Glycosyl transferase IlUGT88A3 and application of glycosyl transferase IlUGT88A3 in flavonoid glycoside synthesis

Abstract

The invention discloses glycosyl transferase IlUGT88A3 and application of the glycosyl transferase IlUGT88A3 in flavonoid glycoside synthesis, and belongs to the technical field of gene engineering and enzyme engineering. The glycosyl transferase IlUGT88A3 can be used for specifically catalyzing hydroxyl at C3 site of quercetin to generate glucosylation and galactosylation, so that the isoquercitrin and the hyperoside are efficiently synthesized. The optimum pH is 7.0-7.5, the optimum temperature is 30 DEG C, and the reaction conditions are mild; the method has good substrate heterogeneity, and can be used for carrying out site-specific glycosylation modification on various flavonoid receptors by utilizing sugar donors such as UDP-glucose, UDP-galactose, UDP-rhamnose, UDP-xylose and the like. According to the method, the blank of shortage of flavonoid 3-O-galactosyl transferase resources is filled, the catalytic specificity is high, the efficiency is high, the product is pure, the method can be used for in-vitro enzymatic synthesis and plant heterologous synthesis of high-value flavonoid glycosides such as hyperoside, and a core enzyme element and a technical support are provided for green and efficient preparation of medical raw materials.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, and in particular to a glycosyltransferase IlUGT88A3 and its application in the synthesis of flavonoid glycosides. Background Technology

[0002] Flavonoid glycosylation is a key modification step that determines the water solubility, stability, and bioactivity of flavonoids. Flavonoid 3-O-glucosylation is common in nature, but there are few reports on glycosyltransferases that can efficiently catalyze the 3-O-galactosylation of quercetin to hyperoside (quercetin-3-O-galactoside).

[0003] Hypericin is a core active ingredient that plays a role in protecting pancreatic β-cells and regulating glucose metabolism, but its plant extraction method suffers from bottlenecks such as low yield and long cycle. The plant UDP-glycosyltransferase family is large and has complex functional differentiation, making it difficult to accurately predict substrate specificity based solely on sequence homology.

[0004] In addition, the plant UGT gene family is exceptionally large and has undergone complex replication and expansion. Although they usually share a highly conserved PSPG motif at the C-terminus to bind sugar donors, their N-terminal sugar receptor binding pockets vary greatly, making it extremely difficult to accurately predict their actual catalytic function based solely on sequence homology. Summary of the Invention

[0005] To address the shortcomings of existing hyperoside preparation technologies, the scarcity of flavonoid 3-O-galactosyltransferase resources, and the unclear mechanism of sugar donor selectivity, this invention provides a glycosyltransferase IlUGT88A3 and its application in flavonoid glycoside synthesis.

[0006] To achieve the above objectives, the present invention provides a glycosyltransferase IlUGT88A3, the amino acid sequence of which is shown in SEQ ID NO.4.

[0007] On the other hand, the present invention provides a gene encoding the above-mentioned glycosyltransferase IlUGT88A3, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] On the other hand, the present invention provides a recombinant expression vector containing the gene encoding the glycosyltransferase IlUGT88A3.

[0009] On the other hand, the present invention provides a recombinant strain comprising the above-described recombinant expression vector.

[0010] On the other hand, the present invention provides the application of the above-mentioned glycosyltransferase IlUGT88A3 in the synthesis of flavonoid glycosides.

[0011] Preferably, the glycosyltransferase IlUGT88A3 uses quercetin as a sugar acceptor and UDP-glucose as a sugar donor to catalyze the synthesis of isoquercitrin.

[0012] Preferably, the glycosyltransferase IlUGT88A3 uses quercetin as a sugar acceptor and UDP-galactose as a sugar donor to catalyze the synthesis of hyperoside.

[0013] Preferably, the sugar donor in the synthesis of flavonoid glycosides includes at least one of UDP-glucose, UDP-galactose, UDP-rhamnose, and UDP-xylose.

[0014] Preferably, the sugar receptors in flavonoid glycoside synthesis include at least one of quercetin, kaempferol, myricetin, apigenin, luteolin, and naringenin.

[0015] Preferably, glycosyltransferase IlUGT88A3 heterologously synthesizes flavonoid glycosides in prokaryotic expression systems or plant transient expression systems.

[0016] Therefore, the glycosyltransferase IlUGT88A3 of the present invention and its application in the synthesis of flavonoid glycosides have the following beneficial effects: (1) For the first time, IlUGT88A3, a glycosyltransferase that can simultaneously catalyze the 3-O-glucosylation and 3-O-galactosylation of quercetin, was identified from broadleaf bamboo, filling the gap in flavonoid 3-O-galactosyltransferase resources.

[0017] (2) Through structural modeling, molecular docking, 100ns molecular dynamics simulation, and site-directed mutagenesis verification, this invention elucidates for the first time that Q386 at the end of the PSPG box is an essential residue for maintaining substrate binding and catalytic conformation, and confirms that it stabilizes the substrate through a hydrogen bond network and ensures the efficient execution of SN2 nucleophilic substitution reactions. The loss-of-function mechanism of the mutant is clear, providing a precise target and structural basis for subsequent rational design, semi-rational modification, improvement of galactose donor preference, improvement of thermal stability, and expansion of substrate range in enzyme engineering optimization, avoiding blind mutation and greatly improving the modification efficiency.

[0018] (3) Excellent catalytic performance: The optimal pH is 7.0-7.5 and the optimal temperature is 30℃. The conditions are mild and suitable for industrial applications. It has high catalytic activity for both UDP-Glc and UDP-Gal.

[0019] (4) Four sugar donors, UDP-Glc, UDP-Gal, UDP-Rha and UDP-Xyl, can be used to catalyze the synthesis of a series of high-value flavonoid monosaccharides from six flavonoid substrates, namely quercetin, kaempferol, myricetin, luteolin, apigenin and naringenin.

[0020] (5) Glycosyltransferase IlUGT88A3 can be expressed efficiently and solublely in the Escherichia coli prokaryotic system, and the protein purification is simple and has a high yield. At the same time, it can stably perform its function in the transient expression system of Nicotiana benthamiana, and is located in the cytoplasm and nucleus, and is highly compatible with the endogenous flavonoid synthesis pathway in plants. It can be used for efficient in vitro enzymatic preparation and for mass production of plant chassis synthetic biology. It has flexible application scenarios and meets the different needs of laboratory research, pilot-scale amplification and industrial production.

[0021] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The relative expression level of IlUGT88A3 in different tissues of *Indocalamus latae* was verified by qRT-PCR. Figure 2 Agarose gel electrophoresis results of CDS clones of the IlUGT88A3 gene; Figure 3 SDS-PAGE electrophoresis analysis of crude enzyme solution induced by IlUGT88A3 recombinant protein expression; Figure 4 SDS-PAGE electrophoresis analysis of purified IlUGT88A3 recombinant protein; Figure 5 HPLC chromatogram of the products of the enzymatic reaction with UDP-Glc as the sugar donor; Figure 6 HPLC chromatogram of the products of the enzymatic reaction with UDP-Gal as the sugar donor; Figure 7 Modeling results for the recombinant IlUGT88A3 protein; Figure 8 It is a superposition of eutectic ligands (cyan) and rearranged conformation (yellow); Figure 9 Cluster heatmap of binding free energies of IlUGT88A3 with quercetin aglycone and two UDP-sugar donors; Figure 10The binding modes of IlUGT88A3 with different UDP-glycan donors and quercetin are shown, where A is the three-dimensional binding mode of the IlUGT88A3-UDP-Gal-quercetin ternary complex; and B is the three-dimensional binding mode of the IlUGT88A3-UDP-Glc-quercetin ternary complex. Figure 11 100 ns molecular dynamics simulation analysis of IlUGT88A3 with different UDP-glycan donors and quercetin ternary complexes. In the figure, a is the root mean square deviation (RMSD); b is the solvent accessible surface area (SASA); c is the root mean square fluctuation (RMSF); d is the radius of gyration (Rg); e is the secondary structure change diagram of IlUGT88A3-UDP-Gal-Que; f is the secondary structure change diagram of IlUGT88A3-UDP-Glc-Que. Figure 12 For overexpression IlUGT88A3 The accumulation of quercetin-3-O-glucoside (Que-3-O-Glc) and quercetin-3-O-galactoside (Que-3-O-Gal) in tobacco leaves of the gene; Figure 13 Subcellular localization of the IlUGTs-eGFP fusion protein in tobacco leaf epidermal cells; Figure 14 This study analyzed the enzymatic properties and kinetics of IlUGT88A3, where a) represents the relative catalytic activity of IlUGT88A3 under different pH conditions; b) represents the relative catalytic activity of IlUGT88A3 under different temperature conditions; c) represents the kinetic fitting curve of the enzymatic reaction of IlUGT88A3 with UDP-Glc as the sugar donor and quercetin as the substrate; and d) represents the kinetic fitting curve of the enzymatic reaction of IlUGT88A3 with UDP-Gal as the sugar donor and quercetin as the substrate. Figure 15 Validation of the in vitro enzyme activity of the recombinant protein IlUGT88A3-Q386H; Figure 16 Structural analysis of the ternary complexes formed by IlUGT88A3 wild-type (green) and IlUGT88A3-Q386H mutant (red) with UDP-Gal and quercetin (Que). Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0026] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0027] Example 1 Screening and cloning of IlUGT88A3: (1) Based on WGCNA analysis of the transcriptome and metabolome of *PacBio* var. *broadleaf* (green module, |r|>0.85), key UGT genes involved in the biosynthesis of quercetin glycoside flavonoids (especially hyperoside) in *PacBio* var. *broadleaf* were screened. Five candidate genes with complete sequences that were highly related to the target metabolites were obtained, one of which was named IlUGT88A3 According to NCBIBLASTp homology comparison, it shows... IlUGT88A3 It has a sequence identity of 97.06% with the functionally validated flavonoid glycosyltransferase in Bambusoideae, making it a direct homolog of flavonoid glycosyltransferase with a well-defined function within the Bambusoideae subfamily.

[0028] RT-qPCR validation results are as follows Figure 1 As shown, IlUGT88A3 It was highly expressed in leaves and expressed at low abundance in other tissues such as roots and stems, consistent with the tissue accumulation pattern of quercetin glycoside metabolites, confirming the accuracy of the transcriptome data.

[0029] (2) Using the cDNA from the leaves of *Indocalamus latae* as a template, amplification was performed. IlUGT88A3 Complete CDS: Total RNA was extracted from fresh leaves of *Inula thunbergii* using the Tiangen Biochemical Total RNA Extraction Kit. After integrity was assessed by 1% agarose gel electrophoresis, Takara PrimeScript was used to extract RNA. TM First-strand cDNA was synthesized by reverse transcription using an RT kit. Based on the transcriptome Unigene sequence, specific amplification primers (IlUGT88A3-F / R) were designed at both ends of the open reading frame (ORF) using Primer Premier 5.0. The downstream primer had the target gene stop codon removed to ensure in-frame translation with the GST tag. Homologous arm sequences matching the pGEX-6p-1 vector after double digestion with BamHI and EcoRI were introduced at the 5′ end of the primers, respectively.

[0030] PCR amplification was performed using high-fidelity DNA polymerase under the following conditions: 98℃ pre-denaturation for 2 min; 35 cycles of 98℃ for 10 s, 58℃ for 15 s, and 72℃ for 20 s, followed by a final extension at 72℃ for 5 min. The amplified product was recovered from the gel and ligated into the pGEX-6p-1 vector, which had been double-digested with BamHI and EcoRI, using a homologous recombinase. The recombinant plasmid was transformed into DH5α competent cells, and positive clones were screened by colony PCR and sent to Qingke Biotechnology for Sanger sequencing verification.

[0031] IlUGT88A3 The CDS sequences are shown in SEQ ID NO.1, IlUGT88A3-F in SEQ ID NO.2, and IlUGT88A3-R in SEQ ID NO.3.

[0032] SEQ ID.NO.1:

[0033] SEQ ID NO. 2: CAACCCCTCCATCACCTTCC.

[0034] SEQ ID.NO.3: GCGCAGAACATGTCGATGAC.

[0035] IlUGT88A3 The results of agarose gel electrophoresis of the CDS clone of the gene are as follows: Figure 2 As shown, the cloned data has been generated. IlUGT88A3 The complete CDS region was verified to be consistent by Sanger sequencing and then heterologously expressed in E. coli.

[0036] Example 2 The correctly sequenced recombinant plasmid and empty vector (EV) were transformed into the BL21(DE3) expression strain, respectively. Single colonies were selected and inoculated into LB broth containing 100 μg / mL ampicillin, and cultured at 37°C with shaking until OD200. 600 The concentration was increased to 0.6-0.8. IPTG was added to a final concentration of 0.2 mM, and the mixture was induced at 16°C for 16 h. After induction, the cells were collected by centrifugation at 8000 rpm for 15 min at 4°C. The cells were resuspended in pre-cooled 50 mM Tris-HCl (pH 7.5) buffer (5 mM DTT, 0.1 mg / mL lysozyme, 0.1% cocktail) and sonicated on ice (200 W, 3 s for disruption, 5 s for pause, for a total of 15 min). The lysate was centrifuged at 8000 rpm for 20 min at 4°C, and the supernatant was collected for protein purification.

[0037] Prepare equilibration / washing buffer (Buffer A: 50mM Tris-HCl (pH 7.5), 150mM NaCl, filtered through a 0.22μm filter and stored at 4℃) and elution buffer (Buffer B: 50mM Tris-HCl (pH 8.0), 150mM NaCl, 10mM MGSH, 1mM DTT, wrapped in aluminum foil to protect from light). Load the crude enzyme solution onto a chromatography column equilibrated with Buffer A (flow rate 0.2-0.5mL / min), wash thoroughly with 10 column volumes (10mL) of Buffer A, then elute stepwise with Buffer B and collect the eluted fractions (E1-E10). Verify the molecular weight and purity by 12% SDS-PAGE gel electrophoresis, combine the fractions as purified samples, and quantify using a Bradford protein assay kit. Finally, add 20% glycerol, aliquot, and store at -80℃ for later use.

[0038] SDS-PAGE electrophoresis analysis of the crude enzyme solution induced by IlUGT88A3 recombinant protein expression is as follows: Figure 3 As shown, the recombinant IlUGT88A3 protein was successfully expressed in *E. coli* after IPTG induction. Purification was performed using a GST affinity chromatography column. Figure 4 (This will allow for subsequent studies such as in vitro enzyme activity verification.)

[0039] The amino acid sequence of IlUGT88A3 is shown in SEQ ID NO.4, SEQ ID NO.4: MEETVVLYPGLGVGHLVPMVELAKVFLQHGLAVTVVLVESPIEAMDSSAAVARAKASNPSITFHVLPPPTPAPAGSSSDSTSHIIQMFQFLNAMNAPLRDFLRSLPSVHAIV IDMFCADVLDVAAELNLPAYFSYTAGASDLAVFLNLPSMRAGMNTSFEELGDSIVSFPGVRPFKASDLPSVICNDGEASKAILRMFDRMTEANGILVNTFESLEKRAVRALRDGFCVPG RATPPVYCIGPLVSGGGDKEHECLRWLDAQPDHSVVFLSFGSMGSFPKKQLEEIAVGLDKSGQRFLWVVRSPPNPDHKFGDPLPEPDLDAILPDGFLERTKDRGLVVKSWAPQVEVLRH RATSAFVTTHCGWNSTMEGITAGLPLLCWPLYAEQRLNKVHIVEEMNLGVEMRGYNEEVVKAEEVEAKVRWVMESEGGQALRERVVAVKDAAAEALKEGGSSDTAFAQFLKDLDTSNGA.

[0040] Example 3 To clarify IlUGT88A3 The catalytic function was investigated using quercetin as a glycosyl acceptor and uridine diphosphate glucose (UDP-Glc) and uridine diphosphate galactose (UDP-Gal) as sugar donors, respectively. The purified recombinant IlUGT88A3 protein was used for in vitro enzymatic reaction analysis.

[0041] The total volume of the in vitro enzymatic reaction system was 100 μL, containing: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 5 mM MTT, 0.1 mM sugar acceptor substrate, and 2 mM sugar donor. The reaction was initiated by adding 4 μg of purified recombinant protein. An equal volume of the purified empty vector protein was added as a control group.

[0042] The system was incubated at 30℃ for 2 hours, then an equal volume of ice-cold methanol was added to terminate the reaction. The mixture was vortexed for 2 minutes to ensure thorough mixing, and centrifuged at 10,000 rpm for 10 minutes. The supernatant was then used for analysis. HPLC analysis was performed using a Waters ACQUITY Arc-PDA detector. The chromatographic column was a YMC Hydrosphere C18 (250 × 4.6 mm, 5 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. The elution gradient was set as follows: 0–8 min, 5%–15% B; 8–10 min, 15%–18% B; 10–50 min, 18%–50% B; 50–55 min, 50%–90% B; 55–60 min, 90% B; 60–60.1 min, 90%–5% B; 60.1–75 min, 5% B, with a flow rate of 1.0 mL / min. The wavelength was collected in the range of 200-500 nm, and the preliminary qualitative analysis was performed by comparing the retention time and ultraviolet absorption spectrum of the product peak with those of the standard.

[0043] Subsequently, analysis was performed using a Thermo Fisher Scientific ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry system. An ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) was used, with a column temperature of 35 °C and an injection volume of 5 μL. Mass spectrometry was acquired in electrospray ionization (ESI±) mode at a voltage of ±3.5 kV, with a mass range of m / z 66.7–1000. The molecular ion peak (m / z) and characteristic fragments of the secondary mass spectrometry of the product were compared, and consistency with standards was confirmed.

[0044] like Figure 5 As shown, when UDP-Glc is used as the sugar donor, the recombinant IlUGT88A3 protein can catalyze site-specific glycosylation of quercetin to generate the corresponding glucosinolate product, while no product peak is generated in the empty vector (EV) control group.

[0045] like Figure 6 As shown, when UDP-Gal is used as the sugar donor, IlUGT88A3 can specifically catalyze the galactosylation modification of quercetin C3-OH to generate hyperoside (Que-3-O-Gal), while no product is generated in the empty vector control group.

[0046] Example 4 The full-length three-dimensional structure of the IlUGT88A3 protein was predicted using AlphaFold2. The modeling results of the recombinant IlUGT88A3 protein are as follows: Figure 7 As shown.

[0047] The model was evaluated using PyMOL software for structural alignment with the plant UGT standard crystal structure (PDB ID: 2c1z) and main chain RMSD calculations. The modeling evaluation of the recombinant IlUGT88A3 protein is shown in Table 1 below.

[0048] Table 1. Modeling and evaluation of recombinant IlUGT88A3 protein

[0049] Note: The PDB ID of the reference crystal structure used for comparison is 2c1z. a. pLDDT: local distance difference test at the residue level; b. pTM score: predicted template modeling score; c. global main chain RMSD (Å); d. RMSD (Å) of PSPG motif.

[0050] Depend on Figure 7 As shown in Table 1, the recombinant IlUGT88A3 protein model exhibits high overall structural confidence, with pTM scores ranging from 0.90 to 0.95, and average pLDDT scores for the catalytic domains exceeding 95, indicating core functionality. The recombinant IlUGT88A3 protein model was structurally compared with the classic plant UGT standard crystal structure (PDB ID: 2c1z). The results showed that the overall main-chain RMSD of IlUGT88A3 compared to 2c1z was 1.645 Å, less than 2.0 Å, indicating that the model possesses a typical and conserved GT-B fold consistent with the functional UGT structure. Furthermore, the RMSD of the PSPG frame of the recombinant IlUGT88A3 protein model was less than 0.6 Å, and the key residues in the cofactor binding pocket were highly conformally identical to those of 2c1z. These findings indicate that the recombinant IlUGT88A3 protein model possesses high structural quality and accurate functional conformation, making it suitable for subsequent molecular docking analysis of substrate binding characteristics.

[0051] Molecular docking simulations were performed using Smina software. The docking center was set to the active pocket of the protein, the grid size was set to 25 Å × 25 Å × 25 Å, and the exhaustiveness was set to 30. The optimal conformation was selected based on the binding energy fraction (kcal / mol).

[0052] like Figure 8 As shown, the recombinant ligand can reproduce the binding conformation of the natural eutectic ligand very well, and its main chain root mean square deviation (RMSD) is less than 2 Å, indicating that the SMINA software is suitable for subsequent molecular docking analysis of IlUGT88A3.

[0053] Molecular docking simulations of IlUGT88A3 were performed using quercetin as the aglycone substrate and UDP-Glc or UDP-Gal as the sugar donor, respectively. The results, combined with the free energy, are as follows: Figure 9As shown, the binding free energies of IlUGT88A3 are -10.3 kcal / mol (UDP-Glc) and -10.6 kcal / mol (UDP-Gal), which is consistent with its high catalytic performance.

[0054] Further analysis was conducted on the binding modes of IlUGT88A3 with different UDP-glycan donors and quercetin, and the results are as follows: Figure 10 As shown in the figure, nine shared amino acid residues (T138, N365, S366, H361, E385, Y383, Q386, S275, and A344) were found to participate in the interaction with the ligands in both UDP-Glc and UDP-Gal binding modes. These residues are mainly located within the conserved GT-B catalytic domain and the PSPG sugar binding cassette specific to plant UGT, forming the core substrate-binding scaffold of IlUGT88A3. These residues likely constitute the core substrate-binding backbone of IlUGT88A3 and are key conserved sites for maintaining the conformation of the catalytic pocket and substrate recognition. Glutamine 386 (Q386) forms extensive hydrogen bond networks with both the UDP-sugar donor and quercetin aglycone, directly stabilizing the binding posture of the substrate in the catalytic pocket. This finding suggests that Q386 plays a crucial role in substrate recognition and catalytic reactions.

[0055] To further evaluate the conformational stability and dynamic characteristics of this enzyme bound to different sugar donors, molecular dynamics (MD) simulations were performed for 100 ns based on the conformations of the IlUGT88A3-UDP-Glc-Que and IlUGT88A3-UDP-Gal-Que ternary complexes obtained by molecular docking. Figure 11 As shown.

[0056] like Figure 11 As shown in Figure A, after conformational adjustment in the first 20 ns, the two complex systems rapidly converged and stabilized within a range of 0.25-0.30 nm, indicating that the overall three-dimensional structure of IlUGT88A3 did not undergo significant unfolding or deviation, and the resulting ternary complex was stable. Figure 11 As shown in Figures B and D, the Rg values ​​of the two systems remained stable at approximately 2.30 nm and 2.25 nm, respectively, throughout the entire 100 ns process, and the SASA values ​​did not show any significant jumps. Figure 11 As shown in Figure C, the RMSF variation trends of the two systems are highly consistent. The highly flexible regions (RMSF>0.4nm) are mainly concentrated around residues 80-100 and 260-280. These regions correspond to the random coils on the protein surface or the flexible loop regions connecting the N-terminal and C-terminal catalytic domains. This inherent flexibility may be beneficial for substrate entry and product release.

[0057] Notably, the RMSF values ​​of the core residues constituting the substrate binding pocket and the highly conserved PSPG box region remained at low levels (<0.15 nm), indicating that the catalytic active site exhibited rigidity and binding stability after binding to two different configurations of UDP-sugars. The DSSP secondary structure changed over time (…). Figure 11 As can be seen from E and F, the α-helix and β-sheet structure of the protein core remained highly intact throughout the simulation, maintaining the typical GT-B type dense folding characteristics of plant UGT.

[0058] Example 5 Will IlUGT88A3 The full-length CDS sequence was cloned into the pC1300-35S-eGFP vector to construct a transient expression vector. The recombinant plasmid and empty vector (EV) were transformed into Agrobacterium GV3101 and inoculated into YEP medium containing 50 μg / mL kanamycin and 25 μg / mL rifampin. Agrobacterium was collected by centrifugation and resuspended in induction buffer (containing 10 mM MES, 10 mM MgCl2, and 200 μM acetylsylcholine) to adjust OD. 600 The concentration was 0.5. After standing in the dark for 2 hours, 4-5 week old tobacco leaves were infiltrated with a needleless syringe. Fluorescence signals were observed under a laser confocal scanning microscope 48 hours after injection. The excitation wavelength of eGFP fluorescence was set to 488 nm, and the emission wavelength was set to 500-530 nm; simultaneously, chloroplast autofluorescence signals (excitation wavelength 640 nm) were collected as a background control. Transiently expressed tobacco leaf tissue was also collected, ground with liquid nitrogen, and then extracted with 80% methanol in an ice bath for 30 min at a material-to-liquid ratio of 1:10. The extractions were repeated three times, and the extracts were combined and extracted three times with an equal volume of petroleum ether. The extracts were then concentrated under vacuum, reconstituted with methanol, filtered through a 0.22 μm filter, and the accumulation of hyperoside (Que-3-O-Gal) and isoquercitrin (Que-3-O-Glc) in the leaves was detected using LC-MS / MS to verify the glycosylation function of IlUGT88A3 in the plant.

[0059] overexpression IlUGT88A3 The accumulation levels of quercetin-3-O-glucoside (Que-3-O-Glc) and quercetin-3-O-galactoside (Que-3-O-Gal) in the leaves of tobacco plants with the gene are as follows: Figure 12 As shown. The results indicate that in quercetin-based catalytic reactions, overexpression... IlUGT88A3 The accumulation levels of quercetin-3-O-glucoside (Que-3-O-Glc) and quercetin-3-O-galactoside (Que-3-O-Gal) in tobacco leaves were 5.48±0.36 μg / g FW and 1.11±0.13 μg / g FW, respectively. This verifies that IlUGT88A3 also possesses highly efficient quercetin glycosylation capabilities in vivo.

[0060] To investigate the spatial distribution characteristics of IlUGT88A3, bioinformatics prediction was performed using the DeepLoc 2.1 tool. The results showed that IlUGT88A3 was predicted as a soluble protein (Soluble score between 0.74 and 0.79), mainly located in the cytoplasm, with secondary prediction of the nucleus.

[0061] To verify this prediction, this embodiment constructed a 35S::IlUGTs-eGFP fusion expression vector and transiently expressed it in tobacco leaf epidermal cells. Specific primers with vector homologous arms were designed using Primer Premier 5.0 to amplify IlUGTs CDS, with the reverse primer having the stop codon (Δstop) removed to ensure it remained within the same open reading frame as eGFP and to achieve C-terminal fusion expression.

[0062] Specific primers: Forward primer, SEQ ID NO.5: tccggacccttcctctatatggtaccATGGCCGGTGTCCAAGAAACA; Reverse primer, SEQ ID NO.6: cttgctcaccatggtggcgatctagaTCACAGAGACTGGATGTGATC.

[0063] Using *Imperata cylindrica* cDNA as a template, PCR amplification was performed using 2×PrimeSTAR Max DNA Polymerase (Takara, R045A). The 25 μL reaction mixture contained 12.5 μL 2×PrimeSTAR Max Premix, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, 1 μL template, and 9.5 μL ddH2O. The PCR program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 20 s, for a total of 32 cycles; final extension at 72℃ for 5 min.

[0064] The amplified products were detected by 1% agarose gel electrophoresis, and the target band was excised. The DNA was then purified using the TaKaRa MiniBESTAgarose Gel DNA Extraction Kit Ver. 4.0 according to the manufacturer's instructions, yielding purified IlUGTs products with homologous arms. Simultaneously, appropriate restriction endonucleases were used. KpnI and XbaIThe pCAMBIA1300-35S::eGFP empty vector was linearized by double enzyme digestion. After gel extraction and purification, the linearized vector was ligated with an IlUGTs fragment containing homologous arms using the ClonExpress II One Step Cloning Kit (Vazyme).

[0065] The 20 μL recombinant reaction system contained: 1 μL linearized vector, 2 μL target gene fragment, 4 μL 5×CE II buffer, 2 μL Exnase II enzyme, and ddH2O to a final volume of 20 μL. After gentle mixing, the mixture was incubated at 37°C for 30 min, followed by cooling on ice. 10 μL of the recombinant product was added to 100 μL of thawed *E. coli* DH5α competent cells, incubated on ice for 30 min, followed by heat shock at 42°C for 90 s, and then rapidly cooled on ice for 3 min. 900 μL of antibiotic-free LB broth was added, and the cells were incubated at 37°C and 200 rpm for 1 h to revive. After centrifugation and discarding some of the supernatant, 200 μL of the bacterial culture was evenly spread onto LB solid medium containing 50 μg / mL kanamycin and incubated upside down at 37°C for 12 h. Single colonies were selected for colony PCR identification. Positive clones were inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm for 12 h. The recombinant plasmid was extracted and verified by the FastPurePlasmid Mini Kit (Vazyme) for 8 min, which was the 35S::IlUGTs-eGFP fusion expression vector.

[0066] Subcellular localization of the IlUGTs-eGFP fusion protein in tobacco leaf epidermal cells, as shown in... Figure 13 As shown, from left to right, the channels are: eGFP green fluorescence channel, chloroplast autofluorescence channel (Chl AF), bright field channel, and merged channel. The results show that the green fluorescence signal of the IlUGT88A3 fusion protein is widely distributed in the cytoplasm and nucleus, and does not overlap with the red chloroplast autofluorescence. This in vivo localization imaging result is highly consistent with bioinformatics predictions, indicating that IlUGT88A3 mainly performs its biological functions in the cytoplasm and nucleus.

[0067] Example 6 To determine the optimal reaction conditions for IlUGT88A3, the relative activity of the enzyme was measured at different pH values ​​and temperatures. In the pH gradient experiment, a gradient range of pH 5.0–8.0 was set, using 50 mM sodium citrate buffer (pH 5.0–6.0), sodium phosphate buffer (pH 6.0–7.0), and Tris-HCl buffer (pH 7.0–8.0), respectively. All reactions were incubated at 30°C, with 15 min for UDP-Glc as the sugar donor and 25 min for UDP-Gal as the sugar donor, to ensure the reaction was in the initial kinetic rate phase. In the temperature gradient experiment, a gradient range of 20–45°C was set, and all reactions were incubated at the optimal pH for the same duration. The reaction system was constructed as in Example 3, with each reaction repeated three times.

[0068] like Figure 14 As shown in Figures A and B, the optimal pH for IlUGT88A3 to catalyze the reaction with two sugar donors and quercetin as a substrate is 7.0-7.5, and the optimal temperature is 30℃. The results indicate that the enzyme reaction conditions are mild and consistent with the physiological characteristics of key enzymes in secondary metabolism in plants.

[0069] Under optimal conditions, the final concentration of the sugar donor (UDP-Glc or UDP-Gal) was fixed at 2 mM, and the final concentration gradient of the acceptor substrate quercetin was adjusted to 5, 10, 20, 30, 40, 50, 60, and 80 μM. The total volume of the reaction system was 100 μL, and the reaction was incubated at 30 °C. The reaction was carried out for 15 min with UDP-Glc as the sugar donor and for 25 min with UDP-Gal as the sugar donor, maintaining the initial rate. After the reaction was completed, an equal volume of ice-cold methanol was added to terminate the reaction, and the product yield was determined by HPLC. The reaction was repeated three times, and the data were analyzed using GraphPad Prism 8.0 software for nonlinear regression analysis. Kinetic parameters were calculated by fitting the data to the Michaelis-Menten equation.

[0070] like Figure 14 As shown in Figures C and D, the fitted curves demonstrate that the enzyme conforms to a typical Michaelis-Menten equation. When UDP-Glc is used as the sugar donor, its Michaelis constant (Km) is 10.71 μM, and its catalytic efficiency (kcat / Km) is 0.0241 s⁻¹. -1 ·μM -1 When UDP-Gal was used as the sugar donor, Km increased to 24.1 μM, and kcat / Km decreased to 0.0104 s. -1 ·μM -1Comparative analysis revealed that IlUGT88A3 exhibits approximately 2.25 times the affinity for UDP-Glc and 2.32 times the catalytic efficiency for UDP-Gal. This indicates that while IlUGT88A3 possesses functional activity in catalyzing the synthesis of hyperoside (quercetin-3-O-galactoside), it kinetically prefers to use UDP-Glc as a donor to synthesize isoquercetin. This may be closely related to the abundance of different sugar donors and the regulatory requirements of secondary metabolism within *Isoquercetin var. thunbergii*.

[0071] Example 7 Based on multiple sequence alignment and molecular docking results, the key amino acid site (Q386) at the end of the PSPG Box affecting sugar donor specificity was selected. Reverse amplification primers carrying the Q→H mutant base were designed using Primer Premier 5.0. Using the original recombinant expression plasmid as a template, whole-plasmid PCR amplification was performed using high-fidelity DNA polymerase. After gel recovery of the PCR product, Dpn I restriction endonuclease was added and digested at 37℃ for 1 h to remove residual wild-type template plasmid. The digested product was transformed into DH5α competent cells, and positive clones were selected by colony PCR screening. After successful mutation confirmation by sequencing, the mutant protein was induced for expression, purified, and its in vitro enzymatic activity was measured to analyze the effect of this site on in vitro catalytic activity.

[0072] In vitro enzyme activity verification of the mutant IlUGT88A3-Q386H recombinant protein is as follows: Figure 15 As shown, the results indicate that the IlUGT88A3-Q386H mutant not only failed to increase its preference for UDP-Gal as expected, but also completely lost its transfer activity to UDP-Glc and UDP-Gal.

[0073] To elucidate the structural basis for the loss of catalytic activity in the mutant, the sugar donor UDP-Gal was docked into the structural models of the IlUGT88A3 wild-type (WT) and IlUGT88A3-Q386H mutant, respectively. Figure 16As shown, there are significant differences in the binding orientation of the galactose groups in the two enzymes. In the wild-type enzyme, the highly conserved terminal glutamine (Q386) in the PSPG box can form a key hydrogen bond network with the galactose ring, thereby achieving stable anchoring of the sugar donor; the distance between the Q386 side chain and the C2-hydroxyl group of galactose is approximately 3.2 Å (green), indicating that it forms a stable binding conformation that is conducive to SN2 nucleophilic substitution reactions. In contrast, Q386H mutates the shorter glutamine side chain into a larger and more rigid histidine imidazole ring. To avoid steric hindrance and adapt to the change in the binding pocket environment, the galactose group undergoes a significant spatial shift, with the distance between the H386 side chain and the C2 position of galactose increasing to 7.4 Å (pink). This physically blocks the nucleophilic attack, and the mutant cannot anchor the sugar donor in the correct catalytic conformation.

[0074] Example 8 To systematically evaluate the recognition ability and catalytic heterogeneity of IlUGT88A3 for different sugar donors and acceptors, four activated sugar donors—UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-rhamnose (UDP-Rha), and UDP-xyl (UDP-Xyl)—were selected. For sugar acceptors, representative flavonols (quercetin, kaempferol, myricetin), flavonoids (apigenin, luteolin), and flavanones (naringenin) were selected as substrates. The qualitative screening results of the catalytic activity of IlUGT88A3 for different flavonoid acceptors and UDP-glycan donors are shown in Table 2 below.

[0075] Table 2. Qualitative screening results of the catalytic activity of IlUGT88A3 against different flavonoid receptors and UDP-glycan donors.

[0076] Note: "+" indicates that a specific catalytic product can be detected. Product verification criteria: ① No corresponding product peak in the EV empty negative control, excluding interference from host endogenous enzyme catalysis; ② The UV spectrum is consistent with the characteristic absorption peak of the corresponding aglycone flavonoid nucleus; ③ High-resolution primary mass spectrometry [M+H] + The deviation from the theoretical mass-to-charge ratio of the corresponding flavonoid monosaccharide glycoside is <5 ppm; ④ Characteristic diagnostic fragment peaks of the corresponding aglycone were detected by secondary mass spectrometry. "-" indicates that no specific product meeting the above criteria was detected.

[0077] The results showed that IlUGT88A3 can utilize UDP-glycan donors with various structures, but the catalytic ranges differed. Among them, UDP-Glc and UDP-Rha had broad receptor coverage, both catalyzing five tested receptors except for naringenin; while UDP-Gal and UDP-Xyl had relatively narrow receptor ranges, each catalyzing only two flavonoid receptors.

[0078] Regarding sugar receptors, IlUGT88A3 exhibits a certain degree of selectivity for the structural features and substitution patterns of the flavonoid core: (1) The C3-hydroxyl group and the B-ring substitution mode may jointly affect substrate recognition. The tested flavonols (containing C3-OH) generally exhibited a broader sugar donor acceptance range than flavones / flavones (without C3-OH). Among the flavonols, quercetin, which has a 3',4'-ortho-dihydroxyl group on the B-ring, can be catalyzed by all four sugar donors; kaempferol, which has a 4'-monohydroxyl group, can utilize three donors (UDP-Glc / Gal / Rha); while myricetin, which has a 3',4',5'-trihydroxyl group, can only utilize UDP-Glc and UDP-Rha. Flavonoid substrates lacking the C3-hydroxyl group (luteolin, apigenin) can only be catalyzed by UDP-Glc and UDP-Rha.

[0079] (2) The saturation of the C2-C3 bond caused changes in catalytic characteristics. Flavonoids and flavonols with C2-C3 double bonds can undergo glycosylation reactions with UDP-Glc and UDP-Rha as donors; while naringenin, a flavanone substrate with C2-C3 single bonds, showed different donor preferences. It could not utilize UDP-Glc, UDP-Gal or UDP-Rha, and only catalytic products with UDP-Xyl as donors were detected.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A glycosyltransferase IlUGT88A3, characterized in that: The amino acid sequence of glycosyltransferase IlUGT88A3 is shown in SEQ ID NO.

4.

2. The gene encoding the glycosyltransferase IlUGT88A3 of claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

3. A recombinant expression vector, characterized in that, It contains the gene described in claim 2.

4. A recombinant bacterial strain, characterized in that, It includes the recombinant expression vector as described in claim 3.

5. The application of the glycosyltransferase IlUGT88A3 of claim 1 in the synthesis of flavonoid glycosides.

6. The application according to claim 5, characterized in that: Glycosyltransferase IlUGT88A3 uses quercetin as a sugar acceptor and UDP-glucose as a sugar donor to catalyze the synthesis of isoquercitrin.

7. The application according to claim 5, characterized in that: Glycosyltransferase IlUGT88A3 uses quercetin as a sugar acceptor and UDP-galactose as a sugar donor to catalyze the synthesis of hyperoside.

8. The application according to claim 5, characterized in that: Sugar donors in flavonoid glycoside synthesis include at least one of UDP-glucose, UDP-galactose, UDP-rhamnose, and UDP-xylose.

9. The application according to claim 5, characterized in that: The sugar receptors involved in flavonoid glycoside synthesis include at least one of quercetin, kaempferol, myricetin, apigenin, luteolin, and naringenin.

10. The application according to claim 5, characterized in that: Glycosyltransferase IlUGT88A3 heterologously synthesizes flavonoid glycosides in microbial or plant expression systems.

Images