A method of biocatalytic generation of c-glycosides
By engineering the Indian mango glycosyltransferase MiCGT into QDP, the problem of low efficiency in catalyzing C-glycosidic bond formation was solved, and the preparation of C-glucosides with high selectivity and high conversion rate was achieved.
Patent Information
- Application Number
- CN202310761371.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-27
- Publication Date
- 2026-01-23
- Estimated Expiration
- 2043-06-27
AI Technical Summary
In nature, there are few glycosyltransferases that catalyze the formation of C-glycosidic bonds, and they have a narrow substrate recognition range and low catalytic efficiency, which limits their practical applications.
Protein engineering was performed on the glycosyltransferase MiCGT from Indian mangoes, which was mutated to QDP (E152Q/V190D/S122P) to improve its catalytic efficiency and chemoselectivity.
The mutant QDP significantly improved the conversion rate and specific enzyme activity of C-glycoside modified products, from 3% to over 99%, and increased TON by 76 times, achieving gram-level preparation of highly selective C-glucosides.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a method for biocatalytic production of C-glycoside, belonging to the field of bioengineering technology. BACKGROUND
[0002] Glycosides are a class of compounds widely existing in nature, with diverse structures and wide physiological activities. The biological activity of this class of compounds is largely determined by the sugar group in the structure, so developing a method that can efficiently catalyze the glycosylation of the corresponding aglycone is a hot spot of concern for organic chemists and medicinal chemists. The glycosylation reaction in the biosynthesis of natural products is catalyzed by glycosyltransferases (GTs; EC 2.4.), which catalyze the transfer of sugar molecules from activated glycosyl donors to substrate acceptors to directly construct glycosylated products in a simple one-step method, avoiding the lengthy protection-deprotection steps in chemical methods. Therefore, this class of enzymes has been widely recognized as a powerful tool for glycosylation.
[0003] Most glycoside natural products are O-glycosylation modification products, in addition to which glycoside compounds also include C-, N-, and S-glycosylation products. From a chemical point of view, C-glycosides have significant resistance to spontaneous, acid, and enzyme-catalyzed hydrolysis, and this property endows the corresponding glycosylation products with a longer in vivo half-life. However, there are relatively few GTs in nature that have the activity of catalyzing the formation of C-glycoside bonds, and the range of substrates they recognize is relatively narrow, and the catalytic efficiency is mostly relatively low, which limits their practical application range. SUMMARY
[0004] The present application aims to improve the productivity and chemical selectivity of the glycosyltransferase MiCGT from Mangifera indica by protein engineering. The present application provides a method for biocatalytic synthesis of different C-glycosides using the glycosyltransferase mutant QDP (E152Q / V190D / S122P). This method has the advantages of mild conditions, environmental friendliness, and catalytic synthesis of glycosylation products with different chemical selectivities.
[0005] The first object of the present application is to provide a glycosyltransferase MiCGT mutant QDP, which mutates glutamic acid at position 152 to glutamine, valine at position 190 to aspartic acid, and serine at position 122 to proline in the amino acid sequence shown in SEQ ID NO. 1.
[0006] In an embodiment of the present application, the nucleotide sequence encoding the glycosyltransferase is shown in SEQ ID NO. 2.
[0007] A second object of the present application is to provide a gene encoding the above-mentioned glycosyltransferase mutant QDP of MiCGT.
[0008] A third object of the present application is to provide an expression vector containing the above-mentioned gene.
[0009] In one embodiment of the present application, the expression vector includes, but is not limited to, pET series vectors, pRSF series vectors or pCDF series vectors.
[0010] A fourth object of the present application is to provide a genetically engineered bacterium expressing the above-mentioned mutant.
[0011] In one embodiment of the present application, the host cell of the genetically engineered bacterium includes, but is not limited to, Escherichia coli.
[0012] In one embodiment of the present application, the construction of the genetically engineered bacterium specifically includes the following steps: using pET28a as a vector, connecting the gene encoding the mutant with the vector, and recombining and expressing the glycosyltransferase mutant in Escherichia coli BL21 (DE3).
[0013] In one embodiment of the present application, the components of the fermentation medium are TB medium, the inducer is 4-6 g / L of α-lactose monohydrate, the preferred temperature is 18-25°C, the rotation speed is 160-200 rpm, and the expression time is 18-20 h.
[0014] In one embodiment of the present application, about 0.05% glucose is added during inoculation.
[0015] In one embodiment of the present application, the protein purification of the genetically engineered bacterium specifically includes the following steps: collecting the expression bacterium with a pre-cooled centrifuge, washing twice with a buffer solution, adding a small amount of lysozyme, quick-freezing with liquid nitrogen, thawing in an ice water bath, ultrasonic crushing, high-speed centrifugation, His-nickel column affinity purification and desalting column desalting.
[0016] A fifth object of the present application is to provide a method for synthesizing various C-glycosides by using the glycosyltransferase mutant QDP of the present application.
[0017] In one embodiment of the present application, the method is carried out in a 40-60 mM NaH2PO4-Na2HPO4 buffer system, the enzyme amount is 40-100 μg / 100 μL or the corresponding whole cell (OD 600nm = 40-60), the pH is 7.0-8.0, the temperature is 30-40°C, the acceptor substrate concentration is 0.1-5 mM, the UDP-glucose concentration is 0.1-10 mM or 2% glucose, the rotation speed is 800-1200 rpm, and the reaction time is 2-6 h.
[0018] A sixth object of the present application is to provide applications of the mutant, the gene, the vector, the genetically engineered bacterium, the method for preparing the mutant, or the method for synthesizing C-glycoside in the fields of food, medicine, etc.
[0019] Advantages:
[0020] The present application is based on the production of morin using sugar transferase MiCGT derived from Mangifera indica for protein engineering, and the mutant QDP (E152Q / V190D / S122P) obtained as a biological catalyst to catalyze BP-2, 2,4-dihydroxypropiophenone, phloretin or 2-phenyl-2',4',6'-trihydroxypropiophenone to generate corresponding C-glucoside modified products.
[0021] (1) Compared with wild enzyme MiCGT, the mutant QDP (E152Q / V190D / S122P) can obtain high-chemical-selectivity C-glucoside modified products using BP-2 as a substrate, the conversion rate is increased from nearly 3% to more than 99%, the TON is increased from 136 to 10375, the specific enzyme activity is increased from 135.8 U / mg to 939.4 U / mg, and the preparation of high-chemical-selectivity C-glucoside modified products in a kilogram level (1.2 g) is achieved in a 1.5 L reaction system.
[0022] (2) Using 2,4-dihydroxypropiophenone as a substrate, high-chemical-selectivity C-glucoside modified products can be obtained, the conversion rate is increased from <3% to 54%; using phloretin as a substrate, high-chemical-selectivity C-glucoside modified products can be obtained, the conversion rate is increased from 17% to more than 99%; using 2-phenyl-2',4',6'-trihydroxypropiophenone as a substrate, high-chemical-selectivity C-glucoside modified products can be obtained, the conversion rate is increased from 11% to more than 99%. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1 HPLC chromatogram (A) and column chart (B) of the reaction mixture of BP-2 and mutant QDP and wild type of sugar transferase MiCGT.
[0024] Figure 2 Mass spectrum and secondary mass spectrum of the products 1a and 1b produced by the reaction of BP-2 and mutant QDP; A) MS of 1a; B) MS of 1a 2 ; C) MS of 1b; D) MS of 1b 2 .
[0025] Figure 3HPLC chromatogram (A) and column chromatogram (B) of the reaction mixture of 2,4-dihydroxyphenylacetone with the mutant QDP and wild-type of glycosyltransferase MiCGT.
[0026] Figure 4 The primary and secondary mass spectra of products 2a and 2b from the reaction of 2,4-dihydroxyphenylacetone with mutant QDP; A) MS of 2a; B) MS of 2a. 2 C) 2b MS; D) 2b MS 2 .
[0027] Figure 5 HPLC chromatogram (A) and column chart (B) of the reaction mixture of phloretin with the mutant QDP and wild-type glycosyltransferase MiCGT.
[0028] Figure 6 : Primary and secondary mass spectra of products 3a and 3b generated by the reaction of phloretin with mutant QDP; A) MS of 3a; B) MS of 3a 2 C) 3b MS; D) 3b MS 2 .
[0029] Figure 7 HPLC chromatogram (A) and column chromatogram (B) of the reaction mixture of 2-phenyl-2',4',6'-trihydroxyacetophenone with the mutant QDP and wild-type of glycosyltransferase MiCGT.
[0030] Figure 8 Primary and secondary mass spectra of products 4a and 4b generated from the reaction of 2-phenyl-2',4',6'-trihydroxyacetophenone with mutant QDP; A) MS of 4a; B) MS of 4a. 2 C) 4-bit MS; D) 4-bit MS 2 . Detailed Implementation
[0031] All the chemicals used in this invention were purchased commercially.
[0032] The culture medium formulation used in the examples is as follows:
[0033] Solid culture medium formula (1L): 5g yeast extract, 10g peptone, 10g sodium chloride, 15g agar, bring to volume with deionized water, and autoclave.
[0034] LB medium formula (1L): 5g yeast extract powder, 10g peptone, 10g sodium chloride, bring to volume with deionized water, and autoclave.
[0035] TB culture medium formula (1L): 24g yeast extract, 12g peptone, 4mL glycerol. Add 900mL deionized water to dissolve, then autoclave. Add 100mL of a 0.17MKH₂PO₄ / 0.72MK₂HPO₄ solution that has undergone the same sterilization process. (Note: Sodium chloride was purchased from Aladdin; all other components were purchased from Sangon Biotech Co., Ltd.)
[0036] Product analysis methods: High performance liquid chromatography (HPLC) and mass spectrometry (MS / MS). The corresponding HPLC detection method used a 5-μm C18 column with a PDA detector. The mobile phases were: Phase A (H₂O containing 0.1% formic acid) and Phase B (methanol containing 0.1% formic acid). The gradient elution program was: 10-50% B for 8 min, 50%-85% B for 2 min, 85% B for 3 min, and 80%-10% B for 7 min.
[0037] The Turnover Number (TON) is defined as the number of catalytic reactions that occur per unit time per unit active site, or the number of target products generated, or the number of reactants consumed, under certain temperature, pressure, reactant ratio, and reaction extent.
[0038] Turnover number (TON) calculation: In 50 mM NaH₂PO₄-Na₂HPO₄ (pH 8.0), 3 / 4 / 5 mM of substrate and different concentrations of purified MiCGT mutant QDP (0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, and 3 μM) were added, and the reaction was carried out at 40 °C for 24 h, with a total volume of 100 μL. The reaction was terminated by adding 300 μL of ice-cold methanol, and the results were detected by liquid chromatography. TON was calculated as the amount of transformed substrate divided by the amount of enzyme.
[0039] Glycosyltransferase activity assay: Glycosyltransferase activity was determined in 200 μL reaction buffer, which included 6 mM UDP-glucose, 50 mM NaH₂PO₄-Na₂HPO₄ (pH 8.0), and approximately 1 μg of the mutant purified enzyme. The reaction was incubated at 50 °C for 5 min, and then terminated by adding the same volume of methanol. Analysis was performed by HPLC. One unit of enzyme activity was defined as the amount of enzyme consuming 1 μmol of acceptor substrate or producing 1 μmol of product per minute.
[0040] Protein content detection: Protein absorbance at 280 nm was measured using a Nano-300 micro spectrophotometer.
[0041] Enzyme activity calculation: Specific activity = 1 U / (mg protein) -1 = 1 μmol·(min·mg protein) -1 .
[0042] Conversion rate calculation: Conversion rate = Amount of raw materials converted / Total amount of raw materials * 100%.
[0043] Example 1: Protein Engineering of Glycosyltransferase MiCGT
[0044] (1) Design and preparation of mutants:
[0045] Using the gene sequence shown in SEQ ID NO.2 as a template, primers were designed and mutations were performed at sites S122, E152 and V190 to obtain the coding gene of the QDP(E152Q / V190D / S122P) mutant.
[0046] Table 1 QDP Mutant Primers
[0047]
[0048] (2) Construction of recombinant plasmids:
[0049] Using commercialized Vazyme The MultiSOneStepCloning Kit ligates the coding gene of the mutant prepared in step (1) to the pET28a vector to obtain the recombinant vector pET28a-MiCGT. mutant For specific steps, please refer to the instruction manual. Using the same method, ligate the wild-type gene shown in SEQ ID NO.2 with the pET28a vector to obtain the recombinant vector pET28a-MiCGT.
[0050] Example 2: Expression and purification of the glycosyltransferase MiCGT mutant QDP
[0051] The recombinant vector pET28a-MiCGT constructed in Example 1 mutant pET28a-MiCGT and pET28a-MiCGT were chemically transformed into competent Rosetta-gamiB(DE3) cells, respectively. A suitable amount of bacterial culture was plated onto solid medium containing kanamycin and chloramphenicol and incubated at 37°C for 12-15 hours. A single colony containing the transformed recombinant plasmid was then picked and added to LB medium (containing 35 μg / mL of the recombinant plasmid). -1 Chloramphenicol and 50 μg / mL -1 Kanamycin was added and cultured overnight at 37°C. 1% LB culture was then inoculated into TB medium (containing 35 μg / mL). -1 Chloramphenicol, 50 μg / mL -1 Kanamycin and 0.05% glucose (sterilized by membrane) were added, and the mixture was then cultured at 37°C to the logarithmic growth phase, i.e., the OD value was 0.6-0.8. 5 g / L α-lactose monohydrate was added as an inducer, and the mixture was transferred to 25°C and 160 rpm for expression for 18-20 h.
[0052] Expression cells were collected separately using a centrifuge pre-cooled to 4°C. They were washed twice with 50 mM (pH 8.0) NaH₂PO₄-Na₂HPO₄ buffer containing 10% glycerol, followed by the addition of a small amount of lysozyme. The cells were then flash-frozen in liquid nitrogen, thawed in an ice-water bath, sonicated, centrifuged at high speed, and purified using His-nickel column affinity purification (washing impurities with 50 mM tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 10% glycerol and 80 mM imidazole, eluting the target protein with 50 mM tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 10% glycerol and 500 mM imidazole, and desalting using a desalting column (desalting buffer was 50 mM NaH₂PO₄ containing 10% glycerol). (NaH2PO4-Na2HPO4 buffer solution). The specific activity of the mutant QDP against BP-2 was determined to be 939.4 U / mg, while the specific activity of the wild-type enzyme against BP-2 was 135.8 U / mg, indicating that the specific activity of QDP against BP-2 was 7-fold higher than that of the wild-type enzyme. The TON of the mutant QDP against BP-2 was determined to be 10375, while that of the wild-type enzyme against BP-2 was 136, indicating that the TON of QDP against BP-2 was 76-fold higher than that of the wild-type enzyme.
[0053] Example 3: Glycosyltransferase MiCGT and its mutant QDP catalyze the synthesis of C-glucosides from BP-2.
[0054] In a 100 μL reaction system, the concentration of UDP-glucose was 4 mM, the concentration of substrate 2,2',4,4'-tetrahydroxybenzophenone (BP-2) was 3 mM, 50 mM NaH2PO4-Na2HPO4 (pH 8.0), DMSO (v / v, 5%), and the final concentration of the biocatalyst, the pure enzyme mutant QDP, was 54 μg. Using the wild-type enzyme as a control, the reaction was carried out at 30 °C for 2 h to obtain the corresponding glycosylated products. The reaction was terminated by adding 3 volumes of ice-cold methanol, and the results were analyzed by high-performance liquid chromatography and mass spectrometry.
[0055] Depend on Figure 1 and Figure 2 It can be seen that the mutant QDP catalyzes BP-2 to obtain C-glucosinolate products with a conversion rate >99%. In contrast, the wild-type enzyme yields a more mixed glycoside product with a conversion rate of 3%. The mutant enzyme exhibits higher chemoselectivity and a significantly improved conversion rate compared to the wild-type enzyme.
[0056] In a 1.5 L reaction system, the substrate 2,2',4,4'-tetrahydroxybenzophenone (BP-2) 1.1 g, 50 mM NaH2PO4-Na2HPO4 (pH 8.0), DMSO (v / v, 5%), and whole cells containing the mutant QDP as the biocatalyst were used. The final catalyst concentration was OD. 600=40, reacted at 30℃ for 24 h. Subsequently, the sample was centrifuged at 4,000 g for 5 min, and the supernatant was collected. Cells were washed with 20 mL ddH2O and centrifuged twice. The supernatant was filtered, preliminarily purified and concentrated by macroporous resin column chromatography, and further purified by silica gel column chromatography to obtain 1.2 g of C-glycoside product.
[0057] Example 4: Glycosyltransferase MiCGT and its mutant QDP catalyze the synthesis of C-glucosinolate from 2,4-dihydroxyphenylacetone.
[0058] In a 100 μL reaction system, the concentration of UDP-glucose was 4 mM, the concentration of the substrate 2,4-dihydroxyphenylacetone was 3 mM, 50 mM NaH2PO4-Na2HPO4 (pH 8.0), DMSO (v / v, 5%), and the final concentration of the biocatalyst, the pure enzyme mutant QDP, was 54 μg. Using the wild-type enzyme as a control, the reaction was carried out at 30 °C for 2 h to obtain the corresponding glycosylated product. The reaction was terminated by adding 3 volumes of ice-cold methanol, and the results were analyzed by high-performance liquid chromatography and mass spectrometry.
[0059] Depend on Figure 3 and Figure 4 It can be seen that the mutant QDP catalyzes 2,4-dihydroxyphenylacetone to yield C-glucoside product with a conversion rate of 54%. In contrast, the wild-type enzyme yields a more mixed glycoside product with a conversion rate of <3%. The mutant enzyme exhibits higher chemoselectivity and significantly improved conversion rate compared to the wild-type enzyme.
[0060] Example 5: Glycosyltransferase MiCGT and its mutant QDP catalyze the synthesis of C-glucosides from 4-fluoro-2',4'-dihydroxybenzophenone.
[0061] In a 100 μL reaction system, the concentration of UDP-glucose was 4 mM, the concentration of the substrate phlorizin (4-fluoro-2',4'-dihydroxybenzophenone) was 3 mM, 50 mM NaH2PO4-Na2HPO4 (pH 8.0), DMSO (v / v, 5%), and the final concentration of the biocatalyst, the pure enzyme mutant QDP, was 54 μg. Using the wild-type enzyme as a control, the reaction was carried out at 30 °C for 2 h to obtain the corresponding glycosylated products. The reaction was terminated by adding 3 volumes of ice-cold methanol, and the results were analyzed by high-performance liquid chromatography and mass spectrometry.
[0062] Depend on Figure 5 and Figure 6 It can be seen that the mutant QDP catalyzes 4-fluoro-2',4'-dihydroxybenzophenone to obtain the C-glucoside product with a conversion rate >99%. While the wild-type enzyme can also obtain the C-glucoside product, the conversion rate is only 17%. The mutant yields a product with different chemoselectivity compared to the wild-type enzyme, and the conversion rate is significantly improved.
[0063] Example 6: Glycosyltransferase MiCGT and its mutant QDP catalyze the synthesis of C-glucosyl from 4'-hydroxyphenylheptanone
[0064] In a 100 μL reaction system, the concentration of UDP-glucose was 4 mM, the concentration of the substrate 4'-hydroxyphenylheptanone was 3 mM, 50 mM NaH2PO4-Na2HPO4 (pH 8.0), DMSO (v / v, 5%), and the final concentration of the purified enzyme mutant QDP biocatalyst was 54 μg. The reaction was carried out at 30 °C for 2 h to obtain the corresponding glycosylated product. The reaction was terminated by adding 3 volumes of ice-cold methanol, and the results were analyzed by high performance liquid chromatography and mass spectrometry.
[0065] Depend on Figure 7 and Figure 8 It can be seen that the mutant QDP catalyzes 4'-hydroxyphenylheptanone to yield C-glucoside product with a conversion rate >99%. While the wild-type enzyme can also yield C-glucoside product, the conversion rate is only 11%. The mutant yields a product with different chemoselectivity than the wild-type enzyme, and the conversion rate is significantly improved.
[0066] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A mutant of the glycosyltransferase MiCGT, characterized in that, The amino acid sequence shown in SEQ ID NO. 1 was modified by mutating glutamic acid at position 152 to glutamine, valine at position 190 to aspartic acid, and serine at position 122 to proline.
2. The gene encoding the mutant of claim 1.
3. An expression vector containing the gene of claim 2, characterized in that, The expression vectors include pET series vectors, pRSF series vectors, or pCDF series vectors.
4. A genetically engineered bacterium expressing the mutant of claim 1, characterized in that, The host cells of the genetically engineered bacteria include Escherichia coli.
5. The method for preparing the mutant of claim 1, characterized in that, Ferment the genetically engineered bacteria as described in claim 4.
6. The method as described in claim 5, characterized in that, An inducer is added during fermentation, wherein the inducer is 4- α - Lactose or IPTG.
7. A method for synthesizing C-glycosides, characterized in that, Using UDP-glucose or glucose as the glycosyl donor, the mutant of claim 1 or the genetically engineered bacteria of claim 4 as the biocatalyst, and BP-2, 2,4-dihydroxyphenylacetone, phloretin or 2-phenyl-2',4',6'-trihydroxyacetophenone as the acceptor, the reaction is carried out.
8. The application of the mutant of claim 1, or the gene of claim 2, or the expression vector of claim 3, or the genetically engineered bacteria of claim 4, or the method of claim 5 or 6, or the method of claim 7, in the food or pharmaceutical fields.