A glycosyltransferase PmUGT7 and its application in the biosynthesis of stilbene glycosides

Abstract

This invention discloses a glycosyltransferase PmUGT7 and its application in the biosynthesis of stilbene glycosides. The glycosyltransferase PmUGT7 can efficiently and specifically glycosylate the C2 hydroxyl group of 2,3,5,4'-tetrahydroxystilbene, producing stilbene glycosides in a one-step process. The reaction conditions are mild, the operation is convenient and rapid, the catalytic efficiency is high, and it has strong sustainability.

Description

Technical Field

[0001] This invention relates to a glycosyltransferase PmUGT7 and its application in the biosynthesis of stilbene glycosides. It belongs to the field of biosynthesis technology. Background Technology

[0002] 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG) is the characteristic main active ingredient in the dried tuberous root of *Polygonum multiflorum* Thunb., a plant in the Polygonaceae family, and is the core material basis for the medicinal efficacy of *Polygonum multiflorum*. Modern pharmacological studies have shown that stilbene glycoside possesses broad-spectrum anti-aging and multi-target pharmacological activities, including lifespan extension, neuroprotection (improving symptoms of neurodegenerative diseases such as Alzheimer's and Parkinson's), cardiovascular protection (inhibiting atherosclerosis and regulating hypertension), delaying gonadal aging, improving osteoporosis, and promoting hair growth. It also has therapeutic effects on inflammatory diseases, hepatic steatosis, depression, diabetes, and kidney disease. Therefore, based on its significant antioxidant, anti-inflammatory, anticancer, antidiabetic, anti-cholesterol, and antimicrobial activities, stilbene glycoside has important value and broad prospects in innovative drug development, functional product development, and clinical applications.

[0003] The shortage of stilbene glycosides is becoming increasingly prominent, with current production primarily relying on extraction from Polygonum multiflorum plants. However, this method suffers from drawbacks such as long production cycles, complex processes, and high resource consumption, and is also limited by plant cultivation, raw material quality, and extraction efficiency. In chemical synthesis, regioselective glycosylation is a crucial step in constructing stilbene glycoside structures and presents significant technical challenges. It typically requires the introduction of protecting groups to achieve site-specific reactions, resulting in complex processes and poor environmental friendliness. In contrast, enzymatic biosynthesis offers advantages such as mild reaction conditions, environmental friendliness, and fewer byproducts. Furthermore, it possesses natural advantages in regioselectivity and stereoselectivity, exhibiting high selectivity for substrate functional groups, making it an ideal pathway for the efficient and green production of stilbene glycosides.

[0004] Glycosylation is a key secondary metabolic modification process in plants, and glycosyltransferases are the core functional enzymes mediating the glycosylation modification of natural products from medicinal plants. They can significantly improve the water solubility and chemical stability of substrates and reduce their reactive toxicity, thus having important application value in the field of natural product biosynthesis. To date, no glycosyltransferases have been reported that can specifically catalyze the production of stilbene glycosides from 2,3,5,4'-tetrahydroxystilbene (THS). These glycosyltransferases are the key rate-limiting enzymes for the one-step biosynthesis of stilbene glycosides, directly determining the synthesis efficiency, and are crucial for establishing a highly efficient, specific, and large-scale biosynthetic system for stilbene glycosides. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a glycosyltransferase PmUGT7 and its application in the biosynthesis of stilbene glycosides.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A glycosyltransferase PmUGT7, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Preferably, the glycosyltransferase PmUGT7 has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity with the amino acid sequence shown in SEQ ID NO.1.

[0008] The gene or codon-optimized gene encoding the aforementioned glycosyltransferase PmUGT7 has nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0009] Preferably, the gene has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity with the nucleotide sequence shown in SEQ ID NO.2.

[0010] Preferably, the codon-optimized gene has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity with the nucleotide sequence shown in SEQ ID NO.3.

[0011] Related biomaterials include: 1) Recombinant expression vectors containing the aforementioned genes or codon-optimized genes; 2) Recombinant bacteria or engineered cell lines containing the recombinant expression vector described in 1).

[0012] The aforementioned application of a glycosyltransferase PmUGT7, a gene or codon-optimized gene, or related biological materials in the biosynthesis of stilbene glycosides.

[0013] Preferably, the glycosyltransferase PmUGT7 specifically catalyzes the glycosylation reaction of the C2-hydroxyl group of 2,3,5,4'-tetrahydroxystilbene to generate stilbene glycoside.

[0014] A biosynthetic method for stilbene glycosides is disclosed, using 2,3,5,4'-tetrahydroxystilbene as a substrate and glycosyltransferase PmUGT7 as a biocatalyst to catalyze the glycosylation reaction of the C2-position hydroxyl group of 2,3,5,4'-tetrahydroxystilbene to specifically generate stilbene glycosides.

[0015] Preferably, the total volume of the biosynthesis reaction system is 100 μL, containing: 50 mM phosphate buffer (PBS, pH 7.0), 1 mM 2,3,5,4′-tetrahydroxystilbene, 2.5 mM uridine diphosphate glucose (UDP-Glucose), and 50 μg glycosyltransferase PmUGT7.

[0016] Preferably, the reaction conditions are: incubation in a water bath at 37°C for 0.5 to 1 hour.

[0017] Preferably, the reaction is terminated by ice-cold methanol, followed by an ice bath for 2 min, centrifugation at 4°C and 12000×g for 1 min, and filtration of the supernatant through a 0.22 μm organic phase microporous membrane to obtain stilbene glycosides.

[0018] The beneficial effects of this invention are: Stilbene glycosides are currently mainly derived from plant extraction and separation. This process is not only time-consuming and labor-intensive, but also requires a huge amount of plant resources. The complexity of their structure and the limitation of their sources greatly restrict their further development. Selective glycosylation is one of the most critical but also the most challenging transformations in the organic synthesis of stilbene glycosides, usually requiring a guiding group or functional group to achieve the chemical reaction. Although chemical synthesis alleviates the resource pressure to some extent, it requires cumbersome group protection and deprotection steps.

[0019] This invention provides a glycosyltransferase PmUGT7 and its application in the biosynthesis of stilbene glycosides. The glycosyltransferase PmUGT7 can efficiently and specifically glycosylate the C2 hydroxyl group of 2,3,5,4'-tetrahydroxystilbene, specifically generating stilbene glycosides in a one-step process. The reaction conditions are mild, the operation is convenient and rapid, the catalytic efficiency is high, and it has strong sustainability. Attached Figure Description

[0020] Figure 1 Map of the pET28a-SUMO-PmUGT7 expression plasmid.

[0021] Figure 2 The reaction structure for the synthesis of THSG from THS catalyzed by PmUGT7 is shown.

[0022] Figure 3SDS-PAGE images of PmUGT7 and its codon-optimized protein expression; M, protein marker; 1, total protein of whole bacteria; 2, total protein of supernatant; 3, purified protein; 4, total protein of whole bacteria after codon optimization; 5, total protein of supernatant after codon optimization.

[0023] Figure 4 This is an HPLC chromatogram; the HPLC chromatogram of stilbene glycosides produced from the substrate THS catalyzed by pET28a-SUMO-PmUGT7.

[0024] Figure 5 High-resolution LC-MS mass spectra; A. Mass spectrum of the standard stilbene glycoside. B. Mass spectrum of the PmUGT7 enzyme-catalyzed product stilbene glycoside. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0026] Example 1: Chemical Synthesis of the Substrate 2,3,5,4'-Tetrahydroxystilbene Ferrous iodide (0.025 mmol), resveratrol (0.05 mmol), α-ketoglutaric acid (0.125 mmol), BCPOM (0.25 mmol), acetonitrile (2 mL), and water (2 mL) were added sequentially to a 25 mL reaction flask. Hydrogen peroxide (0.5 mmol) was then slowly added dropwise, and the reaction mixture was reacted at 80°C for 1 hour. The reaction solution was concentrated under vacuum using a rotary evaporator, centrifuged to dryness, and extracted three times with ethyl acetate (10 mL). The organic phases were combined, the solvent was evaporated under reduced pressure, and the concentrated residue was further separated by column chromatography (ethyl acetate / petroleum ether = 1:4, v / v) to obtain 2,3,5,4'-tetrahydroxystilbene in approximately 40% yield.

[0027] Example 2 Construction of pET28a(SUMO)-PmUGT7 expression plasmid Total RNA was extracted from the root tissue of Polygonum multiflorum using the TRIzol method and then reverse transcribed to synthesize cDNA. Specific primers were designed for the PmUGT6 gene (amino acid sequence as shown in SEQ ID NO.4, nucleotide sequence of the encoding gene as shown in SEQ ID NO.5) and the PmUGT7 gene (Table 1). The target fragments were amplified by falling polymerase chain reaction (PCR) (Table 2) and purified by agarose gel electrophoresis.

[0028] Table 1. Specific primers for PmUGT7 Table 2 High-fidelity PCR program The pET28a(SUMO) vector (Suzhou Junji Biotechnology Co., Ltd.) was double-digested with restriction endonucleases KpnI and BamHI to obtain a linearized vector after purification. The purified PmUGT7 fragment was ligated to the linearized vector via homologous recombination to construct a recombinant plasmid. The ligation product was transformed into *E. coli* TOP10 chemocompetent cells and plated on LB agar plates containing 50 μg / mL kanamycin, incubated overnight at 37°C. Single colonies were picked for amplification culture, and the plasmid was extracted for DNA sequencing verification. The correctly sequenced recombinant plasmid was named pET28a(SUMO)-PmUGT7, and its plasmid map is shown below. Figure 1 .

[0029] Example 3: Protein expression and purification of pET28a(SUMO)-PmUGT7 plasmid The empty vector pET28a-SUMO and the recombinant plasmid pET28a-SUMO-PmUGT7 were transformed into the *E. coli* expression strain Rosetta 2(DE3), respectively. The following day, single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL chloramphenicol, and cultured at 37°C with shaking until the OD600 reached 0.8–1.0. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.15 mM, and expression was induced at 18°C ​​for 16–20 h. The cells were collected (centrifuged at 5000 rpm at 4°C), resuspended in PBS buffer (pH=7.4), and then sonicated (ice-water bath, 180 W, 5–10 min) or subjected to high-pressure cell disruption to obtain the crude enzyme solution. The crude enzyme solution was purified by nickel column affinity chromatography. The purified protein was quantified using a BCA protein assay kit, aliquoted, and stored at -80°C.

[0030] SDS-PAGE analysis was performed on the whole bacterial lysate, supernatant, and purified protein of PmUGT7. The results are as follows: Figure 3 As shown, the PmUGT7 band was prominent in the total protein of the whole bacteria, indicating a high expression level of this protein; the bands of the supernatant and purified protein were clear, indicating that the protein has good solubility, and the expression level of PmUGT7 protein was significantly increased after codon optimization. PmUGT7 is fused with a His tag and also has a SUMO tag, with a molecular weight of approximately 65 kDa.

[0031] Example 4: Activity identification of PmUGT7 in the synthesis of stilbene glycosides like Figure 2As shown, the total volume of the reaction system was 100 μL, containing 50 mM phosphate-buffered saline (PBS, pH 7.0), 1 mM 2,3,5,4′-tetrahydroxystilbene, 2.5 mM uridine diphosphate glucose (UDP-Glucose), and 50 μg of purified PmUGT7 protein. The reaction system was incubated in a 37°C water bath for 0.5–1 h. The reaction was terminated by adding 100 μL of ice-cold methanol, followed by an ice bath for 2 min. The mixture was then centrifuged at 4°C and 12000×g for 1 min. The supernatant was filtered through a 0.22 μm organic phase microporous membrane and analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[0032] The HPLC analysis conditions were as follows: A C18 reversed-phase column (4.6 mm × 150 mm, 5 μm); mobile phase A was ultrapure water, and mobile phase B was acetonitrile; flow rate was 0.2 mL / min; detection wavelength was 300 nm; column temperature was 30℃; and injection volume was 10 μL. The gradient elution program (% are volume percentages) was as follows: 0–7 min, 10%–20% B; 7–15 min, 20%–35% B; 15–18 min, 35%–60% B; 18–20 min, 60%–100% B; 20–21 min, 100% B; and 21–25 min, 100%–10% B.

[0033] The results are as follows Figure 4 As shown, a chromatographic peak with the retention time of stilbene glycoside standard appeared at 10.5 min in the reaction solution. LC-MS analysis ( Figure 5 Further verification of the molecular weight of the product revealed the presence of [M+H] with a mass-to-charge ratio (m / z) of 407.1305 in positive ion mode. + The ion peaks are consistent with the molecular weight of stilbene glycoside standards. These results indicate that PmUGT7 can specifically catalyze the glycosylation of the C2-hydroxyl group of 2,3,5,4′-tetrahydroxystilbene to generate stilbene glycosides, with near-complete conversion at a substrate concentration of 1 mM. Its catalytic efficiency is significantly higher than that of its homologous enzyme PmUGT6. The substrate 2,3,5,4′-tetrahydroxystilbene has a similar molecular structure to resveratrol, and the resveratrol 3-O-glycosyltransferase R3GAT, reported in the literature for the catalytic production of polysaccharide, showed no significant catalytic activity against it.

Claims

1. A glycosyltransferase PmUGT7, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene or codon-optimized gene encoding the glycosyltransferase PmUGT7 of claim 1, characterized in that, Their nucleotide sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

3. Related biomaterials, characterized in that, include: 1) Recombinant expression vectors containing the aforementioned genes or codon-optimized genes; 2) Recombinant bacteria or engineered cell lines containing the recombinant expression vector described in 1).

4. The application of the glycosyltransferase PmUGT7 of claim 1, the gene or codon-optimized gene of claim 2, or the related biological material of claim 3 in the biosynthesis of stilbene glycosides.

5. The application according to claim 4, characterized in that, The glycosyltransferase PmUGT7 specifically catalyzes the glycosylation of the C2-hydroxyl group of 2,3,5,4'-tetrahydroxystilbene to generate stilbene glycoside.

6. A method for the biosynthesis of stilbene glycosides, characterized in that, Using 2,3,5,4'-tetrahydroxystilbene as a substrate and glycosyltransferase PmUGT7 as a biocatalyst, the glycosylation reaction of the C2-hydroxyl group of 2,3,5,4'-tetrahydroxystilbene was catalyzed to specifically generate stilbene glycosides.

7. The method for biosynthesizing stilbene glycoside according to claim 6, characterized in that, The total volume of the biosynthesis reaction system was 100 μL, containing: 50 mM phosphate buffer, 1 mM 2,3,5,4′-tetrahydroxystilbene, 2.5 mM uridine diphosphate glucose, and 50 μg glycosyltransferase PmUGT7.

8. The method for biosynthesizing stilbene glycoside according to claim 6, characterized in that, The reaction conditions are: incubation in a water bath at 37°C for 0.5 to 1 hour.

9. The method for biosynthesizing stilbene glycoside according to claim 6, characterized in that, The reaction was terminated by ice-cold methanol, followed by an ice bath for 2 min, centrifugation at 4℃ and 12000×g for 1 min, and filtration of the supernatant through a 0.22 μm organic phase microporous membrane to obtain stilbene glycosides.

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