Application of glycosyl transferase OsUGT in catalyzing glycosylation of emodin

Abstract

The invention relates to an application of glycosyl transferase in catalyzing glycosylation of emodin. The amino acid sequence of the glycosyl transferase is shown as SEQ ID NO: 2. According to the present invention, the glycosyl transferase capable of catalyzing the glucosylation reaction of the emodin C6-OH and the emodin C8-OH is excavated through the bioinformatics technology, and the new method for preparing the emodin-6-O-glucoside and the emodin-8-O-glucoside through the in-vitro enzyme catalysis is developed; the invention has important significance on research and application development of emodin-6-O-glucoside and emodin-8-O-glucoside, can be used for constructing a microbial cell factory, and can be used for producing the emodin glycoside compound through methods such as fermentation, whole-cell catalysis or cell lysis buffer catalysis, so that the production cost is further reduced, and the method is suitable for industrial production. The problems that the content of the emodin glucoside in plants is low and the extraction cost is high are solved, so that wide application and market popularization of the emodin glucoside are promoted.

Description

Technical Field

[0001] The present invention belongs to the fields of microbial metabolic engineering, synthetic biology and medicinal chemistry, and particularly relates to the application of glycosyltransferases in the modification of natural products. More particularly, it relates to a glycosyltransferase OsUGT capable of catalyzing the glycosylation of emodin and its application. Background Art

[0002] Emodin, with the chemical name of 1,3,8-trihydroxy-6-methylanthraquinone, is a natural anthraquinone compound with the chemical formula C 15 H 10 O 5 , which is almost insoluble in water and widely exists in Polygonaceae plants such as Rheum palmatum, Reynoutria japonica, and Fallopia multiflora. It has pharmacological activities such as antibacterial, laxative, diuretic, blood pressure lowering, anti-inflammatory, antitussive, and anti-tumor. At the same time, as an attractant in aquaculture, it can improve the appetite of fish and shrimp and promote growth.

[0003] However, the low water solubility of emodin limits its development and application. To improve its bioavailability, modifying and transforming emodin is a reasonable approach.

[0004] Studies have shown that after specific glycosylation modification of emodin, it can not only increase solubility and stability but also improve biological activity and even produce new physiological and pharmacological functions. For example, emodin-6-O-glucoside has anti-inflammatory and barrier protection effects and is beneficial for diabetic complications and atherosclerosis; emodin-8-O-glucoside has neuroprotective effects and can improve learning and memory disorders, etc. Therefore, post-modification based on emodin to enhance its biological activity and water solubility is an important research work, and glycosyltransferases capable of glycosylating emodin need to be found. However, current research on glycosylating enzymes of natural products mainly focuses on flavonoids, phenylpropanoids, terpenoids, and steroids, etc. There is relatively little research on glycosylating enzymes of anthraquinone compounds represented by emodin. The enzyme activity also needs to be further improved, the conversion efficiency is unstable, and it is difficult to achieve industrial application. Summary of the Invention

[0005] Our team has discovered through bioinformatics technology an emodin-6-O-glucosyltransferase and an emodin-8-O-glucosyltransferase that can catalyze the glucosylation of C6-OH and C8-OH of emodin ( Figure 1 ).

[0006] Based on the above research, the present invention provides the application of glycosyltransferase in catalyzing the glycosylation of emodin, and the amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO:2.

[0007] In a specific embodiment, the emodin is glycosylated into emodin-6-O-glucoside and / or emodin-8-O-glucosyltransferase.

[0008] The present invention also provides a method for producing glycosylated emodin, which includes the step of mixing a glycosyltransferase with emodin; the amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO:2.

[0009] In a specific embodiment, the glycosyltransferase is mixed with emodin in vitro or in cells.

[0010] In a specific embodiment, the reaction system further contains glucose or its derivatives.

[0011] In a specific embodiment, the glucose derivative is UDP-glucose.

[0012] The present invention has mined a glycosyltransferase that can simultaneously catalyze the glucosylation reaction of C6-OH and C8-OH of emodin through bioinformatics technology, and developed a new method for in vitro enzymatic catalysis to prepare emodin-6-O-glucoside and emodin-8-O-glucoside. This invention is of great significance for the research and application development of emodin-6-O-glucoside and emodin-8-O-glucoside in medicinal natural products.

[0013] By genetically engineering the metabolic pathway of the strain, the above-mentioned glycosyltransferase can be further utilized to construct a microbial cell factory to produce emodin glycosides by fermentation method. It can also be used for whole-cell catalysis by the cell factory, or in vitro catalysis by cell lysate to produce emodin glycosides. Compared with the traditional plant extraction method, these methods can reduce production costs, improve yield and quality, and also help to solve the problems of low content of emodin glucoside in plants and high extraction costs, thus promoting its wide application and market promotion. Description of the Drawings

[0014] Figure 1 Schematic diagram of OsUGT catalyzing emodin into emodin-6-O-glucoside and / or emodin-8-O-glucoside under the supply of UDP-glucose.

[0015] Figure 2 Photo of SDS-PAGE analysis of protein expression and purified product of OsUGT.

[0016] Figure 3 HPLC analysis of in vitro enzyme activity characterization of purified OsUGT protein.

[0017] Figure 4 UV absorption peak analysis of in vitro enzyme activity characterization of purified OsUGT protein.

[0018] Figure 5 It is the result of high-resolution anion mass spectrometry analysis of the reaction product of OsUGT and emodin. Detailed implementation manners

[0019] The principles and features of the present invention will be described below in conjunction with the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0020] 1. Gene synthesis and heterologous expression of glycosyltransferase

[0021] 1.1 Gene synthesis of glycosyltransferase OsUGT

[0022] Our team screened OsUGT (Sequence ID: XP_015645476.1) from rice (Oryza sativa) by bioinformatics methods, which may be able to catalyze the glycosylation of anthraquinone compounds represented by emodin to generate emodin-6-O-glucoside and / or emodin-8-O-glucoside ( Figure 1 ).

[0023] The above gene was synthesized and ligated to the expression vector pET28b. Among them, the DNA sequence of OsUGT contains 1,473 nucleotides, as shown in SEQ ID NO: 1, encoding 490 amino acids, as shown in SEQ ID NO: 2.

[0024] 1.2 Induced expression and protein purification of OsUGT

[0025] The stabbed bacteria of Escherichia coli containing the OsUGT expression vector were inoculated into 5 ml of LB medium containing kanamycin with a final concentration of 50 μg / ml, cultured overnight at 37 °C for 18 h, and the plasmid was extracted using the OMEGA plasmid extraction kit. The quality of plasmid extraction was detected by 1% agarose gel electrophoresis. Take 1 μl of plasmid to transform BL21(DE3), pick the growable transformants on the kanamycin-resistant plate into 10 ml of liquid LB medium containing kanamycin with a final concentration of 50 μg / ml and culture at 37 °C for 18 h, and then transfer it to 1 L of TB medium containing kanamycin with a final concentration of 50 μg / ml and culture at 37 °C until OD 600 reaches 0.6 - 0.8, add IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.2 mM, induce protein expression at 16 °C for 20 h, and collect the bacteria by centrifugation at 5000 × g.

[0026] Resuspend the bacterial cells in 20 ml of lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 10% (w / v) glycerol, 10 mM imidazole, pH 8.0), place them under an ultrasonic crusher for 30 min (5 s on / 5 s off), and then centrifuge at 11000 rpm for 60 min to collect the supernatant of the lysate. Add 1 ml of Ni-NTA Agarose resin to the supernatant, rotate and shake at 4°C for 2 h, and then slowly pack the Ni-NTA Agarose resin into an empty column and wait for the liquid to drain. Next, slowly wash away the non-specifically bound miscellaneous proteins with 100 ml of washing buffer (20 mM Tris-HCl, 300 mM NaCl, 10% (w / v) glycerol, 20 mM imidazole, pH 8.0). Thereafter, elute the bound target protein with 10 ml of elution buffer (20 mM Tris-HCl, 300 mM NaCl, 10% (w / v) glycerol, 250 mM imidazole, pH 8.0). Finally, ultrafilter and concentrate to remove salts through an Amicon Ultra-15 ultrafiltration tube with a cut-off molecular weight of 30 kDa. All the above purification steps are carried out at 4°C.

[0027] As Figure 2 can be seen, SDS-PAGE analysis shows that the target protein OsUGT has been successfully expressed and purified and can be used for the next step of in vitro activity analysis research.

[0028] 2. In vitro Activity and Product Detection of Glycosyltransferase

[0029] Use the above-obtained target protein OsUGT to prepare a catalytic reaction system to study the catalytic activity with emodin as the substrate. The reaction system is as follows:

[0030]

[0031] After reacting at 30°C for 45 min, add 100 μl of methanol, shake vigorously, and then centrifuge at 13000 rpm for 10 min to precipitate the protein. Take the supernatant for the next analysis.

[0032] Analyze the above reaction products on an Agilent 1260 HPLC using an Agilent Eclipse Plus C18 column (5 μm, 4.6 mm × 250 mm). The analysis method is as follows:

[0033] Mobile phase B (100% ACN), mobile phase D (100% H 2O + 0.1% TFA), gradient elution (0 - 10 min 0% - 35% B, 10 - 20 min 35% - 100% B, 20 - 24 min 100% B, 24 - 25 min 100% - 35% B, 25 - 30 min 35% B), flow rate 1 ml / min, detection wavelength 440 nm.

[0034] The results are as Figure 3 shown. The pure OsUGT enzyme can catalyze emodin to produce two new compounds. The retention time of one new compound is exactly the same as that of the emodin - 6 - O - glucoside standard, and the retention time of the other new compound is exactly the same as that of the emodin - 8 - O - glucoside standard. Further research found that the UV absorption peaks of the two new compounds also exactly correspond to those of their standards ( Figure 4 ).

[0035] Using Thermo Fisher LC - MS (LC: Vanquish; MS: Orbitrap Exploris 480), further analysis of the suspected emodin - 6 - O - glucoside and emodin - 8 - O - glucoside produced by the reaction of OsUGT and emodin was carried out on a Waters Symmetry C18 column (5 μm, 2.1 mm × 150 mm) using anion high - resolution mass spectrometry. The analysis method is as follows:

[0036] Mobile phase A (100% H 2 O + 0.1% FA), mobile phase B (100% ACN), gradient elution (0 - 1 min 40% A, 1 - 18 min 40% - 0% A, 18 - 24 min 0% A, 24 - 25 min 0% - 40% A, 25 - 30 min 40% A), flow rate 0.2 ml / min, column temperature 30 °C.

[0037] The results are as Figure 5 shown. Their m / z values are 431.0983 (OsUGT - emodin - 6 - O - glucoside) and 431.0984 (OsUGT - emodin - 8 - O - glucoside) ([M - H] - , and the corresponding molecular formulas are both C 21 H 19 O 10 - ), which are consistent with the theoretical anion mass spectra of emodin - 6 - O - glucoside and emodin - 8 - O - glucoside (calc. 431.0984).

[0038] The above experiments fully demonstrate that OsUGT can catalyze the glycosylation of emodin to produce emodin-6-O-glucoside and emodin-8-O-glucoside. After reacting for 20 minutes with 2 μM enzyme and 100 μM emodin substrate, the yield of emodin-6-O-glucoside is 51%, and the yield of emodin-8-O-glucoside is 8.5%.

[0039] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. The use of glycosyltransferase in catalyzing the glycosylation of rhamnosin, characterized in that: The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO: 2, or a polypeptide having more than 90% homology with SEQ ID NO: 2 and having rhamnosyl glycosylation activity.

2. The use according to claim 1, characterized in that: The rhein is glycosylated to rhein-6-O-glucoside and / or rhein-8-O-glucoside.

3. A method for producing glycosylated rhein, characterized in that: The method comprises the steps of mixing glycosyltransferase or its equivalent variant with rhamnosine, wherein the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO:

2.

4. The method according to claim 3, characterized in that The glycosyltransferase is mixed with rhein in vitro or in cells.

5. The method according to claim 3, characterized in that: The reaction system also contains glucose and / or its derivatives.

6. The method according to claim 5, characterized in that The glucose derivative is UDP-glucose.

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