxymethyltransferase used has an amino acid sequence as shown in SEQ ID NO:4 or 5, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or 5.

[0027] In some specific embodiments, the preparation of the erythroflavonoids uses erythroflavonoids as a substrate, and the erythroflavonoid oxymethyltransferase used has an amino acid sequence as shown in SEQ ID NO:3 or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3.

[0028] In some specific embodiments, the preparation of the Podocarpus flavonoid A uses Taxus chinensis biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has an amino acid sequence as shown in SEQ ID NO:4 or 5 or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or 5.

[0029] In some specific embodiments, the 4',7”-oxymethyltachys biflavonoids are prepared using ginkgo biflavonoids as a substrate, and the biflavonoid oxymethyltransferase used has an amino acid sequence as shown in SEQ ID NO:1 or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1.

[0030] In some specific implementations, the reaction further includes the use of S-adenosine-L-methionine.

[0031] In some embodiments, the volume ratio of biflavonoid oxymethyltransferase to substrate in the reaction is 1-50:1.

[0032] In some specific implementations, the volume ratio of biflavonoid oxymethyltransferase to substrate in the reaction is 10-50:1.

[0033] In some specific implementations, the volume ratio of biflavonoid oxymethyltransferase to substrate in the reaction is 10-49:1.

[0034] In some embodiments, the amount of substrate added in the reaction is 10-100 μM.

[0035] In some embodiments, the amount of substrate added in the reaction is 50-100 μM.

[0036] In some specific implementations, the amount of substrate added in the reaction is 50 μM.

[0037] In some embodiments, the reaction temperature is 30-38°C.

[0038] In some implementations, the reaction temperature is 32-37°C.

[0039] In some specific implementations, the reaction temperature is 37°C.

[0040] In some implementations, the reaction time is 0.5h-2h.

[0041] In some implementations, the reaction time is 1-2 hours.

[0042] In some specific implementations, the reaction time is 1 hour.

[0043] In some embodiments, the amount of S-adenosyl-L-methionine added is 0.01-0.5 mM.

[0044] In some embodiments, the amount of S-adenosyl-L-methionine added is 0.1-0.5 mM.

[0045] In some specific implementations, the amount of S-adenosyl-L-methionine added is 0.1 mM.

[0046] The present invention also provides a reaction system for preparing cyperus tinctoria flavonoids, the reaction system comprising:

[0047] The present invention comprises the biflavonoid oxymethyltransferase or the enzyme combination thereof, and at least one of the following: Taxus chinensis biflavonoid, Ginkgo biflavonoid, Ginkgo biloba extract, Isoginkgo biloba extract, Taxus chinensis biflavonoid, Podocarpus flavonoid A and 4',7”-oxymethyl Taxus chinensis biflavonoid.

[0048] The cypress biflavonoids include ginkgo biflavonoids, ginkgolide, isoginkgolide, yew biflavonoids, podocarpus flavonoid A, and 4',7”-oxymethylcypress biflavonoids.

[0049] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0050] The reagents and raw materials used in this invention are all commercially available.

[0051] The positive and progressive effects of this invention are as follows:

[0052] This invention marks the first discovery and identification of a key enzyme capable of catalyzing the specific oxygen-methylation modification of cypermethrin-based biflavonoids, enabling the selective synthesis of ginkgo biflavonoids, ginkgol, isoginkgolide, redwood biflavonoids, podocarpus flavonoid A, and 4',7"-oxymethylcypermethrin. This discovery provides a new technological pathway for the large-scale production of cypermethrin-based biflavonoids and has significant application value. The discovery of these oxygen-methyltransferases for cypermethrin-based biflavonoids not only provides important genetic elements for the biosynthesis of highly pharmacologically active cypermethrin-based biflavonoids such as ginkgo biflavonoids, ginkgol, isoginkgolide, redwood biflavonoids, podocarpus flavonoid A, and 4',7"-oxymethylcypermethrin, but also offers valuable insights for expanding the production tools and applications of biflavonoids. Attached Figure Description

[0053] Figure 1 The structural formulas of the oxygen methyltransferases GbOMT1, GbOMT2, GbOMT3, GbOMT4 and GbOMT5 catalyzing the reaction of the biflavonoids of Taxus wallichiana provided in the embodiments of the present invention are shown in the figures.

[0054] Figures 2A-2C This invention describes the tissue-specific distribution of Taxodium biflavonoids and the tissue-specific expression of oxymethyltransferase in Ginkgo biloba, as provided in the embodiments of the present invention.

[0055] Figure 3 The plasmid map of pOMT1 provided in this embodiment of the invention; the plasmid maps of pOMT2, pOMT3, pOMT4, and pOMT5 have the same structure.

[0056] Figure 4The HPLC and LC-MS / MS results of the in vitro catalysis of the production of ginkgo biloba flavonoids by GbOMT1 and GbOMT2 proteins provided in the embodiments of the present invention are shown.

[0057] Figure 5 The HPLC and LC-MS / MS results of the GbOMT3 protein catalyzing the in vitro production of ginkgo biloba extract from biflavonoids provided in this embodiment of the invention are shown.

[0058] Figure 6 The HPLC and LC-MS / MS results of the in vitro catalysis of the production of isoginkgo biloba extract from ginkgo biloba by the GbOMT4 and GbOMT5 proteins provided in the embodiments of the present invention are shown.

[0059] Figure 7 The HPLC and LC-MS / MS results of GbOMT3, GbOMT4 and GbOMT5 proteins catalyzing the in vitro production of Podocarpus flavonoids A and Taxus flavonoids from Cephalotaxus difformis flavonoids provided in this embodiment of the invention.

[0060] Figure 8 The HPLC and LC-MS / MS results of the GbOMT1 protein catalyzing the in vitro production of 4',7”-oxymethylcyperazine from ginkgo biloba flavonoids provided in this embodiment of the invention are shown.

[0061] Figure 9 The NOESY NMR spectrum of Podocarpus flavonoid A, a product of the in vitro catalysis of the production of flavonoids from Cirsium japonicum by GbOMT4 and GbOMT5 proteins provided in this embodiment of the invention.

[0062] Figure 10 The NOESY NMR spectrum of red flavonoid, a product of the in vitro catalysis of the production of flavonoids from yew flavonoids by the GbOMT3 protein provided in this embodiment of the invention.

[0063] Figure 11 HPLC comparison chromatograms of the products of isolated and purified GbOMT3, GbOMT4 and GbOMT5 catalyzed by cypress biflavonoids and the standards Podocarpus flavonoids A and redwood biflavonoids provided in the embodiments of the present invention. Detailed Implementation

[0064] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0065] Example 1: Cloning of the coding genes for GbOMT1, GbOMT2, GbOMT3, GbOMT4, and GbOMT5

[0066] In Ginkgo biloba, tissue distribution analysis revealed that flavonoids such as cypermethrin, ginkgo biloba, ginkgol, and isoginkol were all enriched in the leaves. Figure 2A and Figure 2B Based on the compound distribution results, bioinformatics and transcriptome analyses were performed, identifying leaf-specific expression candidate genes GbOMT1, GbOMT2, GbOMT3, GbOMT4, and GbOMT5. (Results are shown below.) Figure 2C As shown.

[0067] Healthy ginkgo leaves were collected, and RNA was extracted using the Tiangen RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit. Specific extraction methods were performed according to the manufacturer's instructions. cDNA was obtained by reverse transcription using the Takara PrimeScript RTMaster Mix (Perfect Real Time) Reverse Transcription Kit, following the manufacturer's instructions.

[0068] The obtained cDNA was used as a template for cloning using primer pairs. The primer pairs were as follows:

[0069] GbOMT1:

[0070]

[0071] GbOMT2:

[0072]

[0073] GbOMT3:

[0074]

[0075] GbOMT4:

[0076]

[0077] GbOMT5:

[0078]

[0079] In this text, the underlined part represents the vector homologous arm required for vector construction, and the bolded part represents the sequence of the enzyme recognition site.

[0080] After agarose gel electrophoresis and gel recovery, the fragment was inserted between the Nde I and EcoR I sites of the pET28a vector using In-Fusion (Vazyme) to transform DH10B competent cells.

[0081] Positive single clones were expanded and cultured. Plasmids were extracted using the Axygen Plasmid Extraction Kit (AxyPrep Plasmid DNA Mini-Kit) and sequenced by Sangon Biotech Co., Ltd. The plasmid extraction method followed the manufacturer's instructions. Plasmids that were correctly sequenced were named pOMT1, pOMT2, pOMT3, pOMT4, and pOMT5 (example figure shown). Figure 3 ), used to express the functional verification after host transformation.

[0082] The amino acid sequence and encoding nucleotide sequence of GbOMT1 are listed in SEQ ID NO:1 and SEQ ID NO:6, respectively; the amino acid sequence and encoding nucleotide sequence of GbOMT2 are listed in SEQ ID NO:2 and SEQ ID NO:7, respectively; the amino acid sequence and encoding nucleotide sequence of GbOMT3 are listed in SEQ ID NO:3 and SEQ ID NO:8, respectively; the amino acid sequence and encoding nucleotide sequence of GbOMT4 are listed in SEQ ID NO:4 and SEQ ID NO:9, respectively; and the amino acid sequence and encoding nucleotide sequence of GbOMT5 are listed in SEQ ID NO:5 and SEQ ID NO:10, respectively.

[0083] Example 2: Functional verification of O-methyltransferase in vitro

[0084] Plasmids pOMT1, pOMT2, pOMT3, pOMT4, and pOMT5 were transformed into competent *E. coli* BL21(DE3) cells using conventional chemical transformation methods. The transformed and revived bacterial cultures were plated on LB agar plates (containing 50 μg / mL Kanamycin, Kan) and incubated overnight at 37°C. Single colonies were selected from the plates and inoculated into 2 mL of LB agar (containing 50 μg / mL Kan), and cultured at 37°C and 220 rpm for 10 h to obtain the seed culture. The seed culture was then inoculated into 20 mL of LB agar (containing 50 μg / mL Kan) at a 1% v / v ratio and cultured at 37°C and 220 rpm until OD (dose elapsed). 600=0.4. After cooling in an ice-water bath for 10 min, IPTG was added to a final concentration of 0.1 mM, and the cells were induced and cultured at 20℃ and 180 rpm for 20 h. After protein induction culture, the cells were collected by centrifugation at 4℃ and 6000 rpm for 5 min, and the supernatant was removed. Subsequently, the cells were resuspended in 1 mL of resuspension buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10% w / v glycerol, 5 mM DTT). A protease inhibitor (Cocktail, Sangon, China) was added, and the cells were sonicated at 30% power in an ice-water bath. After cell lysis, the cells were centrifuged at 4℃ and 8000 rpm for 20 min to remove cell debris, and the supernatant was used for in vitro reactions. The protein obtained by expression of the BL21(DE3) recombinant strain carrying the pET28a empty vector was used as a negative control.

[0085] For the in vitro reaction of the oxymethyltransferase, the total reaction volume was 100 μL. The reaction mixture contained 98 μL of crude enzyme of a single candidate oxymethyltransferase, 50 μM of biflavonoid substrate (caproic acid biflavonoid or ginkgo biflavonoid), and 0.1 mM S-adenosyl-L-methionine (SAM). The reaction system was reacted at 37 °C for 1 h. After the reaction, the mixture was extracted three times with 100 μL of ethyl acetate. The organic phases were combined, the solvent was evaporated, and the mixture was reconstituted with 100 μL of methanol; the mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was used for UPLC and LC-MS / MS detection.

[0086] HPLC detection method: A Dionex Ultimate 3000 high-performance liquid chromatograph (Thermo Fisher Scientific, USA) equipped with an Athena C18 column (4.6 × 250 mm) (ANPEL Laboratory Technology Co., Ltd., China) was used. The mobile phase consisted of solvent A (pure water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). Gradient elution was initiated with solvent A at 95% and solvent B at 5%, maintained for 5 minutes. Subsequently, solvent B was linearly increased from 5% to 100% over 15 minutes, and maintained at 100% solvent B for 10 minutes. Afterward, solvent B was decreased from 100% to 5% over 5 minutes. The flow rate was set to 1 mL / min, and the UV detection wavelength was 280 nm.

[0087] LC-MS / MS detection method: LC-MS / MS detection was performed using a Thermo Q Exactive_UV_small molecule 104 chromatographic column, Phenomenex 00D-4462-AN (…). 2.6μm C18 LC Column (100x2.1mm, Ea). Mobile phase A was ddH₂O + 0.1% formic acid, and mobile phase B was acetonitrile. Column temperature was 45℃, and flow rate was 0.3 mL / min. Gradient elution conditions: 0–1 min 10% B, 1–15 min 10%–99% B, 15–18 min 99% B, 18–21 min 10% B. UV wavelength was 280 nm.

[0088] Mass spectrometry detection conditions: ESI ion source, capillary voltage 3.0 kV, cone voltage 30.0 V, extraction voltage 4.0 V, ion source temperature 100 °C, desolvation gas temperature 300 °C, desolvation gas flow rate 600 L / h, cone gas flow rate 5 L / h. The full scan mass spectrometry mass range is m / z 100–1500 Da, and the MS / MS second-stage scan collision energy is 20.0 V.

[0089] When cypermethrin was used as the substrate, the results showed that all candidate oxymethyltransferases exhibited oxymethylation activity for the cypermethrin substrate. Specifically, GbOMT1 and GbOMT2 catalyzed the 4'-OH oxymethylation of cypermethrin, producing a product identical to the cypermethrin standard (purchased from ChemFaces, catalog number CFN98846) (m / z: 551.09 [MH]). - This study confirmed that GbOMT1 and GbOMT2 can catalyze the 4'-OH oxymethylation modification of paclitaxel flavonoids to generate ginkgo biflavonoids. Figure 4 GbOMT3 can catalyze the formation of an oxymethylated modified product with a parent ion of m / z 551.09 from yew biflavonoids. Figure 7 Through amplification and compound purification, we confirmed the oxymethylation site of the product using NOESY NMR spectroscopy, identifying it as 7-OH site-modified sequoiaflavone. Figure 10 Meanwhile, we used HPLC analysis of the standard of safflower flavonoids (purchased from the source leaves) and the isolated and purified product to confirm that GbOMT3 can catalyze the conversion of yew flavonoids to safflower flavonoids. Figure 11 GbOMT4 and GbOMT5 can catalyze the formation of the same product from cypermethrin by oxymethylation with a parent ion of m / z 551.09. Figure 7 Similarly, we confirmed the oxygen methylation site of this product using NOESY NMR spectroscopy, and confirmed that it was a 4”'-OH site-modified Podocarpus flavone A. Figure 9Meanwhile, we used HPLC analysis of the standard of Podocarpus flavonoid A (purchased from the source leaves) and the isolated and purified product to confirm that GbOMT4 and GbOMT5 can catalyze the production of Podocarpus flavonoid A from Cephalotaxus japonicus flavonoids. Figure 11 ).

[0090] When ginkgo biloba flavonoids were used as the substrate, the results showed that GbOMT1, GbOMT3, GbOMT4, and GbOMT5 all exhibited enzymatic activity for oxymethyl modification of the substrate ginkgo biloba flavonoids, while GbOMT2 did not show catalytic activity. Specifically, GbOMT1 catalyzed the formation of oxymethylation modification of ginkgo biloba flavonoids with a parent ion of m / z 565.11. Figure 8 Unfortunately, due to the low catalytic activity of GbOMT1 for the substrate ginkgo biflavonoids, we were unable to successfully isolate a sufficient amount of product for structural identification through scale-up. However, based on the fact that only the 7-OH, 4”'-OH, and 7”-OH sites in the substrate ginkgo biflavonoids have free and easily modified hydroxyl sites, and that the oxymethyl modification at the 7-OH site is ginkgol, and the oxymethyl modification at the 4”'-OH site is isoginkol, the possibility of ginkgol and isoginkol can be ruled out by liquid chromatography. Figure 8 Therefore, the unknown product is presumed to be 4',7"-O-methylamentoflavoen, an oxygen-methylated derivative at the 7"-OH site. GbOMT3 catalyzes the oxygen-methylation modification of ginkgo biloba flavoen, producing a product identical to that of the ginkgo biloba standard (purchased from ChemFaces, catalog number CFN90173) (m / z: 565.11 [MH)). - This confirms that GbOMT3 can catalyze the production of ginkgo biloba extract from ginkgo biloba flavonoids (Ginkgo biloba extract). Figure 5 GbOMT4 and GbOMT5 catalyze the oxymethylation modification of ginkgo biloba extract, producing a product identical to the isoginkgolide standard (purchased from ChemFaces, catalog number CFN90174) (m / z: 565.11 [MH)). - This confirms that GbOMT4 and GbOMT5 can catalyze the production of ginkgo biloba extract from ginkgo biloba flavonoids (Ginkgo biloba extract). Figure 6 ).

[0091] Catalytic equations for all oxymethyltransferases (reference) Figure 1 .

[0092] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A biflavonoid oxymethyltransferase, characterized in that, The biflavonoid oxymethyltransferase has an amino acid sequence as shown in any one of SEQ ID NO: 1-5 or the amino acid sequence of the biflavonoid oxymethyltransferase has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 1-5.

2. An isolated nucleic acid, characterized in that, The nucleic acid encodes the biflavonoid oxymethyltransferase as described in claim 1; Preferably, the nucleic acid comprises a nucleotide sequence as shown in any one of SEQ ID NO:6-10.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 2; and / or, the backbone of the recombinant expression vector is preferably pET28a.

4. A transformant comprising the nucleic acid as described in claim 2, or the recombinant expression vector as described in claim 3; Preferably, the host cell used in the construction of the transformant is Escherichia coli, such as Escherichia coli BL21(DE3).

5. A method for preparing biflavonoid oxymethyltransferase, characterized in that, The method includes culturing the transformant as described in claim 4 to obtain a fermentation product; Preferably, the culture medium used for the culture is LB liquid medium; More preferably, the method further includes the step of purifying the fermentation product to obtain the biflavonoid oxymethyltransferase.

6. An enzyme combination, characterized in that, The enzyme combination includes at least two of the biflavonoid oxymethyltransferases as described in claim 1.

7. The application of the biflavonoid oxymethyltransferase as described in claim 1, the nucleic acid as described in claim 2, the recombinant expression vector as described in claim 3, the transformant as described in claim 4, or the enzyme combination as described in claim 6 in the preparation of Taxus chinensis biflavonoid compounds; The cypress biflavonoids include ginkgo biflavonoids, ginkgolide, isoginkgolide, yew biflavonoids, podocarpus flavonoid A, and 4',7”-oxymethylcypress biflavonoids; Preferably, the preparation of the ginkgo biflavonoid uses paclitaxel biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:1 or 2, or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or 2; the preparation of the ginkgolide uses ginkgo biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:3, or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:3; the preparation of the isoginkgolide uses ginkgo biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:4 or 5, or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or 5; the preparation of the erythroflavonoid uses paclitaxel biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:3, or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or 5. The amino acid sequence shown in NO:3 has at least 95% sequence identity; the preparation of the Podocarpus flavonoid A uses Taxodium biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:4 or 5 or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or 5; the preparation of the 4',7”-oxymethyl Taxodium biflavonoid uses Ginkgo biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:1 or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

8. A method for preparing cypermethrin-based flavonoid compounds, characterized in that, The method includes: The biflavonoid compounds of Taxus chinensis were prepared by contacting and reacting the biflavonoid oxymethyltransferase as described in claim 1 with a substrate. The cypress biflavonoids include ginkgo biflavonoids, ginkgolide, isoginkgolide, yew biflavonoids, podocarpus flavonoid A, and 4',7”-oxymethylcypress biflavonoids; Preferably, the preparation of the ginkgo biflavonoid uses paclitaxel biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:1 or 2, or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or 2; the preparation of the ginkgolide uses ginkgo biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:3, or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:3; the preparation of the isoginkgolide uses ginkgo biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:4 or 5, or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or 5; the preparation of the erythroflavonoid uses paclitaxel biflavonoid as a substrate, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:3, or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or 5. The amino acid sequence shown in NO:3 has at least 95% sequence identity; the preparation of the Podocarpus flavonoid A uses Taxodium spp. as a substrate, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:4 or 5 or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:4 or 5; the preparation of the 4',7”-oxymethyl Taxodium spp. as a substrate uses Ginkgo biflavonoid, and the biflavonoid oxymethyltransferase used has the amino acid sequence shown in SEQ ID NO:1 or has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:1; And / or, the reaction further includes the use of S-adenosyl-L-methionine.

9. The method as described in claim 8, characterized in that, The volume ratio of biflavonoid oxymethyltransferase to substrate in the reaction is 1-50:1, preferably 10-50:1; And / or, the amount of substrate added in the reaction is 10-100 μM, preferably 50-100 μM; And / or, the reaction temperature is 30-38°C, preferably 32-37°C; And / or, the reaction time is 0.5h-2h, preferably 1-2h; And / or, the amount of S-adenosyl-L-methionine added is 0.01-0.5 mM, preferably 0.1-0.5 mM.

10. A reaction system for preparing cypress biflavonoids, characterized in that, The reaction system includes: The biflavonoid oxymethyltransferase as described in claim 1 or the enzyme combination as described in claim 6, and at least one of the following: cypress biflavonoids, ginkgo biflavonoids, ginkgo biloba extract, isoginkgo biloba extract, yew biflavonoids, podophyllum flavonoid A and 4',7”-oxymethylcypress biflavonoids; The cypress biflavonoids include ginkgo biflavonoids, ginkgolide, isoginkgolide, yew biflavonoids, podocarpus flavonoid A, and 4',7”-oxymethylcypress biflavonoids.

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