Flavone dimerization enzyme and coding gene and application thereof

Abstract

The invention discloses flavone dimerization enzyme as well as a coding gene and application thereof. The amino acid sequence of the flavone dimerization enzyme has at least 95% sequence identity compared with the amino acid sequence as shown in SEQ ID NO: 1, and the flavone dimerization enzyme comprises one or more amino acid residue differences in the site L47, the site L127, the site M140, the site V154, the site V215, the site L382 or the site A426 compared with the amino acid sequence as shown in SEQ ID NO: 1. The flavone dimerization enzyme can catalyze apigenin monomers to be connected through intermolecular C3 '-C8' bonds to generate apigenin biflavone, a new scheme is provided for large-scale production of biflavone compounds, and the flavone dimerization enzyme has important significance on development of novel drugs for treating neurodegenerative diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a flavonoid dimerization enzyme and a coding gene and application thereof. Background Art

[0002] Neurodegenerative diseases have become a global health challenge, projected to affect 139 million people by 2050. Among numerous therapeutic strategies, natural biflavonoids have attracted considerable attention due to their unique multi-target therapeutic properties. Natural biflavonoids are dimers composed of two flavonoid units. To date, nearly 600 natural biflavonoids have been reported. Among them, carbon-carbon bonded biflavones exhibit a rich skeletal structure due to the diversity of flavonoid unit types and linkages. Flavonoid units mainly include: flavan, flavonoid, anthocyanidin, isoflavan, isoflavone, neoflavan, chalcone, aurone and xanthone; the connection sites are also extremely diverse, including 2-3", 2'-2"', 2'-6", 2'-8", 3-3", 3-3"', 3'-3"', 3'-4"', 3'-5", 3-6", 3'-6", 3-7", 3'-7", 3-8", 3'-8", 4-6", 4-8", 4'-8", 5-5", 6-6", 6-γ, 6-8", 7-7" and 8-8". Preparations represented by 761 contain amentoflavone derivatives, which have been shown in clinical trials to have significant cognitive function improvement and neuroprotective effects.

[0003] The key active ingredients of the amentoflavone family of compounds include amentoflavone, bilobetin, ginkgetin, and isoginkgetin. These compounds exhibit significant inhibitory activity against amyloid-β (Aβ40) aggregation (IC50: 5-10 μM). However, the extremely low concentration of these compounds in natural plants (isolated yield <0.001%) severely limits their widespread clinical application.

[0004] Existing chemical synthesis methods often require high temperatures, strong oxidants, and complex multi-step reactions, resulting in low yields and limited practicality. In contrast, biosynthesis offers significant advantages by enabling efficient and selective synthesis of biflavonoids under mild conditions. Unfortunately, despite the discovery of biflavonoids nearly a century ago, the key enzymes responsible for flavonoid dimerization during their biosynthesis have remained largely unidentified, significantly hindering the development of related applications.

[0005] In the field of natural product biosynthesis, a variety of oxidases are known to catalyze cross-coupling reactions, including peroxidases, laccases, polyphenol oxidases, and cytochrome P450. Cytochrome P450, due to its high regio- and stereoselectivity, plays a crucial role in the biosynthesis of compounds such as dinaphthalene, diketopiperazine alkaloids, and dicoumarins. Cytochrome P450 possesses diverse substrate-binding pocket structures, enabling it to recognize specific substrates and catalyze dimerization reactions with precise linkage patterns. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a flavonoid dimerization enzyme and its encoding gene and application.

[0007] Specifically, the present invention provides a flavonoid dimerase, the amino acid sequence of which 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, and comprises one or more amino acid residue differences at positions L47, L127, M140, V154, V215, L382 or A426 compared to the amino acid sequence shown in SEQ ID NO: 1.

[0008] In some embodiments, the amino acid residue difference at position L47 is L47W.

[0009] In some embodiments, the amino acid residue difference at position L127 is L127V.

[0010] In some embodiments, the amino acid residue difference at position M140 is M140L.

[0011] In some embodiments, the amino acid residue difference at position V154 is V154W.

[0012] In some embodiments, the amino acid residue difference at position V215 is V215A.

[0013] In some embodiments, the amino acid residue difference at position L382 is L382F.

[0014] In some embodiments, the amino acid residue difference of A426 is A426P.

[0015] In some specific embodiments, the flavonoid dimerization enzyme has any of the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 1:

[0016] L47W;

[0017] L127V;

[0018] M140L;

[0019] V154W;

[0020] V215A;

[0021] L382F;

[0022] L382F / A426P;

[0023] A426P / M140L;

[0024] A426P / V215A;

[0025] L127V / A426P;

[0026] L382F / A426P / L127V;

[0027] L382F / A426P / V154W;

[0028] A426P / L47W / V154W;

[0029] A426P / L127V / V154W;

[0030] L382F / L47W / V154W;

[0031] L127V / L47W / V154W;

[0032] L382F / L127V / V154W;

[0033] L382F / L127V / L47W;

[0034] A426P / M52F / I146M.

[0035] In the present invention, the symbol " / " indicates that the flavonoid dimerization enzyme has the same amino acid difference before and after the symbol.

[0036] In some specific embodiments, the first 29 amino acid residues at the N-terminus of the flavonoid dimerase are replaced with the amino acid sequence shown in SEQ ID NO:17.

[0037] The present invention also provides an isolated nucleic acid encoding the flavonoid dimerization enzyme of the present invention.

[0038] The present invention also provides a recombinant expression vector comprising the nucleic acid of the present invention.

[0039] In some specific embodiments, the backbone of the recombinant expression vector is pEAQ or pFe00.

[0040] The present invention also provides a transformant comprising the nucleic acid or the recombinant expression vector of the present invention;

[0041] In some embodiments, the host cell used in the construction of the transformant is Escherichia coli or Agrobacterium.

[0042] In some specific embodiments, the Agrobacterium is Agrobacterium GV3101, and the Escherichia coli is Escherichia coli Jm109 (DE3).

[0043] The present invention also provides a method for preparing flavonoid dimerization enzyme, which comprises culturing the transformant of the present invention to obtain a fermentation product.

[0044] In some specific embodiments, the culture medium used is selected from LB liquid medium or TB liquid medium.

[0045] In some specific embodiments, the method further comprises the step of purifying the fermentation product to obtain the flavonoid dimerization enzyme.

[0046] The present invention also provides an enzyme combination, comprising: the flavonoid dimerization enzyme of the present invention and at least two flavonoid dimerization enzymes having a sequence identity of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% compared to the amino acid sequence shown in any one of SEQ ID NOs: 1 and 3-10.

[0047] The flavonoid dimerization enzyme of the present invention, the flavonoid dimerization enzyme having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 1 and 3-10, the nucleic acid of the present invention, the recombinant expression vector, the transformant or the enzyme combination in the preparation of carbon-carbon bond-linked biflavonoids, wherein the preparation of carbon-carbon bond-linked biflavonoids uses flavonoid compounds as substrates.

[0048] In some embodiments, the flavonoid compound is selected from the group consisting of flavanones, flavones, flavonols, anthocyanidins, isoflavans, isoflavones, neoflavans, dihydroflavones, dihydroflavonols, chalcones, aurones, and xanthones.

[0049] In some specific embodiments, the flavonoids include apigenin (CAS: 520-36-5), luteolin (CAS: 491-70-3) and triticum vulgare flavonoids (CAS: 520-31-0), the flavanones include eriodictyol (CAS: 552-58-9) and citronellol (CAS: 480-20-6), the flavonols include kaempferol (CAS: 520-18-3), quercetin (CAS: 117-39-5) and myricetin (CAS: 529-44-2), the dihydroflavonols include taxifolin (CAS: 480-18-2) and dihydromyricetin (CAS: 27200-12-0), and the dihydroflavones include (S)-naringenin (CAS: 480-41-1).

[0050] In some specific embodiments, the carbon-carbon bond-linked biflavonoids include aconitum biflavonoids, naringenin biflavonoids B, naringenin biflavonoids A and triticum vulgare biflavonoids.

[0051] In some embodiments, the attachment sites of the carbon-carbon bond-linked biflavonoids include 2-3", 2'-2"', 2'-6", 2'-8", 3-3", 3-3"', 3'-3"', 3'-4"', 3'-5", 3-6", 3'-6", 3-7", 3'-7", 3-8", 3'-8", 4-6", 4-8", 4'-8", 5-5", 6-6", 6-γ, 6-8", 7-7" and 8-8".

[0052] In some specific embodiments, the carbon-carbon bond-linked biflavonoid has a linking site of 3'-8".

[0053] In some specific embodiments, the preparation of the aconitum biflavonoids uses apigenin as a substrate and uses the flavonoid dimerase described in the present invention or a flavonoid dimerase whose amino acid sequence has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 1 and 3-4.

[0054] In some specific embodiments, the preparation of the naringenin biflavonoid methyl uses (S)-naringenin as a substrate and uses the flavonoid dimerase described in the present invention or a flavonoid dimerase having an amino acid sequence having 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 any one of SEQ ID NOs: 1, 3-4 and 7.

[0055] In some specific embodiments, the preparation of the naringenin biflavonoid ethyl uses (S)-naringenin as a substrate and uses the flavonoid dimerase described in the present invention or a flavonoid dimerase having an amino acid sequence having 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 any one of SEQ ID NOs: 1, 3 and 7.

[0056] In some specific embodiments, the preparation of the triticum biflavonoids uses triticum flavonoids as a substrate and uses the flavonoid dimerizer described in the present invention or a flavonoid dimerizer whose amino acid sequence has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0057] The present invention also provides a method for preparing a carbon-carbon bond-linked biflavonoid, the method comprising:

[0058] The flavonoid dimerase of the present invention or the flavonoid dimerase having an amino acid sequence having 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 any one of SEQ ID NOs: 1 and 3-10 is reacted with a flavonoid compound; the flavonoid compound and the carbon-carbon bond-linked biflavonoid are as defined in the present invention.

[0059] In some embodiments, the flavonoid dimerization enzyme is present in yeast microsomal protein, and the reaction comprises 100-1000 μg of yeast microsomal protein, 0.01-100 μM flavonoid compound, coenzyme, and hydrogen donor and / or hydrogen donor regeneration system per mL of reaction system.

[0060] In some specific embodiments, the coenzymes are FAD and FMN, the hydrogen donor is NADPH, and the hydrogen donor regeneration system includes glucose-6-phosphate and glucose-6-phosphate dehydrogenase.

[0061] In some specific embodiments, the reaction includes 200-500 μg yeast microsomal protein, 0.1-30 μM flavonoid compound, 0.1-5 mM NADPH, 1-10 μM FAD, 1-10 μg FMN, and 1-10 mM glucose-6-phosphate and 0.1-5 U glucose-6-phosphate dehydrogenase per mL reaction system.

[0062] In some embodiments, the temperature of the reaction is 25-35°C.

[0063] In some embodiments, the temperature of the reaction is 30-35°C.

[0064] In some specific embodiments, the reaction temperature is 30°C.

[0065] In some embodiments, the reaction time is 0.5-10 h.

[0066] In some embodiments, the reaction time is 1-5 hours.

[0067] In some specific embodiments, the reaction time is 1 h.

[0068] In some embodiments, the pH of the reaction is 7.0-8.0.

[0069] In some embodiments, the pH of the reaction is 7.0-7.5.

[0070] In some specific embodiments, the pH of the reaction is 7.5.

[0071] In some embodiments, the reaction comprises co-culturing the transformant of the present invention with the flavonoid compound, wherein the amount of the flavonoid compound added is 10-200 μM; and / or the reaction temperature is 20-25° C.; and / or the reaction time is 12 h-144 h; and / or the reaction system further comprises a solvent.

[0072] In some embodiments, the amount of the flavonoid compound added is 100-200 μM; and / or the reaction temperature is 22-25° C.; and / or the reaction time is 48-96 h.

[0073] In some specific embodiments, the amount of the flavonoid compound added is 100 μM; and / or the reaction temperature is 22°C.

[0074] In some specific embodiments, the co-solvent is a methyl-β-cyclodextrin solution.

[0075] In some embodiments, the methyl-β-cyclodextrin solution is added at a concentration of 1-20 mM.

[0076] In some embodiments, the methyl-β-cyclodextrin solution is added at a concentration of 4.75-10 mM.

[0077] In some specific embodiments, the added concentration of the methyl-β-cyclodextrin solution is 4.75 mM.

[0078] In some specific embodiments, the preparation of the aconitum biflavonoids uses apigenin as a substrate and uses the flavonoid dimerase described in the present invention or a flavonoid dimerase whose amino acid sequence has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1 and 3-4.

[0079] In some specific embodiments, the preparation of the naringenin biflavonoid methyl uses (S)-naringenin as a substrate and uses the flavonoid dimerase described in the present invention or a flavonoid dimerase having an amino acid sequence having 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 any one of SEQ ID NOs: 1, 3-4 and 7.

[0080] In some specific embodiments, the preparation of the naringenin biflavonoid ethyl uses (S)-naringenin as a substrate and uses the flavonoid dimerase described in the present invention or a flavonoid dimerase having an amino acid sequence having 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 any one of SEQ ID NOs: 1, 3 and 7.

[0081] In some specific embodiments, the preparation of the triticum biflavonoids uses triticum flavonoids as a substrate and uses the flavonoid dimerizer described in the present invention or a flavonoid dimerizer whose amino acid sequence has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0082] The present invention also provides a reaction system for preparing a carbon-carbon bond-linked biflavonoid, the reaction system comprising:

[0083] The flavonoid dimerization enzyme of the present invention, the flavonoid dimerization enzyme having an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 and 3-10, or the enzyme combination, and at least one of a flavonoid compound and a carbon-carbon bond-linked biflavonoid;

[0084] The flavonoid compound and the carbon-carbon bond-linked biflavonoid are as defined in the present invention.

[0085] In some embodiments, the reaction system further comprises a cytochrome P450 enzyme redox partner, and the cytochrome P450 enzyme redox partner is NADPH-cytochrome P450 reductase and / or cytochrome b5.

[0086] In some specific embodiments, the NADPH-cytochrome P450 reductase is AtCPR2 protein; and / or the cytochrome b5 is RsCYB5 protein.

[0087] In the present invention, the sequence identity of at least 95% is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

[0088] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0089] The reagents and raw materials used in the present invention are commercially available.

[0090] The positive progress effect of the present invention is:

[0091] The present invention invents flavonoid dimerization enzymes (aconitum flavonoid synthases). The flavonoid dimerization enzymes encoded thereby can regioselectively catalyze the polymerization of two molecules of apigenin monomers through C3'-C8" intermolecular linkage to produce aconitum flavonoids. The discovery of these flavonoid dimerization enzymes not only provides important genetic elements for the biosynthesis of aconitum flavonoids, but also has reference significance for the exploration and application of other functional enzymes that catalyze flavonoid dimerization. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 : is the formula of the reaction catalyzed by flavonoid dimerization enzyme (aconitoflavone synthase) GbCYP90J6, ChCYP90J7, MgCYP90J1 and SmCYP90E3v1 in the examples.

[0093] Figure 2 This is an illustration of the tissue-specific distribution of aegyptiflavone in Ginkgo biloba and the tissue-specific expression of GbCYP90J6 provided in the examples.

[0094] Figure 3 Figure 3 is the plasmid map of pEAQ-GbCYP90J6 provided in the examples.

[0095] Figure 4 The LC-MS results of GbCYP90J6 catalyzing the polymerization of apigenin to produce acetotaxel in tobacco chassis are provided in the examples.

[0096] Figure 5 Phylogenetic tree of CYP90 family genes from plants.

[0097] Figure 6 The plasmid map of pFe00 provided in the embodiment

[0098] Figure 7The plasmid maps of pFe00-trGbCYP90J6, pFe00-trTcCYP90J7, pFe00-trChCYP90J7, pFe00-trCpCYP90J8, pFe00-trMgCYP90J1, pFe00-trTpCYP90J1v1, pFe00-trTpCYP90J9, pFe00-trTpCYP90J10, and pFe00-trSmCYP90E3v1 provided in the examples are shown.

[0099] Figure 8 These are the HPLC and LC-MS results of the biotransformation of GbCYP90J6, TcCYP90J7, CpCYP90J8, MgCYP90J1, TpCYP90J1v1, TpCYP90J9, TpCYP90J10, ChCYP90J7, and SmCYP90E3v1 provided in the examples, catalyzing the polymerization of apigenin to produce aegyptiflavonoids in recombinant Escherichia coli.

[0100] Figure 9 LC-MS results of the biotransformation catalysis of (S)-naringenin polymerization to generate bi-naringenin a and bi-naringenin b by GbCYP90J6, MgCYP90J1, ChCYP90J7, and SmCYP90E3v1 in recombinant Escherichia coli provided in the examples.

[0101] Figure 10 The following are the substrates selected in the in vitro substrate diversity enzymatic reaction of GbCYP90J6 provided in the examples, the LC-MS results of positive dimerization products, and the enzyme kinetic curve of GbCYP90J6 for the substrate apigenin.

[0102] Figure 11 The catalytic activities of the mutants of GbCYP90J6 provided in the Examples. DETAILED DESCRIPTION

[0103] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0104] Example 1: Cloning of the gene encoding GbCYP90J6

[0105] In Ginkgo biloba L., compound tissue distribution analysis revealed that flavonoids were enriched in leaves ( Figure 2Based on the compound distribution results, bioinformatics analysis and transcriptome analysis were performed to identify the candidate gene GbCYP90J6 that is specifically expressed in leaves. Figure 2 As shown in Part B.

[0106] Healthy Ginkgo biloba leaves were collected and RNA was extracted using the Tiangen RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit, following the manufacturer's instructions. RNA was reverse-transcribed using the Takara PrimeScript RTMaster Mix (Perfect Real Time) Reverse Transcription Kit to generate cDNA, following the manufacturer's instructions. The resulting cDNA was used as a template for cloning using the primer pair 1.3004-f: 5'-ATGCAGATCGAGTGGCAAATG-3' (SEQ ID NO: 11) and 1.3004-r: 5'–TTATATATATTGGAGAGGTTTCACAGTAATGG–3' (SEQ ID NO: 12).

[0107] After agarose gel electrophoresis and gel recovery, the target fragment was added with homology arms using the primer pair EAQ-SmaI-1.3004-F: 5'-CACCATCACCATCATCCCGGGATGCAGATCGAGTGGCA-3' (SEQ ID NO: 13) and EAQ-XhoI-1.3004-R: 5'-GAAACCAGAGTTAAAGGCCTCGAGTTATATATATTGGAGAGGTTTCACAGTAATGG-3' (SEQ ID NO: 14). The fragment was inserted between the SmaI and XhoI sites of the pEAQ-HT vector using In-Fusion (Vazyme) and transformed into DH10B competent cells.

[0108] Positive single clones were expanded and cultured, and plasmids were extracted using the Axygen Plasmid Extraction Kit (AxyPrep Plasmid DNA Mini Kit) and sequenced by Sangon Biotechnology Engineering Co., Ltd. The plasmid extraction method followed the manufacturer's instructions. The plasmid that was confirmed to be correct by sequencing was named pEAQ-GbCYP90J6 (see Figure 1). Figure 3 ), used for Agrobacterium transformation.

[0109] According to sequence similarity analysis, the target gene was named GbCYP90J6, which contains 1434 bp of nucleotides and encodes a protein of 477 amino acids. The protein was named GbCYP90J6, and its amino acid sequence and encoding nucleotide sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0110] Example 2: Functional verification of GbCYP90J6 in tobacco

[0111] The plasmid pEAQ-GbCYP90J6 was transformed into Agrobacterium GV3101 competent cells.

[0112] The specific conversion method is:

[0113] 1) Take the GV3101 competent cell stored at -80°C, add 4 μL of the pEAQ-GbCYP90J6 plasmid, mix gently, and place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0114] 2) Add 1 mL of sterile LB medium to the centrifuge tube, mix well, and place in a shaker at 30°C and 180 rpm for 2-3 hours to recover the culture.

[0115] 3) Apply 100 μl of bacterial solution to the Kan R +Rif R +Gen R The plate was placed upside down on the antibiotic-containing LB plate and cultured in a 28°C incubator for 2 days.

[0116] Monoclonal colonies were selected from the plates for colony PCR verification to screen out positive monoclones.

[0117] Transient transformation of tobacco leaves with Agrobacterium

[0118] 1) Agrobacterium positive clones were inoculated into 2 ml of Kan R +Rif R +Gen R The culture medium was LB (100 mL Erlenmeyer flask) containing antibiotics and cultured at 30°C and 220 rpm for 24 h.

[0119] 2) 0.2% v / v inoculation ratio: transfer fresh Agrobacterium to 20 mL of Kan R +Rif R +Gen R Culture overnight in LB liquid medium containing antibiotics until OD 600 =1.0.

[0120] 3) Centrifuge at 6000 rpm for 5 minutes, discard the supernatant, and wash the cells once with resuspension buffer. (Resuspension buffer: 1 mL 1M MgCl2, 1 mL 1M MES, 100 μL 200 mM Acetosyringone per 100 mL)

[0121] 4) Centrifuge at 6000 rpm for 5 minutes, discard the supernatant, and add resuspension buffer to adjust the bacterial OD 600 =0.8.

[0122] 5) Leave at room temperature for 2-3 hours.

[0123] 6) Draw Agrobacterium into a 1 mL syringe without a needle. Use your thumb to press the syringe back to inject the liquid into a 4-week-old tobacco leaf through the lower epidermis of the leaf. For each sample, inject one leaf from each of three plants as replicates.

[0124] 7) The GV3101 strain carrying the pEAQ-HT empty vector was used as a negative control, and the rest of the procedures were the same as those of the experimental group.

[0125] 8) Three days after the Agrobacterium injection, perform substrate injection. The substrate dissolution system is as follows: add the apigenin stock solution to a high-concentration methyl-β-cyclodextrin solution. After complete dissolution, dilute with water to a final concentration of 100 μM apigenin and 4.75 mM methyl-β-cyclodextrin, respectively.

[0126] 8) Two days after substrate injection, leaves were harvested for compound extraction and detection.

[0127] Tobacco leaf collection and compound extraction

[0128] 1) Weigh 0.3 g of each leaf sample, freeze it in liquid nitrogen, and grind it using a tissue grinder;

[0129] 2) Extract with 1 mL of water-saturated n-butanol three times, evaporate the solvent, and reconstitute with 200 μL of methanol;

[0130] 3) Centrifuge at 12000 rpm for 10 min;

[0131] 4) The supernatant was used for LC-MS detection

[0132] 5) LC–MS / MS detection was performed using Thermo Q Exactive_UV_small molecule104 and Phenomenex column 00D-4462-AN ( 2.6μm C18 LC column (100 x 2.1 mm, Ea). Mobile phase A was ddH2O + 0.1% formic acid, and mobile phase B was acetonitrile. Column temperature was 45°C, flow rate was 0.3 mL / min. Gradient elution conditions were: 10% B (0-1 min), 10%-99% B (10-15 min), 99% B (15-18 min), and 10% B (18-21 min). UV wavelength was 280 nm.

[0133] 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 temperature 300°C, desolvation gas flow rate 600 L / h, cone gas flow rate 5 L / h. The full-scan mass spectrometry mass scan range was m / z 100 to 1500 Da, and the MS / MS secondary scan collision energy was 20.0 V.

[0134] The test results showed that the reaction equation catalyzed by GbCYP90J6 was as follows Figure 1 GbCYP90J6 catalyzes the dimerization of apigenin monomers to form aconitol (m / z: 537.08 [MH] - ), negative control aconitum biflavonoids product. Figure 4 shown.

[0135] Example 3: Screening of GbCYP90J6 isozymes and verification in Escherichia coli

[0136] GbCYP90J6 is the first plant-derived P450 enzyme discovered to date that can catalyze the formation of intermolecular carbon-carbon bonds; it is also the first site-specific flavonoid dimerization enzyme. To discover more flavonoid dimerization enzyme elements, we used Orthofinder to search for orthologous genes and constructed a phylogenetic tree ( Figure 5 ). The results showed that GbCYP90J6 belongs to the CYP90J subfamily, clustered in a separate branch with CYP90J members from other gymnosperms, and adjacent to CYP90E subfamily members from lycopods, which are more upstream in the evolution of gymnosperms. At the same time, both CYP90J and CYP90E are far away from the known CYP90 subfamily members with steroid hydroxylase activity (CYP90A, CYP90B, CYP90C, CYP90D). The discovery of the flavonoid dimerization functional activity catalyzed by GbCYP90J6 has expanded the inherent understanding that the CYP90 subfamily only catalyzes steroid hydroxylation modifications, and speculates that the unique branch formed by CYP90J and CYP90E subfamily members has a rare flavonoid dimerization function, which is responsible for catalyzing the intermolecular polymerization of flavonoid monomers.

[0137] To verify our bioinformatics hypothesis, we selected ChCYP90J7 from Cephalotaxus harringtonia, SmCYP90E3v1 from Selaginella moellendorffii, TcCYP90J7 from Taxus chinensis, CpCYP90J8 from Cycas panzhihuaensis, MgCYP90J1 from Metasequoia glyptostroboides, and TpCYP90J1v1, TpCYP90J9, and TpCYP90J10 from Thuja pilcata (amino acid sequences shown in SEQ ID NOs: 3-10, respectively) as candidate flavone dimerases for validation. All protein coding sequences were codon-optimized and obtained by gene synthesis (Universal Biotechnology). Similarly, apigenin was used as a representative flavonoid unit to verify the activity of flavonoid dimerization enzyme.

[0138] For the candidate enzymes screened, we used the in vivo biotransformation method in E. coli to verify them. Specifically, pFe00 (such as Figure 6 The plasmid (shown) was used as a negative control for the functional verification of cytochrome P450 enzymes in Escherichia coli. First, TMHMM was used to predict and remove the N-terminal transmembrane regions of all cytochrome P450 proteins (sequence SEQ ID NO: 1, the underlined region of SEQ ID NO: 3-10), and then the 17α solubility tag (protein sequence: MALLLAVF (SEQ ID NO: 17) was fused to the N-terminus. The obtained sequence was inserted into the NcoI and BamHI sites of the pFe00 plasmid to construct plasmids pFe00-trGbCYP90J6, pFe00-trChCYP90J7, pFe00-trTcCYP90J7, pFe00-trCpCYP90J8, pFe00-trMgCYP90J1, pFe00-trTpCYP90J1v1, pFe00-trTpCYP90J9, pFe00-trTpCYP90J10 and pFe00-SmCYP90E3v1 (as shown in Figure 2). Figure 7 shown).

[0139] These recombinant plasmids were transformed into Escherichia coli JM109 (DE3) strains for functional characterization, with the strain carrying the pFe00 plasmid as a negative control. These engineered bacteria were named JM109(DE3):pFe00-trGbCYP90J6, JM109(DE3):pFe00-trChCYP90J7, JM109(DE3):pFe00-trTcCYP90J7, JM109(DE3):pFe00-trCpCYP90J8, JM109(DE3):pFe00-trMgCYP90J1, JM109(DE3):pFe00-trTpCYP90J1v1, JM109(DE3):pFe00-trTpCYP90J9, JM109(DE3):pFe00-trTpCYP90J10 and JM109(DE3):pFe00-trSmCYP90E3v1. First, the engineered bacteria were inoculated into 2 mL LB (containing 50 μg / mL spectinomycin ( R )) and cultured overnight at 37°C and 220 rpm to obtain seed solution. The seed solution was inoculated into 10 mL TB medium (containing 2% w / v glycerol, 50 μg / mL Spec R ), culture at 37°C, 220 rpm until OD 600 =0.6-0.8, ice-bath for 10 minutes, add IPTG to a final concentration of 0.1 mM and apigenin dissolved in 50 mM methyl-β-cyclodextrin (final concentration of apigenin is 100 μM, final concentration of methyl-β-cyclodextrin is 4.75 mM), and continue culturing at 22°C, 220 rpm for 96 h.

[0140] After the incubation period, samples were post-processed and analyzed by LC-MS / MS. Specifically, 1 mL of bacterial culture was ultrasonically disrupted at 40% power, followed by extraction three times with equal volumes of ethyl acetate. The organic phases were combined, the solvent evaporated, and then reconstituted with 200 μL of methanol. The mixture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was analyzed by LC-MS. The detection method was the same as described in Example 2.

[0141] The results of in vivo validation in Escherichia coli showed that GbCYP90J6, ChCYP90J7 and SmCYP90E3v1 could catalyze the 3'-8" site-specific dimerization of apigenin monomer to produce aconitol (m / z: 537.08 [MH] - ), negative control pFe00 aconitum biflavonoids product. The other test genes did not show dimerization activity for the substrate apigenin. LC-MS results are as follows Figure 8The above protein catalyzes the carbon-carbon bond between C3' and C8" of apigenin molecules to form the apigenin dimer aconitum biflavonoids. The reaction structure is shown in Figure 1 shown.

[0142] Due to the large number of biflavonoid skeleton types, we also selected (S)-naringenin ((S)-naringenin), which is upstream of flavonoid biosynthesis, as a substrate and tested the flavonoid dimerase activity of candidate CYP90J and CYP90E family members. The results showed that some of the tested enzymes had flavonoid dimerase activity for (S)-naringenin. Specifically, GbCYP90J6, ChCYP90J7 and MgCYP90J1 could catalyze (S)-naringenin to produce two polymerization products, bi-naringenin a (structure to be identified) and bi-naringenin b (structure to be identified), while SmCYP90E3v1 could catalyze (S)-naringenin to produce bi-naringenin a ( Figure 9 )). The reaction structure is as follows Figure 1 shown.

[0143] Example 4: Evaluation of the catalytic activity of GbCYP90J6 for apigenin substrate and exploration of substrate diversity

[0144] Since GbCYP90J6 is the first discovered plant-derived flavonoid dimerase that catalyzes the formation of site-specific carbon-carbon bonds between molecules, we used GbCYP90J6 as a representative of this newly discovered flavonoid dimerase to explore substrate diversity. Based on the biosynthetic pathway of flavonoids, we screened the flavonoids for testing according to the structural classification of different flavonoids. Excluding the verified (S)-naringenin and apigenin, we specifically selected luteolin and triticum vulgare flavonoids, which belong to flavonoids, eriodictyol and citronellin, which belong to flavanones, kaempferol, quercetin and myricetin, which belong to flavonols, and taxifolin and dihydromyricetin, which belong to dihydroflavonols (such as Figure 10 To avoid false negative experimental results caused by the difference in permeability of E. coli cells to different substrates, the exploration of substrate diversity was carried out using an in vitro enzymatic reaction protocol.

[0145] The specific protocol was as follows: using the pEAQ-GbCYP90J6 vector as a template, the coding region of GbCYP90J6 was amplified by PCR using the following primer pair: ESC-GbJ6-F / -R (ESC-GbJ6-F: 5'-GTAAGAATTTTTGAAAATTCGAATTCATGCATCATCATCATCATCACCAGATCGAGTGGCAAATGGCAC-3' (SEQ ID NO: 15); ESC-GbJ6-R: 5'-GTAATCCATCGATACTAGTGCGGCCGCTTATATATATTGGAGAGGTTTCACAGTAATGGGAAAG-3' (SEQ ID NO: 16)). The PCR product was digested with EcoRI and NotI (NEB, USA) and ligated into the pre-digested pESC / -HIS vector to construct the recombinant plasmid pESC-GbCYP90J6. The correctly constructed recombinant plasmid was transformed into Saccharomyces cerevisiae WAT11. The transformed recombinant yeast strain was cultured on a histidine-deficient synthetic medium (SD / -His) containing 2% glucose at 30°C for 48 hours. A single colony was picked and inoculated into 200 mL of SD / -His culture medium containing 2% glucose and cultured until the OD 600 After reaching 1.0, the medium was replaced with SD / -His medium containing 2% galactose and the expression was induced for 12 hours. Yeast microsomes were extracted according to the existing method.

[0146] The in vitro enzymatic reaction system used for kinetic analysis consisted of 1 mL of a 1% lysate containing 500 μg of yeast microsomal protein, 50 mM Tris-HCl buffer (pH 7.5), 1 mM NADPH, 5 μM FAD, 5 μg FMN, 4 mM glucose-6-phosphate, 1 U glucose-6-phosphate dehydrogenase, and apigenin at concentrations ranging from 0.1 to 30 μM. Three biological replicates were performed for each reaction. After incubation at 30°C for 1 hour, the reaction was terminated by extraction three times with 1 mL of ethyl acetate. The supernatants were combined, dried, and reconstituted in 100 μL of methanol. Product quantification was performed by high-performance liquid chromatography (HPLC). Curve fitting analysis was performed using GraphPad Prism 10 software based on the Michaelis-Menten model. In addition, to study the in vitro catalytic ability of GbCYP90J6 towards various flavonoid substrates, each substrate was added to the reaction system at a concentration of 25 μM. After the reaction, the mixture was extracted three times with 1 mL of water-saturated n-butanol. The supernatants were combined and dried, and then redissolved in 100 μL of methanol. The products were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0147] The test results are as follows Figure 10 As shown, the K of GbCYP90J6 for the substrate apigenin m The value was 0.4925 μM, indicating its high substrate affinity for apigenin. In addition, the substrate diversity exploration of GbCYP90J6 showed that in addition to exhibiting flavonoid dimerization activity on (S)-naringenin and apigenin, it also exhibited flavonoid dimerization activity on the substrate triticum flavonoids, generating triticum bi-flavonoids (bi-tricetin, structure to be determined) ( Figure 10 ). The reaction structure is as follows Figure 1 shown.

[0148] These functional validation results indicate that members of the CYP90J and CYP90E subfamilies, represented by GbCYP90J6 and SmCYP90E3v1, are specifically evolved enzymes with flavonoid dimerization activity. Although some of the CYP90Js selected for testing did not exhibit dimerization activity against the substrates (S)-naringenin and apigenin, given the diversity of flavonoid monomers and polymerization sites in biflavonoid compounds, our testing range is far from covering all flavonoid monomer combination patterns. Therefore, based on the evolutionary characteristics of enzyme function, they are still considered to be members of the CYP90 subfamily—CYP90J and CYP90E—with unique flavonoid dimerization activity.

[0149] Example 5: Design and activity verification of GbCYP90J6 mutants

[0150] In order to analyze the key amino acids related to the activity of GbCYP90J6 and improve the activity of its flavonoid dimerization enzyme, we constructed a series of mutants of GbCYP90J6. The expression form of the listed mutant names is: amino acid before mutation-amino acid site-amino acid after mutation. All mutants were obtained by reverse PCR using primers using the pFe00-trGbCYP90J6 plasmid as a template and verified by sequencing. The activity detection of the mutants was carried out with reference to the scheme described in Example 3, and the wild-type GbCYP90J6 protein (WT) was used as a control for normalized analysis and the yield of flavonoids from GbCYP90J6 was used as the evaluation standard for quantitative analysis. All the mutants and their activities are shown in Figure 3. Figure 11 The mutation sites of all mutants are listed in Table 1.

[0151] Table 1. Mutation site information of each mutant

[0152]

[0153] Note: The symbol “_” indicates that the flavonoid dimerization enzyme has the same amino acid difference before and after the symbol.

[0154] Sequence information:

[0155] GbCYP90J6 amino acid sequence (SEQ ID NO: 1), the N-terminal transmembrane region is underlined

[0156] MQIEWQMALLYIFGSLLAVLIGSLYVAML WAGQLNRPKRAPPGSFGLPVIGEMLDFVSDHKSEFPQAYAEKRRAKYGDVFTTHHMCYPTVMSVDPEVNKFVLQNEGKYFISRYPKSLQNLFGEGAILRAHGDLHKRLHNMA LSFINSTKLKEYVMADVEQFVTSAMTNWVGRTVYVEDEAKTIAFQVIIKYLLGLMPGEETAYLMHEYRKLIEGVISIPVQIPGTQYYKSMRARENVIKIVKKIIEERRRHPD AEHRDILAALMAETNENGEKFSDNVICSNILNFMGAGEETSPMIMMCAVKYISECSKAYDELREEHMTIRKTKRAGEGLLTWSDYTSMTFTHQVINETLRLVNPASGVLREAI RDVEVKGYFIPKGWLVLPYFRSVHVDETVYPDPLSFNPWRWKEKIPSTYFSPFGGGTRLCAGQELAKLEVAIFLHHLVTQFTWEAHGDEIINFPAVTFKNLFPITVKPLQYI

[0157] GbCYP90J6 encoding gene sequence (SEQ ID NO: 2)

[0158]

[0159] ChCYP90J7 amino acid sequence (SEQ ID NO: 3), the N-terminal transmembrane region is underlined

[0160] MFQRFCEILTLMMITVLQVVGAVVTGVLGCLWLAM LWAGQLNRPKRAPAGSLGLPVIGEMLEFVIDAKSKSPHVYAEKRKAKYGPVYTTHHMGYPTVMSVDPEVNKFIMQNEGKYFVSNYPRSLRNLFGEGAIVRVYGDLHKRIHNV VLSFINTTKLKDSVLADVEHMIKTPMSNWEGRTVYVEDECKTMAFNIITKYLLGLMPGEETSYLMTEYRKFIEGIISIPIKLPGTPYYRSMKAKEKLVKVMKRIVEERRRQP PSEPRDIIGALMAESGENGEKLPDTIIYSNIMNLWGAGEETSPMAMMCAVKYITECPRALDQLKEEHFAIKRTKAPGESLTWNDYANRMTFTHQVINETLRLVNPASGVLRR AIKDVEVKGYFFPKDWLVFPYIRSVHLTESVYPDPLTFNPWRWEKKIPSMYFTPFGGGLRVCAGQELAKLEISVFLHYLVTQFTWILQDDEQINFPAVTFKNQLPMTVKALQH

[0161] SmCYP90E3v1 amino acid sequence (SEQ ID NO: 4), the N-terminal transmembrane region is underlined

[0162] MVDSRIMVWTSIAGIAGALWLVALVYWRSWRFRKLERLPPGSMGWPLIGELIPYVTIARSEAPYIFPMEREKKYGTVFKTSLLTGKTIMITDVEGVKFVLHNEGVLFESGYPRSLKDVLGEHAMLFQHGELHKRMHAMLKRFVSSTPLKKHLTREMELLTKQGMSTWSRGTKILLQDEIQRITHDFLMKQLFGLEPGKLSATIFEEFNRLMGGIIGIPLMIPGTPYHRAMKARKKLTKIISGMVAFRRSRPDIEHKDILNALIEEIKQEERDADQIIVDNALINIANAEGIPAVIIAFAVKNLSENPKALEQIREENLAIRKGKDPSEGLTWNEYMSLDFTHAVFNETLRLANGAQGVMRKALKDVEFRGYVIPKGWTVLPYFLNIHFDEKMFPQPTKFHPWRWLEVLSTTSHSCNFATWLQQRSIPSSYVLPFGGGARLCPGQELAKVQTAVFLHHFVTQFRWEAEPEKVINFPKISTKNHLPVVLHDL

[0163] The amino acid sequence of TcCYP90J7 (SEQ ID NO: 5), with the N-terminal transmembrane region marked by an underline

[0164] MAVLQILGAVIAGIAGFLWLVMLWAGQLNRPKRAPPGSFGLPGIGETLQFAIDGRSLTPHVYADKRKAKYGPVYTTHHMGYPAVMSVDPEVNKFIMQNEGKYFVSNYPRSLRNLFGEGAIVRVYGDMHKRIHNVVLSFINTTKLKEYVLADVERMIKTPMTNWAGRTVYVEDECKTMAFNIVTKYLLGLMPGDETTYLMAEYRKFIEGIISIPIKLPGTQYYRSMKAKEKCVKVMEKIMQEKRNQPQSEANDIISALMAESSENGEKLSDTIICNNIMNLLGAGEETSPMAMMCAVKYITECPKALEQLREEHLAIKKSKGPGESLTWNDYTNRMTFTHYVINETLRLVNPASGVLRRAIKDVEFKGYFFPKDWMAFPYIRSVHLEESVYPDPFTFNPWRWEKKIPSMYFTPFGGGLRVCAGQELAKLEISVFLHYLVTQFTWTLHDDEQINFPAVTFKKQLPMT

[0165] The amino acid sequence of CpCYP90J8 (SEQ ID NO:6), with the N-terminal transmembrane region underlined

[0166] MGLLLQIFAVVVAGVVALAWLIMLWAGQL NRHKRAPPGSLGLPLIGETLEFVLDSKSKTPQIYAEKRRAKYGDVFTTHHMGYPTVMSVDAEVNKFILQNEGKYFESNYPRSLRNLFGEGAILRTYGETHKKLHNLVLSFINTAKLKEYVMADVEFLIKRAMANWEGRTVYVEDEAKTLAFNVIINYLIGVMPGKETTYLMQEYRKLIEGVISIPIRIPGTQFARSMTAKENVVRELQKIIDERRKQPDVPHTDILAAMMAERDEKGQPVYSDTVICNNMVNFMGAGEETSPMAIMLSVKYLSDCPKALDQLREEHLSIRKTKQPGEGLTWSDYTSMTFTSMVVINETLRIANPASGVLRRATKDVEVKGYFIPKDWLVFPYFRSVHYDETVFPDPLTFNPWRWEKKIPSAYFCPFGGGQRICAGQELARLEIALFLHFLVTQFTWEVHEDELINYPAVHFKHNLPITVKALHCH

[0167] MgCYP90J1 amino acid sequence (SEQ ID NO: 7), the N-terminal transmembrane region is underlined

[0168] MAVLQILGAAVAGILGCLWLAMLWAG QLNRPKRAPPGSFGLPVIGETLQFVIDGRSKSPQVYADKRKAKYGQAVYTTHHMGYPTVMSIDPEVNKFILQNEGKYFQSNYPRSLRNLFGEGAIASTHGDLHKRIHNVVL SFINTTKLKEIVMADVEYIIKTAMANWEGRTVYVEDECKTMAFNIITKYLLGLKPGEETTYLMMEYRKFIEGIISIPIKIPGTYYYRSIKCKENCVKIMKKIIEERRRKSQS EHKNILDALLADTDKNGEKIPDSIIYNNIMNLYGAGEETSPMAMMCAVKYIYECPKALDQLREEHFAIRRTKRPEEPLSWNDYTNCMTFTHQVINETLRLVNPAAGVNRKA VQDVEFKGYFFPKGWLVFPYFRNVHLDESVYPDPLTFNPWRWEKKIPSMYFTPFGGGPRMCAGQELAKLEIAIFLHYLVTQFTWIHHDDEIINFPAVSFKNKLPITVKALQH

[0169] TpCYP90J1v1 amino acid sequence (SEQ ID NO: 8), the N-terminal transmembrane region is underlined

[0170] MAVLQILGAVVAGILGFLWLAMLWAGQLHRPKRAPPGSFGLPVIGETLEFIRDSRSKNPQVYADKRKAKYGQPVFTTHHMGYPTVMSIDPEVNKFILQNEGKYFLSNYPRSVRSLFGEGSIASTYGDLHKRIHNVVLSFINTTKLKEIVMADVEYIIKTSMSNWEGRTVYVEDDCKMMAFNIITKYLLGLRPGEETKYLMKEYKKFIEGIISLPIRIPGNYYNRSMTSKENCIKVMEKIVEERRKNPHIENKDILDALMNDSDQNGGKIPDHIIYNNIMNLFGAGDETSPMAMMCAVKYVSECPKALDQLRVINETLRLVNPAAGVNRKVVQDVEFKGYFFPKGWLAFPYFRNVHLDESVYPDPLTFNPWRWEKKIPSIYFTPFGGGPRICAGQELGKLEIAILLHYLVTQFTWVHHEDEIINFPAVSFVNKLPITVKPLQ

[0171] The amino acid sequence of TpCYP90J9 (SEQ ID NO:9), with the N-terminal transmembrane region underlined

[0172] M LWAGQLNRPKRALPGSFGLPIIGETYQFARDGRSKTPHVYADKRKIKYGQPVFTTHHMGYPTVMSIDPEVNKFILQNEGKYFVSNYPRSLRDLFGKGSIASTNGELHKRIHNVVLSFINTTKLKEFVMADVVHVIKTVLRKWGRRTVGRDPYLMMEYQKFIEGIISIPIKIPGTSYYRSLKCKENCVKVMKRIVEEKRRKPHSEQKDILDALLADSDQNGEKIPDSIIYNNIMNLFGAGEETSPVAMMCAAKYMYECPKALDQLWEEHCAIIRMKGQGEPLTWNDYTNHMTFTHQVINETLCLVNPASGVNRKVVQEVEFKGYFFSKGWLAFPYFRNVHLDESVYLDPLTFNPWRWEKKIPSMYYTPFGGGPRICAGQELAKLEVAIFLHYLVTQLMHKSNPFLHISEYIWIHHDDEIINFLAMSFKNKLPITVEALRC

[0173] TpCYP90J10 amino acid sequence (SEQ ID NO: 10), the N-terminal transmembrane region is underlined

[0174] MAVLQILGAVVAGILGFLWLAMLWAG QLHRPKRAPPGSFGLPVIGETLEFIRDSRSKNPQVYADKRKAKYGQPVFTTHHMGYPTVMSIDPEVNKFILQNEGKYFLSNYPRSVRSLFGEGSIASTYGDLHKRIHNVVLSFINTTKLKEIVMADVEYIIKTSMSNWEGRTVYVEDDCKMMAFNIITKYLLGLRPGEETKYLMKEYKKFIEGIISLPIRIPGNYYNR SMTSKENCIKVMEKIVEERRKNPHIENKDILDALMNDSDQNGGKIPDHIIYNNIMNLFGAGDETSPMAMMCAVKYVSECPKALDQLRVINETLRLVNPAAGVNRKVVQDVEFKGYFFPKGWLAFPYFRNVHLDESVYPDPLTFNPWRWEKKIPSIYFTPFGGGPRICAGQELGKLEIAILLHYLVTQFTYKSNPFLHIS

[0175] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may 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 flavonoid dimerization enzyme, characterized in that The amino acid sequence of the flavonoid dimerase has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and comprises one or more amino acid residue differences at positions L47, L127, M140, V154, V215, L382 or A426 compared to the amino acid sequence shown in SEQ ID NO:

1.

2. The flavonoid dimerization enzyme according to claim 1, wherein The amino acid residue difference at position L47 is L47W; and / or the amino acid residue difference at position L127 is L127V; and / or the amino acid residue difference at position M140 is M140L; and / or the amino acid residue difference at position V154 is V154W; and / or the amino acid residue difference at position V215 is V215A; and / or the amino acid residue difference at position L382 is L382F; and / or the amino acid residue difference at position A426 is A426P; Preferably, the flavonoid dimerization enzyme has any of the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 1: L47W; L127V; M140L; V154W; V215A; L382F; L382F / A426P; A426P / M140L; A426P / V215A; L127V / A426P; L382F / A426P / L127V; L382F / A426P / V154W; A426P / L47W / V154W; A426P / L127V / V154W; L382F / L47W / V154W; L127V / L47W / V154W; L382F / L127V / V154W; L382F / L127V / L47W; A426P / M52F / I146M; And / or, the first 29 amino acid residues at the N-terminus of the flavonoid dimerase are replaced with the amino acid sequence shown in SEQ ID NO:

17.

3. An isolated nucleic acid, characterized in that The nucleic acid encodes the flavone dimerization enzyme according to claim 1 or 2.

4. A recombinant expression vector comprising the nucleic acid according to claim 3, wherein the backbone of the recombinant expression vector is preferably pEAQ or pFe00.

5. A transformant comprising the nucleic acid according to claim 3 or the recombinant expression vector according to claim 4; Preferably, the host cell used in the construction of the transformant is Escherichia coli or Agrobacterium, the Agrobacterium is more preferably Agrobacterium GV3101, and the Escherichia coli is more preferably Escherichia coli Jm109 (DE3).

6. A method for preparing flavonoid dimerization enzyme, comprising culturing the transformant according to claim 5 to obtain a fermentation product; Preferably, the culture medium used is selected from LB liquid medium or TB liquid medium; More preferably, the method further comprises the step of purifying the fermentation product to obtain the flavonoid dimerization enzyme.

7. An enzyme combination, characterized in that The enzyme combination comprises: the flavonoid dimerization enzyme according to claim 1 or 2 and at least two flavonoid dimerization enzymes having at least 95% sequence identity compared with the amino acid sequence shown in any one of SEQ ID NOs: 1 and 3-10.

8. Use of the flavonoid dimerization enzyme according to claim 1 or 2, a flavonoid dimerization enzyme having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 1 and 3-10, the nucleic acid according to claim 3, the recombinant expression vector according to claim 4, the transformant according to claim 5, or the enzyme combination according to claim 7 in preparing carbon-carbon bond-linked biflavonoids, wherein the preparation of the carbon-carbon bond-linked biflavonoids uses a flavonoid compound as a substrate; Preferably, the flavonoid compound is selected from flavanones, flavones, flavonols, anthocyanidins, isoflavans, isoflavones, neoflavans, dihydroflavones, dihydroflavonols, chalcones, aurone and xanthones; the flavones preferably include apigenin, luteolin and triticum vulgare flavonoids, the flavanones preferably include eriodictyol and citronellol, the flavonols preferably include kaempferol, quercetin and myricetin, the dihydroflavonols preferably include taxifolin and dihydromyricetin, and the dihydroflavones preferably include (S)-naringenin; And / or, the carbon-carbon bond-linked biflavonoids include aconitum biflavonoids, naringenin biflavonoids B, naringenin biflavonoids A and triticum vulgare biflavonoids; and / or, the linking sites of the carbon-carbon bond-linked biflavonoids include 2-3", 2'-2"', 2'-6", 2'-8", 3-3", 3-3"', 3'-3"', 3'-4"', 3'-5", 3-6", 3'-6", 3-7", 3'-7", 3-8", 3'-8", 4-6", 4-8", 4'-8", 5-5", 6-6", 6-γ, 6-8", 7-7" and 8-8", preferably 3'-8"; More preferably, the preparation of the aconitum biflavonoids uses apigenin as a substrate, using the flavonoid dimerizer according to claim 1 or 2, or a flavonoid dimerizer having an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1 and 3-4; the preparation of the naringenin biflavonoids uses (S)-naringenin as a substrate, Using the flavone dimerization enzyme according to claim 1 or 2 or a flavone dimerization enzyme having an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1, 3-4 and 7; preparing the naringenin biflavonoid ethyl using (S)-naringenin as a substrate, Using the flavonoid dimerization enzyme as described in claim 1 or 2 or a flavonoid dimerization enzyme whose amino acid sequence has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1, 3 and 7; preparing the triticum biflavonoids using triticum flavonoids as a substrate and using the flavonoid dimerization enzyme as described in claim 1 or 2.

9. A method for preparing carbon-carbon bond-linked biflavonoids, characterized in that: The method comprises: reacting the flavonoid dimerization enzyme according to claim 1 or 2 or the flavonoid dimerization enzyme having an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1 and 3-10 with a flavonoid compound; the flavonoid compound and the carbon-carbon bond-linked biflavonoid are as defined in claim 8; Preferably, the flavonoid dimerization enzyme is present in yeast microsomal protein, and the reaction comprises 100-1000 μg yeast microsomal protein, 0.01-100 μM flavonoid compound, coenzyme, and hydrogen donor and / or hydrogen donor regeneration system in each mL reaction system, wherein the coenzyme is preferably FAD and FMN, the hydrogen donor is preferably NADPH, and the hydrogen donor regeneration system preferably comprises glucose-6-phosphate and glucose-6-phosphate dehydrogenase; the reaction preferably comprises 200-500 μg yeast microsomal protein, 0.1-30 μM flavonoid compound, 0.1-5 mM NADPH, 1-10 μM FAD, 1-10 μg FMN, and 1-10 mM glucose-6-phosphate and 0.1-5 U glucose-6-phosphate dehydrogenase; and / or the reaction temperature is 25-35°C, preferably 30-35°C; and / or the reaction time is 0.5-10 h, preferably 1-5 h; and / or the reaction pH is 7.0-8.0, preferably 7.0-7.5; And / or, the reaction comprises co-culturing the transformant according to claim 5 with the flavonoid compound, wherein the amount of the flavonoid compound added is 10-200 μM, preferably 100-200 μM; and / or, the reaction temperature is 20-25° C., preferably 22-25° C.; and / or, the reaction time is 12 h-144 h, preferably 48-96 h; and / or, the reaction system further comprises a cosolvent, the cosolvent is preferably a methyl-β-cyclodextrin solution, and the added concentration of the methyl-β-cyclodextrin solution is 1-20 mM, more preferably 4.75-10 mM; More preferably, the preparation of the aconitum biflavonoids uses apigenin as a substrate, using the flavonoid dimerizer according to claim 1 or 2, or a flavonoid dimerizer having an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1 and 3-4; the preparation of the naringenin biflavonoids uses (S)-naringenin as a substrate, Using the flavone dimerization enzyme according to claim 1 or 2 or a flavone dimerization enzyme having an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1, 3-4 and 7; preparing the naringenin biflavonoid ethyl using (S)-naringenin as a substrate, Using the flavonoid dimerization enzyme as described in claim 1 or 2 or a flavonoid dimerization enzyme whose amino acid sequence has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1, 3 and 7; preparing the triticum biflavonoids using triticum flavonoids as a substrate and using the flavonoid dimerization enzyme as described in claim 1 or 2.

10. A reaction system for preparing carbon-carbon bond-linked biflavonoids, characterized in that: The reaction system comprises: The flavonoid dimerization enzyme according to claim 1 or 2, a flavonoid dimerization enzyme having an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1 and 3-10, or the enzyme combination according to claim 7, and at least one of a flavonoid compound and a carbon-carbon bond-linked biflavonoid; The flavonoid compound and the carbon-carbon bond-linked biflavonoid are as defined in claim 8; Preferably, the reaction system further comprises a cytochrome P450 enzyme redox partner, and the cytochrome P450 enzyme redox partner is NADPH-cytochrome P450 reductase and / or cytochrome b5; more preferably, the NADPH-cytochrome P450 reductase is AtCPR2 protein; and / or, the cytochrome b5 is RsCYB5 protein.

Images