Application of glycosyl transferases FmUGT and UGT75 in catalyzing glycosylation of emodin or polyphenol compound

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 or 4. According to the invention, two kinds of glucoside transferases capable of catalyzing the generation of glucosylation of emodin C6-OH are excavated, and the glucosylation of the emodin and a wide range of polyphenol compounds can be catalyzed, so that the emodin-6-O-glucoside and the glycosylated polyphenol compounds are obtained. The glycosyl transferase can be used for constructing a microbial cell factory, the glycoside compounds of the emodin and polyphenol compounds can be produced through a fermentation method, and the emodin and polyphenol glycoside compounds can also be produced through whole-cell catalysis or in-vitro catalysis of a cell lysis solution by using the cell factory, so that the yield is remarkably increased, the extraction cost is reduced, and the method is suitable for industrial production. Meanwhile, the method has sustainability. The technical prospect paves a road for wide application of the emodin and polyphenol compounds and the derivatives thereof in the fields of medicine and industry.

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 FmUGT and UGT75 that can catalyze the glycosylation of emodin and polyphenolic compounds and their applications. Background Art

[0002] Emodin, with the chemical name of 1,3,8-trihydroxy-6-methylanthraquinone, is a natural anthraquinone compound with the chemical formula of C 15 H 10 O5, which is almost insoluble in water and widely exists in Polygonaceae plants such as Rheum palmatum, Polygonum cuspidatum, and Fallopia multiflora. Emodin has a variety of pharmacological activities, such as antibacterial, laxative, diuretic, blood pressure lowering, anti-inflammatory, antitussive, and antitumor effects. At the same time, as a feeding attractant in aquaculture, it can improve the appetite of fish and shrimp and promote growth. However, due to its poor water solubility, the wide application of emodin is limited. To improve its bioavailability, structural modification of emodin is a reasonable improvement strategy.

[0003] Studies have shown that after glycosylation modification, emodin can not only significantly increase its solubility and stability, but also enhance its biological activity and have a detoxifying effect. For example, emodin-6-O-glucoside has anti-inflammatory and barrier protection effects and has significant benefits for diabetic complications and atherosclerosis; emodin-8-O-glucoside has neuroprotective effects and helps to improve learning and memory disorders, etc.

[0004] Therefore, glycosylation modification of emodin to enhance its water solubility and biological activity is a very promising research direction, and glycosyltransferases that can glycosylate emodin need to be found. However, current research on natural product glycosylating enzymes mainly focuses on flavonoids, phenylpropanoids, terpenoids, and steroids, etc., and there is relatively little research on glycosylating enzymes for 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 that can catalyze the glucosylation of the C6-OH of emodin ( Figure 1 ).

[0006] Based on the above research, the present invention provides the application of glycosyltransferases in catalyzing the glycosylation of emodin or polyphenolic compounds, and the amino acid sequences of the glycosyltransferases are as shown in SEQ ID NO:2 or 4.

[0007] In a specific embodiment, the emodin is glycosylated to emodin-6-O-glucoside.

[0008] In a specific embodiment, the polyphenol compound is selected from one or more combinations of dihydroquercetin, quercetin, luteolin, fisetin, daidzein, kaempferol, genistein, resveratrol, apigenin, and naringenin.

[0009] The present invention also provides a method for producing glycosylated emodin or glycosylated polyphenol compounds, including the step of mixing a glycosyltransferase with emodin or polyphenol compounds; the amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO:2 or 4.

[0010] In a specific embodiment, the glycosyltransferase is mixed with emodin or polyphenol compounds in vitro or in cells.

[0011] In a specific embodiment, the reaction system further contains glucose or its derivatives.

[0012] In a specific embodiment, the glucose derivative is UDP-glucose.

[0013] In a specific embodiment, the polyphenol compound is selected from one or more combinations of dihydroquercetin, quercetin, luteolin, fisetin, daidzein, kaempferol, genistein, resveratrol, apigenin, and naringenin.

[0014] The present invention has mined two glucosyltransferases that can catalyze the glucosylation of emodin C6-OH through bioinformatics technology, heterologously expressed the enzyme, and purified the expression product. Through enzyme-catalyzed reaction experiments, it is shown that the glycosyltransferase can catalyze the glycosylation of emodin and a wide range of polyphenol compounds to obtain emodin-6-O-glucoside and glycosylated polyphenol compounds. This invention is of great significance for the research and application development of emodin-6-O-glucoside and glycosylated polyphenol compounds in medicinal natural products.

[0015] After further research, the above glycosyltransferase can be used to construct a microbial cell factory to produce glycoside compounds of emodin and polyphenol compounds by fermentation. It can also be used for whole-cell catalysis by the cell factory or in vitro catalysis by cell lysates to produce glycoside compounds of emodin and polyphenol compounds. Compared with traditional plant extraction methods, these production methods can significantly increase the yield, reduce the extraction cost, and are sustainable. This technical prospect paves the way for the wide application of emodin and polyphenol compounds and their derivatives in the pharmaceutical and industrial fields. Description of the Drawings

[0016] Figure 1 Schematic diagram of the catalysis of emodin to emodin-6-O-glucoside by FmUGT and UGT75 under UDP-glucose supply.

[0017] Figure 2 Photo of SDS-PAGE analysis of protein expression and purified products of FmUGT and UGT75.

[0018] Figure 3 HPLC analysis of in vitro enzymatic activity characterization of purified proteins of FmUGT and UGT75 with emodin.

[0019] Figure 4 UV absorption peak analysis of in vitro enzymatic activity characterization of purified proteins of FmUGT and UGT75.

[0020] Figure 5 Results of anion high-resolution mass spectrometry analysis of reaction products of FmUGT and UGT75 with emodin.

[0021] Figure 6 HPLC analysis chart of reaction products of purified proteins of FmUGT and UGT75 catalyzing 10 polyphenolic compounds in vitro.

[0022] Figure 7 and 8 HR-LCMS analysis chart of reaction products of purified proteins of FmUGT and UGT75 catalyzing 10 polyphenolic compounds in vitro. Detailed implementation manners

[0023] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0024] 1. Gene synthesis and heterologous expression of glycosyltransferase

[0025] 1.1 Gene synthesis of glycosyltransferases FmUGT and UGT75

[0026] Our team screened glycosyltransferase FmUGT (Sequence ID: ATO91433.1) from Fallopia multiflora and glycosyltransferase UGT75 (Sequence ID: BAP90370.1) from Fagopyrum esculentum by bioinformatics methods, which may have the activity of catalyzing the formation of emodin-6-O-glucoside from emodin.

[0027] The above genes were synthesized and ligated to the expression vector pET28b. Among them, the DNA sequence of FmUGT contains 1,431 nucleotides as shown in SEQ ID NO:1, encoding 476 amino acids as shown in SEQ ID NO:2; the DNA sequence of UGT75 contains 1,416 nucleotides as shown in SEQ ID NO:3, encoding 471 amino acids as shown in SEQ ID NO:4.

[0028] 1.2 Induced expression and protein purification of FmUGT and UGT75

[0029] The stabbed bacteria of Escherichia coli containing the expression vectors of FmUGT and UGT75 were inoculated into 5 ml of LB medium containing kanamycin at a final concentration of 50 μg / ml, and cultured overnight at 37 °C for 18 h. The plasmid was extracted using the OMEGA plasmid extraction kit, and the quality of the plasmid extraction was detected by 1% agarose gel electrophoresis. Take 1 μl of the plasmid and transform it into BL21(DE3). Pick the viable transformants on the kanamycin-resistant plate into 10 ml of liquid LB medium containing kanamycin at a final concentration of 50 μg / ml and culture at 37 °C for 18 h, then transfer it to 1 L of TB medium containing kanamycin at a final concentration of 50 μg / ml and culture at 37 °C until the OD 600 reached 0.6 - 0.8, add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.2 mM, and induce protein expression at 16 °C for 20 h. Collect the bacteria by centrifugation at 5,000 × g.

[0030] Resuspend the bacteria with 20 ml of lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 10% (w / v) glycerol, 10 mM imidazole, pH 8.0), place it under an ultrasonic crusher for 30 min (5 s on / 5 s off), and then centrifuge at 11,000 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, ultrafiltration and concentration to remove salts through an Amicon Ultra-15 ultrafiltration tube (Millipore, Ireland) with a cut-off molecular weight of 30 kDa. All the above purification steps were carried out at 4 °C.

[0031] AsFigure 2 It can be seen that SDS-PAGE analysis shows that the target proteins FmUGT and UGT75 have been successfully expressed and purified. Among them, the purity of FmUGT exceeds 70%, and the purity of UGT75 exceeds 50%, which can be used for the next step of in vitro activity analysis research.

[0032] 2. In vitro activity and product detection of glycosyltransferase

[0033] The above-obtained target proteins FmUGT and UGT75 were used to prepare a catalytic reaction system to study the catalytic activities of the above three enzymes using emodin as a substrate. The reaction system is as follows:

[0034]

[0035] After reacting at 30 °C for 45 min, 100 μl of methanol was added. After vigorous shaking, the protein was precipitated by centrifugation at 13000 rpm for 10 min, and the supernatant was taken for the next analysis.

[0036] The above reaction products were analyzed using Agilent 1260 HPLC on an Agilent Eclipse Plus C18 column (5 μm, 4.6 mm × 250 mm). The analysis method is as follows:

[0037] Mobile phase B (100% ACN), mobile phase D (100% H2O + 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.

[0038] The results are as Figure 3 shown. Both the pure enzymes of FmUGT and UGT75 can catalyze emodin to produce a new compound, and the retention time of the new compound is exactly the same as that of the emodin-6-O-glucoside standard.

[0039] Further research found that the ultraviolet absorption peak of the new compound is exactly the same as that of the emodin-6-O-glucoside standard ( Figure 4 ).

[0040] Using Thermo Fisher LC-MS (LC: Vanquish; MS: Orbitrap Exploris 480), the suspected emodin-6-O-glucosides produced by the reactions of FmUGT and UGT75 with emodin were further analyzed by anion high-resolution mass spectrometry on a Waters Symmetry C18 column (5 μm, 2.1 mm × 150 mm). The analysis method was as follows:

[0041] Mobile phase A (100% H2O + 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.

[0042] The results were as Figure 5 shown. Their m / z values were 431.0983 (FmUGT) and 431.0982 (UGT75) ([M-H] - , and the corresponding molecular formulas were both C 21 H 19 O 10 - ), which was consistent with the theoretical anion mass spectrum of emodin-6-O-glucoside (calc. 431.0984).

[0043] Under the conditions of 2 μM enzyme, 100 μM substrate emodin, and a reaction time of 20 minutes, the conversion rates of emodin-6-O-glucoside by FmUGT and UGT75 were 61% and 63% respectively.

[0044] 3. Substrate broadness analysis of FmUGT and UGT75

[0045] Using the above-mentioned target proteins FmUGT and UGT75, an enzyme-catalyzed reaction system was constructed to study their catalytic activities when using 11 polyphenolic compounds (dihydroquercetin, quercetin, naringenin, luteolin, fisetin, daidzein, kaempferol, genistein, apigenin, resveratrol, and epicatechin) as substrates. The reaction system was as follows:

[0046]

[0047] After reacting at 30 °C for 45 min, 100 μl of methanol was added. After vigorous shaking, the protein was precipitated by centrifugation at 13,000 rpm for 10 min, and the supernatant was taken for the next analysis.

[0048] The extracted compounds were analyzed using an Agilent 1260 HPLC on an Agilent Eclipse Plus C18 column (5 μm, 4.6 mm × 250 mm), and the analysis method was as follows:

[0049] Mobile phase B (100% H2O + 0.1% TFA), mobile phase D (100% ACN), gradient elution (0 - 1 min 5% B, 1 - 20 min 5% - 100% B, 20 - 24 min 100% B, 24 - 25 min 100% - 5% B, 25 - 30 min 5% B), flow rate 1 ml / min, detection wavelength 340 nm.

[0050] The results were as Figure 6 shown, and glycosylated products of 10 polyphenolic compounds were detected, namely glycosylated dihydroquercetin, quercetin, luteolin, fisetin, daidzein, kaempferol, genistein, resveratrol, apigenin, and naringenin. Only the glycosylated product of 1 substrate, epicatechin, was not detected.

[0051] The extracted compounds were analyzed using a Thermo Fisher LC - MS (LC: Vanquish; MS: Orbitrap Exploris 480) on a Waters Symmetry C18 column (5 μm, 2.1 mm × 150 mm), and the analysis method was as follows:

[0052] Mobile phase A (100% H2O + 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.

[0053] The results were as Figure 7 and 8 shown, and the molecular weights of the monoglycosylated and diglycosylated products of 10 polyphenolic compounds were detected. Only the molecular weight of the glycosylated product of 1 compound was not detected. This indicates that FmUGT and UGT75 can catalyze the glycosylation of compounds such as dihydroquercetin, quercetin, luteolin, fisetin, daidzein, kaempferol, genistein, resveratrol, apigenin, and naringenin.

[0054] The above experiments fully demonstrate that both FmUGT and UGT75 can catalyze the glycosylation of emodin to produce emodin-6-O-glucoside. Moreover, the substrates of these two enzymes have broad-spectrum properties and can catalyze the glycosylation of compounds such as dihydroquercetin, quercetin, luteolin, fisetin, daidzein, kaempferol, genistein, resveratrol, apigenin, and naringenin.

[0055] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, 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 or polyphenolic compounds, characterized in that: The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO: 2 or 4, or a polypeptide having more than 90% homology with SEQ ID NO: 2 or 4 and having glycosylation activity of rhamnosine or polyphenolic compounds.

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

3. The use according to claim 1, characterized in that: The polyphenol compound is selected from one or more combinations of dihydroquercetin, quercetin, luteolin, fisetin, daidzein, kaempferol, genistein, resveratrol, apigenin and naringenin.

4. A method for producing glycosylated rhamnosine or glycosylated polyphenol compounds, characterized in that: The method comprises the steps of mixing glycosyltransferase or its equivalent variant with rhamnosine or polyphenolic compounds, wherein the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO: 2 or 4.

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

6. The method according to claim 4, characterized in that The reaction system also contains glucose or its derivatives.

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

8. The method according to claim 4, characterized in that The polyphenol compound is selected from one or more combinations of dihydroquercetin, quercetin, luteolin, fisetin, daidzein, kaempferol, genistein, resveratrol, apigenin and naringenin.

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