Glycosyltransferase bs-yjic mutants and uses thereof

Abstract

The application discloses a glycosyltransferase Bs-YjiC mutant and application thereof, wherein the glycosyltransferase Bs-YjiC mutant is one of the following: M1: in the amino acid sequence of the glycosyltransferase Bs-YjiC shown in the gene bank number NP_389104, glutamine at the 66th position is mutated into valine, serine at the 277th position is mutated into tryptophan, and serine at the 292nd position is mutated into threonine; M2: in the amino acid sequence of the glycosyltransferase Bs-YjiC shown in the gene bank number NP_389104, glutamine at the 66th position is mutated into valine, serine at the 277th position is mutated into tryptophan, and alanine at the 230th position is mutated into valine. The mutant of the application takes triterpenoid compounds and flavonoid compounds as substrates, and catalyzes synthesis of various novel glycosides beneficial to human health.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and botany, and relates to the Bs-YjiC mutant of glycosyltransferase and its applications. Background Technology

[0002] Glycosylation is an attractive method for modifying natural products, expanding their chemical dimensionality and improving their low water solubility. Traditionally, most glycosylation reactions to generate glycosides are carried out through chemical synthesis, often requiring many complex steps to obtain the target molecule. Enzymatic glycosylation by glycosyltransferases can provide an alternative strategy for generating novel glycosides. Nature contains a diverse array of glycosides, including ginsenosides, notoginsenosides, and flavonoid glycosides, among other active ingredients. Ginsenosides Rg3, Rg2, and glycyrrhizin, for example, have been reported to possess antiviral, anti-inflammatory, and anticancer pharmacological activities. Therefore, adding glycosyl groups to drug leads to synthesize new compounds is a valuable tool for drug discovery.

[0003] Currently, numerous glycosyltransferases have been discovered from plants and microorganisms, and these natural glycosyltransferases can catalyze the synthesis of a variety of glycosides. However, most of these natural glycosyltransferases use UDP-Glc as a donor, and glycosyltransferases with UDP-Xyl sugar donor activity are extremely rare. Studies have shown that xylosides possess unique pharmacological activities, including the hypoglycemic effect of astragaloside IV, the angiogenic activity of notoginsenoside R1, and the potential for inhibiting cancer metastasis by ophiopogon japonicus saponin C. Therefore, discovering xylosyltransferases with substrate heterogeneity to synthesize a variety of xylosides could greatly aid in new drug development. Enzyme engineering is known to modify natural glycosyltransferases to expand the range of glycosylation products. Recent work on glycosyltransferase engineering based on semi-rational design has yielded significant results, including achieving sugar donor selectivity in enzymes.

[0004] However, there are currently no reports on the Bs-YjiC mutant glycosyltransferase that catalyzes sugar chain elongation and its role in xyloside synthesis. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Bs-YjiC mutant glycosyltransferase.

[0006] A second objective of this invention is to provide a gene encoding the aforementioned glycosyltransferase Bs-YjiC mutant.

[0007] A third objective of this invention is to provide plasmids containing the aforementioned genes.

[0008] A fourth objective of this invention is to provide genetically engineered strains containing the aforementioned plasmids.

[0009] The fifth objective of this invention is to provide the application of the above-mentioned glycosyltransferase Bs-YjiC mutant in the enzymatic synthesis of xylosinolates.

[0010] The technical solution of this invention is summarized as follows:

[0011] A glycosyltransferase Bs-YjiC mutant, wherein the glycosyltransferase Bs-YjiC mutant is one of the following:

[0012] M1: In the amino acid sequence of the glycosyltransferase Bs-YjiC shown in gene bank number NP_389104, glutamine at position 66 is mutated to valine, serine at position 277 is mutated to tryptophan, and serine at position 292 is mutated to threonine; the amino acid sequence of M1 is shown in SEQ ID NO.1:

[0013] M2: In the amino acid sequence of the glycosyltransferase Bs-YjiC shown in gene bank number NP_389104, glutamine at position 66 is mutated to valine, serine at position 277 is mutated to tryptophan, and alanine at position 230 is mutated to valine. The amino acid sequence of M2 is shown in SEQ ID NO.2.

[0014] The gene encoding the Bs-YjiC mutant of the above-mentioned glycosyltransferase.

[0015] Plasmids containing the above-mentioned genes.

[0016] Genetically engineered strains containing the above plasmids.

[0017] Application of the above-mentioned Bs-YjiC mutant glycosyltransferase in the enzymatic synthesis of xylosinolates.

[0018] Advantages of this invention:

[0019] Experiments have demonstrated that the Bs-YjiC mutant glycosyltransferase of this invention catalyzes the synthesis of various glycosides using triterpenoids (ginsenoside Rh1, ginsenoside CK, ginsenoside Rg3) and flavonoids (glycyrrhizin) as substrates. Given the important pharmacological effects of glycosides, the Bs-YjiC mutant glycosyltransferase of this invention can be used to catalyze the synthesis of novel glycosides beneficial to human health. Attached Figure Description

[0020] Figure 1 The reaction formulas for the formation of glycosylated products of triterpenoid saponins (ginsenoside Rh1, ginsenoside CK, ginsenoside Rg3) and flavonoids (glycyrrhizin) under the catalysis of M1 or M2.

[0021] in:

[0022] Figure A shows the reaction of ginsenoside Rh1 to ginsenoside Rh1 3-O-β-D-xyloside (product 1) under the catalysis of M1 (or M2);

[0023] Figure B shows the reaction of ginsenoside CK with 3-O-β-D-xyloside (product 2) under the catalysis of M1 (or M2).

[0024] Figure C shows the reaction of ginsenoside Rg3 to ginsenoside Rg3 12-O-β-D-xyloside (product 3) under the catalysis of M1 (or M2);

[0025] Figure D shows the reaction of glycyrrhizin with M1 (or M2) to produce glycyrrhizin 7-O-β-D-xyloside (product 4).

[0026] Figure 2 The diagram shows the high-performance liquid chromatography (HPLC) detection of triterpenoid saponins and flavonoids in the formation of their glycosylation products under the catalysis of M1 or M2, where:

[0027] A1 is the high-performance liquid chromatography of product 1 generated from ginsenoside Rh1 catalyzed by M1;

[0028] A2 is the high-performance liquid chromatography of product 1 generated from ginsenoside Rh1 by M2 catalysis;

[0029] B1 is the high-performance liquid chromatography of product 2 generated from ginsenoside CK catalyzed by M1;

[0030] B2 is the high-performance liquid chromatography of product 2 generated from ginsenoside CK catalyzed by M2;

[0031] C1 is the high performance liquid chromatography of product 3 generated from ginsenoside Rg3 by M1 catalysis;

[0032] C2 is the high-performance liquid chromatography of product 3 generated from ginsenoside Rg3 by M2 catalysis;

[0033] D1 is the high-performance liquid chromatography of product 4, which is generated from glycyrrhizin by M1 catalysis;

[0034] D2 is the high-performance liquid chromatography of product 4, which is generated from glycyrrhizin by M2 catalysis.

[0035] Figure 3 For NMR detection of product 1 catalyzed by M1, in which:

[0036] A represents product 1, which is the glycosylation of ginsenoside Rh1 catalyzed by M1. 1 H NMR spectral analysis;

[0037] B represents the product 1 generated by the M1-catalyzed glycosylation of ginsenoside Rh1. 13 C NMR spectral analysis;

[0038] C represents the HMQC spectroscopic analysis of product 1 generated by the glycosylation of ginsenoside Rh1 catalyzed by M1.

[0039] D represents the HSBC spectroscopic analysis of product 1, which is generated by the glycosylation of ginsenoside Rh1 catalyzed by M1.

[0040] E represents the COSY spectral analysis of product 1, which is the glycosylation product of ginsenoside Rh1 catalyzed by M1.

[0041] Figure 4 For NMR detection of product 2 catalyzed by M1, in which:

[0042] A represents the product 2 generated by the M1-catalyzed glycosylation of ginsenoside CK. 1 H NMR spectral analysis;

[0043] B represents the product 2 generated by the M1-catalyzed glycosylation of ginsenoside CK. 13 C NMR spectral analysis;

[0044] C represents the HMQC spectroscopic analysis of the product 2 generated by the glycosylation of ginsenoside CK catalyzed by M1;

[0045] D represents the HSBC spectroscopic analysis of ginsenoside CK glycosylation product 2 catalyzed by M1;

[0046] E represents the COSY spectral analysis of product 2, which is the glycosylation of ginsenoside CK catalyzed by M1.

[0047] Figure 5 For NMR detection of product 3 catalyzed by M1, in which:

[0048] A represents product 3, which is the glycosylation of ginsenoside Rg3 catalyzed by M1. 1 H NMR spectral analysis;

[0049] B represents product 3, which is the glycosylation of ginsenoside Rg3 catalyzed by M1. 13 C NMR spectral analysis;

[0050] C represents the HMQC spectroscopic analysis of product 3 generated by the glycosylation of ginsenoside Rg3 catalyzed by M1.

[0051] D represents the HSBC spectroscopic analysis of product 3, which is generated by the glycosylation of ginsenoside Rg3 catalyzed by M1.

[0052] E represents the COSY spectral analysis of product 3, which is the glycosylation product of ginsenoside Rg3 catalyzed by M1.

[0053] Figure 6 For NMR detection of product 4 catalyzed by M1, in which:

[0054] A represents product 4, which is produced by the M1-catalyzed glycyrrhizin glycosylation process. 1H NMR spectral analysis;

[0055] B represents product 4, which is produced by the M1-catalyzed glycyrrhizin glycosylation process. 13 C NMR spectral analysis;

[0056] C represents the HMQC spectroscopic analysis of product 4 generated by M1-catalyzed glycyrrhizin glycosylation;

[0057] D represents the HSBC spectroscopic analysis of product 4, which is generated by the M1-catalyzed glycyrrhizin glycosylation.

[0058] E represents the COSY spectral analysis of product 4, which is generated by the M1-catalyzed glycyrrhizin glycosylation. Detailed Implementation

[0059] The glycosyltransferase Bs-YjiC of this invention is derived from Bacillus subtilis 168, Genebank number NP_389104.

[0060] The glycosyltransferase Bs-YjiC can be expressed and purified by constructing an expression vector and transforming it into a host strain, specifically *Escherichia coli*. Site-directed mutagenesis was performed using the Tiangen Rapid Site-Directed Mutagene kit (KM101).

[0061] The present invention will be further described in detail below with reference to specific embodiments.

[0062] It is understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0063] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0064] The enzyme activity assay method in this invention is as follows:

[0065] Glycosyltransferase activity assay: A 100 μL reaction system containing substrate (0.5 mM), xyluridine diphosphate (5 mM), 100 mM phosphate buffer (pH 7.5), and 80 μg of glycosyltransferase Bs-YjiC mutant was prepared and reacted in a water bath at 37 °C for 12 h. The reaction was terminated by adding 100 μL of methanol. The amount of substrate consumed and the amount of product generated were determined by high performance liquid chromatography.

[0066] Example 1: Obtaining, expressing, and purifying the Bs-YjiC mutant glycosyltransferase

[0067] The GeneBank accession number for the glycosyltransferase Bs–YjiC is NP_389104. The Bs–YjiC gene was synthesized by Jiangsu Genewise Biotechnology Co., Ltd.

[0068] Mutation was performed using the Tiangen Rapid Site-Directed Mutation Kit (KM101).

[0069] The amino acid sequence of glycosyltransferase Bs-YjiC was modified by mutating glutamine (Q) at position 66 to valine (V), serine (S) at position 277 to tryptophan (W), and serine (S) at position 292 to threonine (T), resulting in glycosyltransferase Bs-YjiC mutant M1 (Q66V / S277W / S292T), abbreviated as M1; the amino acid sequence of M1 is shown in SEQ ID NO.1.

[0070] The amino acid sequence of glycosyltransferase Bs-YjiC was modified by mutating glutamine (Q) at position 66 to valine (V), serine (S) at position 277 to tryptophan (W), and alanine (A) at position 230 to valine (V), resulting in the glycosyltransferase Bs-YjiC mutant M2 (Q66V / S277W / A230V), abbreviated as M2. The amino acid sequence of M2 is shown in SEQ ID NO.2.

[0071] The gene encoding M1 was inserted into the pET-28a(+) expression vector treated with the same restriction endonuclease using the Seamless Cloning Kit (RK21020) from Aibotek Biotechnology Co., Ltd., to construct the recombinant plasmid pET28-Bs-YjiC-M1.

[0072] Using the seamless cloning kit from Ibotek Biotechnology Co., Ltd., the gene encoding M2 was inserted into the pET-28a(+) expression vector treated with the same restriction endonuclease to construct the recombinant plasmid pET28-Bs-YjiC-M2.

[0073] The recombinant plasmid pET28-Bs-YjiC-M1 was transformed into Escherichia coli BL21(DE3) competent cells to obtain genetically engineered strain 1 containing the recombinant plasmid, and the expression of the target protein was induced.

[0074] The recombinant plasmid pET28-Bs-YjiC-M2 was transformed into Escherichia coli BL21(DE3) competent cells to obtain genetically engineered strain 2 containing the recombinant plasmid, and the expression of the target protein was induced.

[0075] The induction conditions for M1 or M2 are:

[0076] Genetically engineered strain 1 (or genetically engineered strain 2) was cultured at 37°C in LB liquid medium containing 50 mg / L kanamycin (10 g / L NaCl, 10 g / L peptone, and 5 g / L yeast extract) until the bacterial culture reached OD. 600 The concentration of the target protein was 0.6–0.8. IPTG was added to a final concentration of 0.5 mM, and the cells were cultured at 18°C ​​and 180 r / min for 18 h to induce protein expression. The cells were then collected by centrifugation and resuspended in a 10 mM imidazole aqueous solution. The cells were lysed by sonication, and the protein-containing supernatant was loaded onto a Ni-NTA purification column for purification. A 40–400 mM imidazole gradient elution was used to obtain purified protein M1 (or M2). The amino acid sequence of M1 is shown in SEQ ID NO.1, and the amino acid sequence of M2 is shown in SEQ ID NO.2.

[0077] The glycosyltransferase mutant of the present invention can be used to prepare a variety of non-natural compounds in vitro.

[0078] Example 2

[0079] The M1-catalyzed glycosylation system for triterpenoid compound ginsenoside Rh1 includes:

[0080] The 100 μL glycosylation reaction system included 0.5 mM substrate ginsenoside Rh1, 5 mM xylose diphosphate, 80 μg M1, and 100 mM phosphate buffer (pH 7.5). The reaction was carried out at 37 °C for 12 h.

[0081] After the reaction was complete, an equal volume of methanol was added to terminate the reaction. The mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected and added to a liquid chromatography vial. The glycosylated products were analyzed and identified by high performance liquid chromatography (see [link to liquid chromatography]). Figure 2 A1). The analytical conditions for high performance liquid chromatography (HPLC) were as follows: mobile phase A was distilled water, mobile phase B was chromatographic acetonitrile, the chromatographic column was a reversed-phase C18 column (4.6 mm × 150 mm, 5 μm, Agilent), the injection volume was 10 μL, the gradient elution conditions were 0–15 min, the eluent was 10%–90% B, the UV detector was used, the detection wavelength was 203 nm, the flow rate of the mobile phase was 1 mL / min, and the column oven temperature was 35 °C.

[0082] And according to 1D-NMR ( 1 H NMR and 13 Its structure was identified by 1C NMR and 2D-NMR (HMBC, HSQC, and COSY) spectroscopy (see [link to article]). Figure 3 A, Figure 3 B. Figure 3 C Figure 3 D and Figure 3 E), is ginsenoside Rh1 3-O-β-D-xyloside (product 1). Figure 1 A.

[0083] Example 3

[0084] By replacing M1 in Example 2 with M2, and otherwise remaining the same as in Example 2, product 1 was obtained.

[0085] The high-performance liquid chromatography (HPLC) of glycosylated product 1 generated from ginsenoside Rh1 by M2 catalysis is shown in the figure. Figure 2 A2.

[0086] M2-catalyzed glycosylation product 1 of ginsenoside Rh1 1 H NMR spectroscopy analysis, 13 The results of C NMR spectroscopy, HMQC spectroscopy, HSBC spectroscopy, and COSY spectroscopy were similar to the above-mentioned spectra of glycosylated product 1 generated by M1 catalyzing ginsenoside Rh1 in Example 2.

[0087] Example 4

[0088] M1 catalyzes the CK glycosylation reaction of the triterpenoid compound ginsenoside.

[0089] The substrate ginsenoside CK was used to replace ginsenoside Rh1 in Example 2, and the eluent was replaced with 30%-90% B instead of 10%-90% B. All other steps were the same as in Example 2 to obtain the glycosylated product. The glycosylated product was analyzed and identified by high-performance liquid chromatography (see [link to example]). Figure 2 B1)

[0090] And according to 1D-NMR ( 1 H NMR and 13 Its structure was identified by 1C NMR and 2D-NMR (HMBC, HSQC, and COSY) spectroscopy (see [link to article]). Figure 4 A, Figure 4 B. Figure 4 C Figure 4 D and Figure 4 E), is ginsenoside CK 3-O-β-D-xyloside (product 2). Figure 1 B.

[0091] Example 5

[0092] M2-catalyzed CK glycosylation reaction of triterpenoid compound ginsenosides

[0093] By replacing M1 in Example 4 with M2, and otherwise remaining the same as in Example 4, product 2 was obtained.

[0094] The high-performance liquid chromatography (HPLC) of glycosylated product 2 generated from ginsenoside CK by M2 catalysis is shown in the figure. Figure 2 B2.

[0095] M2 catalyzes the formation of glycosylated product 2 from ginsenoside CK. 1 H NMR spectroscopy analysis, 13 C NMR spectroscopy, HMQC spectroscopy, HSBC spectroscopy, and COSY spectroscopy showed that the results were similar to the above-mentioned spectra of glycosylated product 2 generated by M1 catalyzing ginsenoside CK in Example 4.

[0096] Example 6

[0097] M1 catalyzes the glycosylation reaction of the triterpenoid compound ginsenoside Rg3.

[0098] The substrate ginsenoside Rg3 was used instead of ginsenoside Rh1 in Example 2, and the eluent was changed from 30%-90% B to 10%-90% B. All other steps were the same as in Example 2 to obtain the glycosylated product. The glycosylated product was analyzed and identified by high-performance liquid chromatography (see [link to example]). Figure 2 C1).

[0099] And according to 1D-NMR ( 1 H NMR and 13 Its structure was identified by 1C NMR and 2D-NMR (HMBC, HSQC, and COSY) spectroscopy (see [link to article]). Figure 5 A, Figure 5 B. Figure 5 C Figure 5 D and Figure 5 E), is ginsenoside Rg312-O-β-D-xyloside (product 3). Figure 1 C.

[0100] Example 7

[0101] By replacing M1 in Example 6 with M2, and otherwise remaining the same as in Example 6, product 3 was obtained.

[0102] High performance liquid chromatography of glycosylated product 3 of ginsenoside Rg3 catalyzed by M2, see Figure 2 C2.

[0103] M2 catalyzes the formation of glycosylated product 3 from ginsenoside Rg3. 1 H NMR spectroscopy analysis, 13 C NMR spectroscopy, HMQC spectroscopy, HSBC spectroscopy, and COSY spectroscopy showed results similar to the spectrum of product 3 obtained by M1-catalyzed glycosylation of ginsenoside Rg3 in Example 6.

[0104] Example 8

[0105] M1 catalyzes the glycosylation reaction of glycyrrhizin, a flavonoid compound.

[0106] By replacing the substrate ginsenoside Rh1 in Example 2 with glycyrrhizin, and otherwise following the same procedure as in Example 2, glycosylated products were obtained. These products were then analyzed and identified by high-performance liquid chromatography (see [link to example]). Figure 2 D1)

[0107] And according to 1D-NMR ( 1 H NMR and 13 Its structure was identified by 1C NMR and 2D-NMR (HMBC, HSQC, and COSY) spectroscopy (see [link to article]). Figure 6 A, Figure 6 B. Figure 6 C Figure 6 D and Figure 6 E), is glycyrrhizin 7-O-β-D-xyloside (product 4). Figure 1 D.

[0108] Example 9

[0109] M2 catalyzes the glycosylation reaction of glycyrrhizin, a flavonoid compound.

[0110] By replacing M1 in Example 8 with M2, and otherwise remaining the same as in Example 8, product 4 was obtained.

[0111] High performance liquid chromatography of M2-catalyzed glycyrrhizin to glycosylated product 4, see Figure 2 D2;

[0112] M2 catalyzes the formation of glycyrrhizin glycosylation product 4. 1 H NMR spectroscopy analysis, 13 C NMR spectroscopy, HMQC spectroscopy, HSBC spectroscopy, and COSY spectroscopy analysis showed that the results were similar to the above-described spectra of glycosylated product 4 generated by M1 catalysis of glycyrrhizin in Example 8.

Claims

1. A Bs-YjiC mutant glycosyltransferase, characterized in that... The glycosyltransferase Bs-YjiC mutant is one of the following: M1: In the amino acid sequence of the glycosyltransferase Bs-YjiC shown in gene bank number NP_389104, glutamine at position 66 is mutated to valine, serine at position 277 is mutated to tryptophan, and serine at position 292 is mutated to threonine; the amino acid sequence of M1 is shown in SEQ ID NO.1: M2: In the amino acid sequence of the glycosyltransferase Bs-YjiC shown in gene bank number NP_389104, glutamine at position 66 is mutated to valine, serine at position 277 is mutated to tryptophan, and alanine at position 230 is mutated to valine. The amino acid sequence of M2 is shown in SEQ ID NO.

2.

2. The gene encoding the Bs-YjiC mutant glycosyltransferase of claim 1.

3. A plasmid containing the gene of claim 2.

4. A genetically engineered strain containing the plasmid described in claim 3.

5. The application of the Bs-YjiC mutant glycosyltransferase of claim 1 in the enzymatic synthesis of xylose, wherein the xylose is ginsenoside Rh1 3-O-β-D-xylose, ginsenoside CK 3-O-β-D-xylose, ginsenoside Rg3 12-O-β-D-xylose or glycyrrhizin 7-O-β-D-xylose.

Images