A glycosyltransferase thugt1 and uses thereof

Abstract

This invention provides a glycosyltransferase ThUGT1, the nucleotide sequence of which is shown in SEQ ID NO.1. This invention also provides a method for synthesizing flavonoid compounds. This invention provides the flavonoid glycosyltransferase ThUGT1 gene from *Trifolium repens*, which can serve as a regulatory gene for the biosynthesis of components such as luteolin, kaempferol, and genistein, and can be applied to the preparation of luteolinoside, kaempferol-7-O-glucoside, and genistein. This invention utilizes a heterologous expression method to biosynthesize glycosides through microbial biosynthesis, and also verifies its function and enzymatic properties. The method exhibits strong site specificity, reacting only at position 7, good solubility, and a broad substrate scope, binding to most flavonoid compounds, especially the relatively scarce 7-O-glucoside transferase. The resulting mutant can improve the yield, enabling industrial production, promoting the development of *Trifolium repens* glycosides, and providing a foundation for the widespread application of *Trifolium repens*.

Description

Technical Field

[0001] This invention relates to a glycosyltransferase ThUGT1 and its use in the synthesis of flavonoids, belonging to the field of biotechnology. Background Technology

[0002] Natural products, especially plant secondary metabolites, are often used in the production of pharmaceuticals, nutritional supplements, and cosmetics due to their many pharmacological activities. In traditional Chinese medicine, plant glycosides such as puerarin and glycyrrhizic acid possess antibacterial, antitumor, and anti-inflammatory functions. However, the actual human utilization effect of purely natural products is not significant, mainly because the special chemical structure of natural products makes them unstable and water-soluble, resulting in low bioavailability.

[0003] To effectively address this issue, current research often utilizes glycosylation, methylation, hydroxylation, and isopentenylation reactions to enhance the complexity and diversity of natural product structures. Among these, glycosylation is the most widely applied chemical reaction, employing glycosyltransferases (GTs) to form specific glycosidic bonds between sugars and natural products to synthesize glycosides. Glycosyltransferases are key enzymes catalyzing the formation of glycosides from sugar donors and substrates, making them a focal point of research in traditional Chinese medicine resource science.

[0004] CN202410099420.3, Invention Title: Application and Preparation Method of a Glycosyltransferase and its Mutants in the Preparation of Trifolin, provides an application and preparation method of a glycosyltransferase and its mutants in the preparation of trifolin. Through screening, six genes with P4'-OGT activity were obtained, and PT577, with the highest conversion efficiency, was obtained, expanding the library of bio-factors for trifolin synthesis. This patent studied the ubiquitous enzyme catalytic activity in P4'-OGT and used virtual mutation, site-directed mutagenesis, and half-saturation point mutagenesis to obtain mutants with better specificity, laying an important foundation for the large-scale biosynthesis of trifolin and providing a reference for the functional exploration and computational simulation of other glycosyltransferases.

[0005] CN202410880641.4, Invention Title: Application of a Glycosyltransferase Mutant in the Catalytic Synthesis of Flavonoid Glycosides. This invention provides a mutant AAVT of the glycosyltransferase VcUGT1 screened from blueberries, which, compared to glycosyltransferase VcUGT1, exhibits point mutations: S367A, V274A, F82V, and I132T. The mutant significantly enhances the catalytic activity of glycosyltransfer reactions, efficiently catalyzing the formation of corresponding 7-O-glucoside compounds from substrates.

[0006] CN202110046315.X, Invention Title: A Method for Biocatalytic Synthesis of Multiple Flavonoid Glycosides. This invention utilizes protein engineering to modify glycosyltransferases to obtain mutants with higher yields and regioselectivity. The resulting mutant VFAH serves as a biocatalyst, using quercetin, kaempferol, luteolin, or isorhamnetin as acceptor substrates and UDP-glucose as donor substrates to catalyze the synthesis of multiple flavonoid glycosides. When quercetin is used as the acceptor substrate, the yield is approximately 90 times higher than the wild type, and the regioselectivity is greater than 98%. For the substrates kaempferol, luteolin, or isorhamnetin, while maintaining high regioselectivity and conversion rates, the main glycosylation products are altered.

[0007] CN201711454429.8, entitled "Glycosyltransferase CtGT-I, its encoding gene, derivatives, and uses," discloses a glycosyltransferase CtGT-I, its encoding gene, derivatives, and uses. Glycosyltransferase CtGT-I can catalyze the reaction of a glycosyl donor with various acceptors of different structural types to generate glycosylated products, exhibiting a high degree of heterogeneity in donor, acceptor, and catalytic forms. It can serve as a universal glycosyltransferase tool enzyme for the enzymatic synthesis of monosaccharide / polysaccharide glycosides of different structural types.

[0008] Currently reported glycosyltransferases have the following problems in the synthesis of glycosides:

[0009] 1. It has broad site selectivity but weak specificity.

[0010] 2. It has limited substrate selectivity and can only bind to a few substrates.

[0011] 3. Plant proteins mostly appear in the form of inclusion bodies and have low solubility.

[0012] 4. Few mutants can increase yield; most mutants decrease yield.

[0013] *Tetrastigma hemsleyanum* Diels et Gilg, a plant belonging to the genus *Tetrastigma* in the family Vitaceae, is a distinctive She ethnic minority medicinal herb, mainly distributed in Fujian, Zhejiang, and Jiangxi provinces. Both its tuberous roots and the whole plant can be used medicinally. It primarily contains flavonoids, phenolic acids, and volatile oils, possessing properties of clearing heat and detoxifying, promoting blood circulation and removing blood stasis, reducing swelling and relieving pain. Clinically, it is often used to treat viral pneumonia and jaundice hepatitis. In particular, the tuberous roots are traditionally used to treat high fever in children and are known as a "plant antibiotic." Currently, more than 20 glycosides, including quercetin glycoside, vitexin, and gentianin, have been isolated and identified from *Tetrastigma hemsleyanum*. These glycosides are mainly C-glycosides and O-glycosides with diverse glycosyl types. Therefore, it is of great significance to effectively and rapidly screen UGT genes that catalyze C-glycosides or highly catalyze O-glycosides, construct "UGTs-substrate" complex models, rationally modify or site-directedly mutate non-conserved amino acid residues of UGTs, and improve their catalytic activity and stereoselectivity. Summary of the Invention

[0014] The technical solution of the present invention is to provide a glycosyltransferase ThUGT1, and the present invention also provides the uses of the glycosyltransferase.

[0015] The flavonoid glycosyltransferase ThUGT1 gene described in this invention was extracted from Tripterygium wilfordii and identified after extensive screening and analysis using transcriptome sequencing and bioinformatics techniques. Total RNA was extracted using the Trizol method, reversed to cDNA, and then amplified by PCR.

[0016] This invention provides a glycosyltransferase ThUGT1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0017] It comes from the plant *Trifolium repens*.

[0018] This invention provides a recombinant plasmid containing the glycosyltransferase ThUGT1 gene. It is obtained by homologous recombination of the glycosyltransferase ThUGT1 gene with the pET-28a(+) vector, yielding the pET-28a(+)-UGTs recombinant plasmid.

[0019] The present invention provides a transgenic engineered bacterium containing the recombinant plasmid described above, or the genome of the genetically engineered bacterium is integrated with an exogenous glycosyltransferase ThUGT1 as described in claim 1 or 2.

[0020] This invention provides the use of the glycosyltransferase ThUGT1, the recombinant plasmid, and the transgenic engineered bacteria in the synthesis of flavonoids; wherein the flavonoids are as shown in Formula I or Formula II:

[0021]

[0022] In formula I: R5, R 3’ R 4’ R 5’ = Hydroxyl (-OH), methoxy (-OCH3), methyl (-CH3), glycoside (Glc), halogen (-F, -Cl, -Br, -l), alkyl (-R), amino (-NH2).

[0023] In Equation II: R3, R5, R 3’ R 4’ R6, R8 = hydroxyl (-OH), methoxy (-OCH3), methyl (-CH3), glycoside (Glc), halogen (-F, -Cl, -Br, -l), alkyl (-R), amino (-NH2).

[0024] The flavonoids mentioned include luteolin, kaempferol-7-O-glucoside, genistein, quercetin-7-O-glucoside, oroxylin A, styracin A-7-O-glucoside, apigenin-β-D-7-O-glucoside.

[0025] This invention provides a method for synthesizing flavonoids, which uses UDP-G as a glycosyl donor, flavonoids and flavonols of Formula III or IV as substrates, and glycosyltransferase ThUGT1 as a biocatalyst to synthesize flavonoids of Formula I or II.

[0026]

[0027] In formula I: R5, R 3’ R 4’ R 5’ = Hydroxyl (-OH), methoxy (-OCH3), methyl (-CH3), glycoside (Glc), halogen (-F, -Cl, -Br, -l), alkyl (-R), amino (-NH2).

[0028] In Equation II: R3, R5, R 3’ R 4’ R6, R8 = hydroxyl (-OH), methoxy (-OCH3), methyl (-CH3), glycoside (Glc), halogen (-F, -Cl, -Br, -l), alkyl (-R), amino (-NH2).

[0029] Specifically, the synthetic route is as follows:

[0030]

[0031] The substrates include kaempferol, luteolin, quercetin, styracin A, genistein, baicalin, physalisin, apigenin, and apigenin as substrates; the products are luteolinoside, kaempferol-7-O-glucoside, genistein, quercetin-7-O-glucoside, oroxylonoside A, physalisin A-7-O-glucoside, physalisin, and apigenin-β-D-7-O-glucoside.

[0032] Preferably, the substrate is kaempferol, and the synthesis conditions are: pH = 8; temperature 40°C.

[0033] The present invention also provides a method for detecting the flavonoids, which uses HUPLC and UPLC-TOF-MS / MS for detection;

[0034] UHPLC detection conditions are as follows:

[0035] Agilent HPLC 1290 system (Agilent Technologies, SantAClara, CA, USA); column: CORTECS UPLC C18 (2.1 × 100 mm, 1.6 μm); mobile phase: acetonitrile (A) - 0.1% formic acid water (B); gradient elution: 0.0 → 3.0 min, 5% A → 8% A; 3.0 → 8.0 min, 8% A → 12% A; 8.0 → 12.0 min, 12% A → 15% A; 12.0 → 14.0 min, 15% A → 16% A; 14.0 → 20.0 min, 16% A → 20% A; 20.0 → 30.0 min, 20% A → 80% A; 30.0 → 35.0 min, 80% A → 5% A; injection volume: 2 μL; flow rate: 0.20 mL / min. -1 Kaempferol, quercetin, and luteolin were detected at a wavelength of 350 nm; phloroglucin A, genistein, baicalin, astragalin, physalisin, apigenin, and salicumin were detected at a wavelength of 327 nm.

[0036] UPLC-TOF-MS / MS detection conditions:

[0037] Column: CORTECS™ UPLC C18 (2.1×100mm, 1.6μm); Mobile phase: Acetonitrile (A) - water containing 0.1% formic acid (B), gradient elution (0-3 min, 5%–5% A; 3.0–6.0 min, 5%–12% A; 6.0–13.0 min, 12%–12% A; 13.0–20.0 min, 12%–32% A; 20.0–25.0 min, 32%–42% A); Flow rate: 0.20 mL / min -1 Column temperature 45℃, injection volume 2μL;

[0038] The mass spectrometer used an ESI ion source, positive ion mode scanning, mass number: 50-1500; capillary voltage 2.5kV; nebulizer pressure 0.2Bar; desolventizing gas N2, desolventizing temperature 500℃, volumetric flow rate: 800L / h; cone gas N2, volumetric flow rate: 50L / h; ion source temperature: 120℃.

[0039] This invention provides the ThUGT1 gene, a flavonoid glycosyltransferase from *Tripterygium wilfordii*, which can serve as a regulatory gene for the biosynthesis of components such as luteolin, kaempferol, and genistein, and can be applied to the preparation of luteolinoside, kaempferol-7-O-glucoside, and genistein. This invention utilizes a heterologous expression method to biosynthesize glycosides through microbial biosynthesis, and also verifies its function and enzymatic properties. The method exhibits strong site specificity, reacting only at position 7, good solubility, and a broad substrate range, binding to most flavonoid compounds, especially the relatively scarce 7-O-glucoside transase. The resulting mutant can improve the yield, enabling industrial production and promoting the development of *Tripterygium wilfordii* glycosides, thus providing a foundation for the widespread application of *Tripterygium wilfordii*. Attached Figure Description

[0040] Figure 1 Candidate gene for *Trifolium repens* flavonoid glycoside glycosyltransferase;

[0041] Figure 2 Clustering heatmap of leaf component content of *Trifolium repens* under different light intensities (Note: L represents light intensity of 75 μmol·m) -2 ·s -1 M represents a light intensity of 150 μmol·m. -2 ·s -1 H represents a light intensity of 300 μmol·m. -2 ·s -1 (parallel three groups);

[0042] Figure 3 Clustering heatmap of candidate gene expression levels in Trifoliate orange;

[0043] Figure 4 ThUGT1-catalyzed conversion of luteolin to luteolinoglycoside: UHPLC and UPLC-MS detection chromatograms;

[0044] Figure 5 ThUGT1-catalyzed conversion of kaempferol to kaempferol-7-O-glucoside: UHPLC and UPLC-MS detection chromatograms;

[0045] Figure 6 UHPLC and UPLC-MS detection images of ThUGT1 catalyzing the formation of genistein from genistein;

[0046] Figure 7ThUGT1-catalyzed quercetin to quercetin-7-O-glucoside UHPLC and UPLC-MS detection chromatograms;

[0047] Figure 8 AUHPLC and UPLC-MS detection images of ThUGT1 catalyzing the formation of oroxylin from baicalin;

[0048] Figure 9 ThUGT1-catalyzed conversion of pectin A to pectin A-7-O glucoside: UHPLC and UPLC-MS detection chromatograms;

[0049] Figure 10 ThUGT1-catalyzed astragaloside UHPLC and UPLC-MS detection chromatograms;

[0050] Figure 11 ThUGT1-catalyzed salicylic acid UHPLC and UPLC-MS detection chromatograms;

[0051] Figure 12 UHPLC and UPLC-MS detection images of ThUGT1 catalyzing the formation of thyrotropin from thyrotropin;

[0052] Figure 13 ThUGT1-catalyzed conversion of apigenin to apigenin-β-D-7-O glucoside: UHPLC and UPLC-MS detection images;

[0053] Figure 14 pH-relative yield variation curve;

[0054] Figure 15 Temperature-relative yield curve;

[0055] Figure 16 Standard curve of kaempferol-7-O-glucoside;

[0056] Figure 17 Curves showing the changes in product content and reaction time;

[0057] Figure 18 Double reciprocal curve. Detailed Implementation

[0058] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0059] The *Tetrastigma hemsleyanum* used in the embodiments of this invention are all experimental seedlings from the light incubator in the seedling cultivation room of Fujian University of Traditional Chinese Medicine. They were identified by Fan Shiming, a senior experimentalist at the Medicinal Plant Laboratory of Fujian University of Traditional Chinese Medicine, as *Tetrastigma hemsleyanum* Diels et Gilg, a plant of the Vitaceae family.

[0060] Example 1: Screening of flavonoid glycosyltransferase genes

[0061] Using the Unigenes obtained from previous sequencing by our research group, we analyzed the annotation results from seven major databases (Nr, Nt, SwissProt, KEGG, PFAM, COG, and GO) to identify annotated UDP-glycosyltransferases and genes with Pfam annotation information of PF00201. This initial screening yielded 100 candidate glycosyltransferase genes related to flavonoid glycoside synthesis. Further analysis was conducted... Figure 1 .

[0062] All GT1 family glycosyltransferases have a special conserved sequence (PSPG Box) of 44 amino acids at their C-terminus, forming a UDP-glycodon donor binding "pocket." Taxonomically, GT1 belongs to the UGT (UDP-glycosyltransferase) superfamily. Therefore, we compared the sequenced genes with the PSPG Boxes of plant glycosyltransferases using the BLAST function in the NCBI database, screening for 73 protein sequences with a Percentage Identity (PERI) of over 60%. Based on literature review, glycosyltransferase sequences from grapes, Scutellaria baicalensis, and Andrographis paniculata were mostly between 1000-2000 bp in length, with the 442-507 amino acid range being the most favorable. Genes with CDS regions shorter than 1000 bp or longer than 2000 bp were removed, and 59 genes were obtained within the favorable range. The results are shown in Table 1.

[0063] Table 1. Alignment results of *Trifolium repens* gene protein sequences and PSPG Box protein sequences.

[0064]

[0065]

[0066]

[0067] Table 2 shows the removal of unqualified *Trifolium repens* flavonoid glycosyltransferase genes.

[0068]

[0069] Previous studies have confirmed that *Trifolium repens* has higher levels of active ingredients under high light intensity, which is more conducive to gene discovery. The 59 genes obtained through screening were tested under three different light intensities (75 / 150 / 300 μmol·m⁻¹). -2· s -1 Expression levels were calculated and compared to identify 7 candidate genes for *Trifolium repens* glycosyltransferase. The results are shown below. Figure 2 and Figure 3 (Them09G1235, Them12G00992, Them15G00856, Them19G00876, Them22G00575, Them22G00585, ThUGT1). Previous bioinformatics analysis and activity verification by our research group revealed that only ThUGT1 was active. Among them, Them26G00299.1 is ThUGT1.

[0070] Example 2: Heterologous expression of trifoliate flavonoid glycosyltransferase

[0071] The instruments are listed in Table 3, and the reagents are listed in Table 4.

[0072] Table 3 Experimental Instruments

[0073]

[0074] Table 4 Experimental Reagents

[0075]

[0076]

[0077]

[0078] The *Tetrastigma hemsleyanum* Diels et Gilg sample used in this experiment was identified by Senior Experimentalist Fan Shiming of the Medicinal Plant Laboratory at Fujian University of Traditional Chinese Medicine as a plant of the Vitaceae family. The sample was collected from a light incubator in the Medicinal Botanical Garden of Fujian University of Traditional Chinese Medicine. After fresh sample collection, it was rinsed with water, then the moisture was gently absorbed with absorbent paper, and the sample was flash-frozen in liquid nitrogen. Liquid nitrogen was then added to grind the sample into powder, and the powder was stored in an ultra-low temperature freezer at -80℃ for later use.

[0079] 1. Constructing vector plasmids

[0080] Fresh *Tripterygium wilfordii* leaves were collected, flash-frozen in liquid nitrogen, ground into powder, and RNA was extracted using the Trizol method. RNA concentration and quality were assessed using UV spectrophotometry and agarose gel electrophoresis. After confirming RNA quality, cDNA was synthesized via reverse transcription using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. Primers were designed and synthesized based on the screened *Tripterygium wilfordii* UGT gene sequences. PCR amplification was performed using the *Tripterygium wilfordii* cDNA as a template. PCR products were separated and purified using agarose gel electrophoresis. The Gel Extraction Kit is used to recover the target fragment from the PCR product; the product with the required mass concentration is ligated into the pET-28a(+) vector to construct the homologous recombinant plasmid pET-28a(+)-UGTs.

[0081] 2. Transformation of Escherichia coli and screening of positive clones

[0082] The recombinant plasmid pET-28a(+)-UGTs was transformed into E. coli BL21(DE3) competent cells using a chemical reagent method with 0.1 mol / L CaCl2. The UGTs recombinant bacterial solution was plated on LB agar plates containing ampicillin using a drug resistance screening method. Positive clones were selected and verified by Sanger sequencing. The positive clones with correct sequencing were identified as recombinant strain T1. After mixing with 50% glycerol, the mixture was stored at -80°C.

[0083] 3. Expression and purification of the target gene

[0084] The correctly sequenced strain T1 was cultured in LB medium at 37°C and 220 rpm. When the OD600 reached 0.4-0.6, IPTG was added to induce protein expression at 16°C and 220 rpm. After about 18 hours of expression, the bacterial cells were collected by centrifugation. The bacterial cells were then lysed and the supernatant was collected. The supernatant was purified according to the GST fusion protein purification kit. The protein purity and molecular weight were detected by SDS-PAGE vertical electrophoresis, and the protein concentration in the purified and concentrated protein solution was detected by BCA kit.

[0085] Example 3: Verification of Trifolium glycosyltransferase activity

[0086] 1. In vitro enzyme activity reaction

[0087] The catalytic reaction system consisted of 200 μL (see Table 5) and was reacted at 37℃ and 220 rpm for 12 h. The reaction was divided into two groups: a sample group (UDP-G, substrate, and purified enzyme) and a negative control group (UDP-G, substrate, and purified enzyme, inactivated at 100℃ for 10 min).

[0088] Table 5 Enzyme activation system

[0089]

[0090] 2. Detection Method

[0091] 2.1 Preparation of reference solution

[0092] Accurately weigh appropriate amounts of the following reference standards: kaempferol, luteolin, quercetin, styracin A, genistein, baicalin, astragalin, apigenin, kaempferol 7-O glucoside, luteolin, quercetin 7-O glucoside, oroxylumin A, genistein, styracin A-7-O glucoside, styracin, and apigenin 7-O-β-D-glucopyranoside. Dissolve in 50% methanol to prepare a single reference stock solution of approximately 10 mM. Accurately transfer an appropriate amount to a 1 ml volumetric flask and dilute to volume with 50% methanol to obtain a single reference solution of approximately 0.5 mM. Filter through a 0.22 μm filter membrane to obtain the reference solution. Further dilute to below μg / ml to obtain the reference solution for mass spectrometry.

[0093] 2.2 Sample Preparation

[0094] The reaction was terminated with 200 μL of methanol, immediately vortexed, centrifuged at 12000 rpm for 10 min, filtered through a 0.22 μm microporous membrane, and the liquid sample was prepared for use in the instrument.

[0095] 2.3 UHPLC detection conditions

[0096] The chromatographic system used was an Agilent HPLC 1290 system (Agilent Technologies, SantAClara, CA, USA). The column was a CORTECS UPLC C18 (2.1 × 100 mm, 1.6 μm). The mobile phase was acetonitrile (A) – 0.1% formic acid in water (B). Gradient elution was used: 0.0 → 3.0 min, 5% A → 8% A; 3.0 → 8.0 min, 8% A → 12% A; 8.0 → 12.0 min, 12% A → 15% A; 12.0 → 14.0 min, 15% A → 16% A; 14.0 → 20.0 min, 16% A → 20% A; 20.0 → 30.0 min, 20% A → 80% A; 30.0 → 35.0 min, 80% A → 5% A. Injection volume: 2 μL; Flow rate: 0.20 mL / min. -1 Kaempferol, quercetin, and luteolin were detected at a wavelength of 350 nm, while genistein A, baicalin, astragalin, physalisin, apigenin, and salicumin were detected at a wavelength of 327 nm.

[0097] 2.4 UPLC-TOF-MS / MS Detection Conditions

[0098] Chromatographic column: CORTECS™ UPLC C18 (2.1×100mm, 1.6μm); Mobile phase: Acetonitrile (A) - water containing 0.1% formic acid (B), gradient elution (0-3 min, 5%–5% A; 3.0–6.0 min, 5%–12% A; 6.0–13.0 min, 12%–12% A; 13.0–20.0 min, 12%–32% A; 20.0–25.0 min, 32%–42% A); Flow rate: 0.20 mL / min -1 The column temperature was 45℃ and the injection volume was 2μL.

[0099] The mass spectrometer used an ESI ion source, positive ion mode scanning, mass number: 50-1500; capillary voltage 2.5kV; nebulizer pressure 0.2Bar; desolventizing gas N2, desolventizing temperature 500℃, volumetric flow rate: 800L / h; cone gas N2, volumetric flow rate: 50L / h; ion source temperature: 120℃.

[0100] Using kaempferol, luteolin, quercetin, styracin A, genistein, baicalin, astragalin, calciferol, apigenin, and salicumin as substrates, and UDP-G as the glycosyl donor, the results were obtained by HUPLC and UPLC-TOF-MS / MS. The results showed that ThUGT1 reacted with all substrates, exhibiting broad reactivity, while also demonstrating specificity, preferentially reacting with the hydroxyl group at the 7-position to form new products (see [detection results]). Figures 4-13 .

[0101] 2.5 Preparation of the product

[0102] The scale-up reaction was carried out in a 50 mL system containing 5 mL of enzyme solution, 20 mL of Tris-HCl buffer (pH 7.5), 5 mL of apigenin, apigenin, astragalin, 1 mM guarbanol, and 25 mL of UDPG (5 mM). The reaction was carried out at 37 °C for 12 h. The reaction was terminated by adding 100 mL of methanol, centrifuged at 12000 rpm for 10 min, rotary evaporated, reconstituted with methanol, and filtered through a 0.22 μm microporous membrane. The products were separated and purified using a Shimadzu LC-20A analytical and semi-preparative high-performance liquid chromatography (HPLC). The separated products were identified by 1H-NMR and 13C-NMR.

[0103] Example 4 Enzymatic properties of *Trifolium repens* flavonoid glycoside glycosyltransferase

[0104] 1. Determination of optimal pH and optimal temperature

[0105] Under the above reaction conditions, using UDP-G as the sugar donor and kaempferol as the acceptor, the effects of pH and temperature on the activity of ThUGT1 enzyme in different enzymatic systems were investigated.

[0106] 1.1 Determination of optimal pH: The reaction was carried out in buffer solutions with pH 5, 6, 7, 7.5, 8, and 9, with other conditions remaining unchanged. The reaction was repeated three times. The reaction products were detected by HUPLC under the same conditions as in Example 3 "2.3". The highest yield was set to 100%. The relative yield was calculated, and a pH-relative yield curve was plotted.

[0107] The peak areas of glycosylation products under different pH conditions were detected and compared (see...). Figure 14 It was found that the peak area of ​​the glycosylated product was the largest at pH 8. Therefore, the optimal pH for using kaempferol as a substrate is 8.

[0108] 1.2 Determination of optimal temperature: Under the optimal pH conditions, the temperature gradient was set to 20, 25, 30, 37, 40, and 45°C, with other conditions remaining unchanged. The reaction was repeated three times. The reaction products were detected by HUPLC under the same detection conditions as in Example 3 "2.3". The highest yield was set to 100%. The relative yield was calculated, and the temperature-relative yield curve was plotted.

[0109] Detection and comparison of peak areas of glycosylation products under different temperature conditions (see...) Figure 15 It was found that the peak area of ​​the glycosylation product was the largest at 40℃. Therefore, the optimal temperature for using kaempferol as a substrate is 40℃.

[0110] 2. Determination of catalytic kinetic parameters

[0111] Preparation of kaempferol-7-O-glucoside standard curve: Prepare kaempferol-7-O-glucoside standard solutions with concentrations of 12.5, 25, 50, 100, 200, and 400 μg / mL, measure the peak area using HPLC, and fit the standard curve (see...). Figure 16 ).

[0112] When determining enzyme activity, kaempferol was used as an excess substrate, and the mixture was incubated for 5, 10, 15, 20, 40, and 60 min, respectively, while other components remained unchanged. The reaction products were detected by HPLC, and curves showing the changes in product content versus reaction time were plotted (see [link to relevant documentation]). Figure 17 ).

[0113] Enzyme kinetic parameters were determined by incubating at different substrate concentrations (100, 200, 300, 400, 500, and 600 mM) for 60 min, with other conditions remaining constant. This was repeated three times. The reaction products were detected by HPLC. Lineweaver-Burk plots were used to generate double reciprocal curves with 1 / [S] as the x-axis and 1 / v as the y-axis (see [link to plot]). Figure 18 ), calculate the values ​​of Km and Vmax.

[0114] The results showed that, at 40℃ and pH 8, using kaempferol as a substrate, Km = 141.52 ± 10.12 μM and Vmax = 2 ± 0.041 nmol·s⁻¹ -1 ·mg -1 .

[0115] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0116] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0117] SEQ ID NO.1:

[0118]

Claims

1. A glycosyltransferase ThUGT1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

1.

2. The glycosyltransferase ThUGT1 according to claim 1, characterized in that: It comes from the plant *Trifolium repens*.

3. A recombinant plasmid, characterized in that: It contains the glycosyltransferase ThUGT1 gene as described in claim 1 or 2; it is obtained by homologous recombination of the glycosyltransferase ThUGT1 gene with the pET-28a(+) vector to obtain the pET-28a(+)-UGTs recombinant plasmid.

4. A genetically engineered bacterium, characterized in that: It contains the recombinant plasmid as described in claim 3, or the genome of the genetically engineered bacterium contains an exogenous glycosyltransferase ThUGT1 as described in claim 1 or 2.

5. The use of the glycosyltransferase ThUGT1 according to claim 1 or 2, the recombinant plasmid according to claim 3, and the transgenic engineered bacteria according to claim 4 in the synthesis of flavonoids; wherein the flavonoids are as shown in Formula I or Formula II: in, In formula I: R5, R 3’ R 4’ R 5’ = Hydroxyl (-OH), methoxy (-OCH3), methyl (-CH3), glycoside (Glc), halogen (-F, -Cl, -Br, -l), alkyl (-R), amino (-NH2); In Equation II: R3, R5, R 3’ R 4’ R6, R8 = hydroxyl (-OH), methoxy (-OCH3), methyl (-CH3), glycoside (Glc), halogen (-F, -Cl, -Br, -l), alkyl (-R), amino (-NH2).

6. The use according to claim 5, characterized in that: The flavonoids mentioned include luteolin, kaempferol-7-O-glucoside, genistein, quercetin-7-O-glucoside, oroxylin A, styracin A-7-O-glucoside, apigenin-β-D-7-O-glucoside.

7. A method for synthesizing flavonoids, characterized in that: It uses UDP-G as a glycosyl donor and flavonoids and flavonols as substrates as described in Formula III or Formula IV, through... The glycosyltransferase ThUGT1 described in claim 1 or 2 is used as a biocatalyst to synthesize flavonoids of formula I or II; In Equation I: R5, R 3’ R 4’ R 5’ = Hydroxyl (-OH), methoxy (-OCH3), methyl (-CH3), glycoside (Glc), halogen (-F, -Cl, -Br, -l), alkyl (-R), amino (-NH2); In Equation II: R3, R5, R 3’ R 4’ R6, R8 = hydroxyl (-OH), methoxy (-OCH3), methyl (-CH3), glycoside (Glc), halogen (-F, -Cl, -Br, -l), alkyl (-R), amino (-NH2).

8. The method for synthesizing flavonoids according to claim 7, characterized in that: The synthetic route is as follows:

9. The method for synthesizing flavonoids according to claim 7 or 8, characterized in that: The substrates include: kaempferol, luteolin, quercetin, styracin A, genistein, baicalin, astragalin, physalisin, apigenin, and apigenin as substrates; the products are luteolinin, kaempferol-7-O-glucoside, genistein, quercetin-7-O-glucoside, oroxylonin A, physalisin A-7-O-glucoside, physalisin, and apigenin-β-D-7-O-glucoside. Preferably, the substrate is kaempferol, and the synthesis conditions are: pH = 8; temperature 40°C.

10. A method for detecting the flavonoids described in any one of 7-9, characterized in that: It uses HUPLC and UPLC-TOF-MS / MS detection; UHPLC detection conditions are as follows: Agilent HPLC 1290 system (Agilent Technologies, SantAClara, CA, USA); column: CORTEC SUPLC C18 (2.1 × 100 mm, 1.6 μm); mobile phase: acetonitrile (A) - 0.1% formic acid water (B); gradient elution: 0.0 → 3.0 min, 5% A → 8% A; 3.0 → 8.0 min, 8% A → 12% A; 8.0 → 12.0 min, 12% A → 15% A; 12.0 → 14.0 min, 15% A → 16% A; 14.0 → 20.0 min, 16% A → 20% A; 20.0 → 30.0 min, 20% A → 80% A; 30.0 → 35.0 min, 80% A → 5% A; injection volume: 2 μL; flow rate: 0.20 mL / min. -1 Kaempferol, quercetin, and luteolin were detected at a wavelength of 350 nm; phloroglucin A, genistein, baicalin, astragalin, physalisin, apigenin, and salicumin were detected at a wavelength of 327 nm. UPLC-TOF-MS / MS detection conditions: Chromatographic column: CORTECS™ UPLC C18 (2.1×100mm, 1.6μm); Mobile phase: Acetonitrile (A) - water containing 0.1% formic acid (B), gradient elution (0-3 min, 5%–5% A; 3.0–6.0 min, 5%–12% A; 6.0–13.0 min, 12%–12% A; 13.0–20.0 min, 12%–32% A; 20.0–25.0 min, 32%–42% A); Flow rate: 0.20 mL / min -1 Column temperature 45℃, injection volume 2μL; The mass spectrometer used an ESI ion source, positive ion mode scanning, mass number: 50-1500; capillary voltage 2.5kV; nebulizer pressure 0.2Bar; desolventizing gas N2, desolventizing temperature 500℃, volumetric flow rate: 800L / h; cone gas N2, volumetric flow rate: 50L / h; ion source temperature: 120℃.

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