A method of biocatalytic production of resorcinolic o-glycosides
By screening and expressing the glycosyltransferase CaGT83 from industrial hemp, the problems of low catalytic activity and poor selectivity in existing technologies have been solved, achieving highly efficient catalysis of resorcinol compounds and glucose to generate O-glycosides with high selectivity and high yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-02-09
- Publication Date
- 2026-06-09
AI Technical Summary
Existing glycosyltransferases exhibit low catalytic activity and poor selectivity for resorcinol substrates, limiting their application in the synthesis of resorcinol O-glycosides.
A glycosyltransferase CaGT83 derived from industrial hemp was screened and prepared. The enzyme was then expressed in Escherichia coli via genetic engineering. A recombinant vector and genetically engineered bacteria were constructed to catalyze the reaction of resorcinol compounds with glucose, generating O-glycosides with high selectivity and high yield.
This study achieved highly efficient catalysis of resorcinol compounds with glucose, with a yield of over 97%, significantly improving the synthesis efficiency of resorcinol O-glycosides.
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Figure CN122168561A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for biocatalytically generating resorcinol-based O-glycosides, belonging to the field of bioengineering technology. Background Technology
[0002] Resorcinol is a key structural unit in many drug molecules, with typical examples including the HSP90 inhibitors Ganetespib and Onalespib, the cosmetic skin-whitening ingredient 4-butylresorcinol, and the natural product cannabidiol with various pharmacological activities. These compounds have wide applications in food, pharmaceuticals, and cosmetics. However, despite their significant value, their practical application is limited by factors such as poor stability, low water solubility, and short lifespan under photosensitivity. These shortcomings hinder the metabolic stability and formulation development of these compounds, significantly reducing their overall bioavailability and clinical translation potential. To overcome these limitations, developing structural modification strategies that improve solubility and stability is crucial. Commonly used structural modification techniques include isopentenylation, acetylation, methylation, glycosylation, and hydroxylation. Among these, glycosylation—that is, covalently linking glycosyl groups to the molecule—has proven to be an effective method. This modification can significantly regulate the physicochemical properties of the aglycone moiety, often resulting in higher metabolic stability and better bioavailability.
[0003] To obtain these glycosylated products with wide applications, scientists have developed various methods, among which plant extraction, chemical synthesis, and biocatalytic synthesis are the three main approaches. Plant extraction is cost-effective for natural glycosides abundant in plants, such as hesperidin and steviol glycosides. However, this traditional process is time-consuming and labor-intensive, limited by the time and space constraints of plant cultivation, requires large amounts of natural plant resources, and is unsuitable for obtaining natural glycosides with low abundance or those not found in nature. Traditionally, chemical synthesis has also been a major method of glycosylation; however, chemical methods are often limited by the complex skeletal structure and diverse functional group modifications of natural products, often requiring cumbersome protection-deprotection steps. In recent years, with the continuous development of synthetic biology, enzymatic glycosylation has gradually gained widespread attention from scientists due to its simplicity, high stereoselectivity, and regioselectivity. Glycosyltransferases (GTs; EC 2.4.) are key enzymes that catalyze the transfer of glycosyl groups from activated sugar donors to acceptor molecules, and can be used for the sustainable and environmentally friendly synthesis of glycosides.
[0004] However, glycosyltransferases still have certain limitations in practical applications, such as susceptibility to inhibition by high substrate concentrations, low catalytic efficiency, the need for pure enzymes, and the relatively high cost of glycosyl donors. It is noteworthy that with the continuous reduction in gene sequencing technology and gene synthesis costs, the scale of enzyme resources available for exploration and utilization is rapidly expanding, providing a rich potential resource base for the discovery and development of novel high-performance glycosyltransferases. However, for resorcinol substrates with significant application value, the catalytic efficiency of currently reported glycosyltransferases is still relatively poor, making it difficult to meet the needs of practical production and large-scale applications. Therefore, it is urgent to screen a glycosyltransferase with high catalytic activity, high regioselectivity, and suitability for industrial applications from existing massive databases to achieve the green and efficient synthesis of resorcinol O-glycosides, thereby providing an excellent biocatalytic material and technical approach for the sustainable production of active glycoside compounds. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a method for the biocatalytic synthesis of resorcinol O-glycosides. The aim is to overcome the limitations of currently reported glycosyltransferases, such as low catalytic activity and poor selectivity for resorcinol substrates, which restrict the synthetic applications of glycosyltransferases. This method offers advantages such as mild conditions, environmental friendliness, and high-efficiency catalytic synthesis of O-glycosides.
[0006] This invention provides a glycosyltransferase CaGT83 containing the amino acid sequence shown in SEQ ID NO.1.
[0007] The present invention also provides a gene encoding the glycosyltransferase CaGT83.
[0008] In one embodiment, the nucleotide sequence of the gene is as shown in SEQ ID NO.2.
[0009] The present invention also provides a recombinant vector carrying the said gene.
[0010] In one embodiment, the recombinant vector uses plasmid pET28a as the expression vector.
[0011] The present invention also provides host cells that express the glycosyltransferase CaGT83, or contain the gene, or are transformed with the recombinant vector.
[0012] In one embodiment, the host cell is Escherichia coli, including but not limited to Escherichia coli BL21(DE3).
[0013] The present invention also provides a genetically engineered bacterium, using Escherichia coli as a host and pET series plasmids as vectors to express the glycosyltransferase CaGT83.
[0014] In one embodiment, the genetically engineered bacteria are constructed by ligating pET28a as an expression vector into *Escherichia coli*. E. coli The glycosyltransferase CaGT83 is expressed in BL21(DE3).
[0015] The present invention also provides a biocatalyst containing the glycosyltransferase CaGT83 or a live cell of the genetically engineered bacteria.
[0016] The present invention also provides a method for preparing the biocatalyst, comprising: culturing the genetically engineered bacteria, and collecting glycosyltransferase CaGT83 or live cells of the genetically engineered bacteria from the fermentation broth.
[0017] In one embodiment, the fermentation medium for culturing the genetically engineered bacteria includes, but is not limited to, TB medium.
[0018] In one embodiment, lactose is used to induce the expression of the glycosyltransferase CaGT83 during the culture process.
[0019] In one embodiment, the culture temperature is 18–25 °C, the rotation speed is 200–220 rpm, and the culture time is 18–20 h.
[0020] The present invention also provides a method for preparing resorcinol O-glycosides, using the genetically engineered bacteria as a catalyst and resorcinol compounds as acceptors to generate resorcinol O-glycosides.
[0021] In one embodiment, the resorcinol compounds include, but are not limited to, 3,5-dihydroxypropylbenzene, 3,5-dihydroxytoluene, resorcinol, 3,5-dihydroxybenzonitrile, methyl 3,5-dihydroxybenzoate, and 6-hydroxyindole.
[0022] In one embodiment, the reaction system contains glucose; the concentration of said glucose is 15-25 g / L.
[0023] In one embodiment, the reaction system uses 90-110 mM KH2PO4. K2HPO4 is used as a buffer solution, and the pH of the reaction system is 7.0 to 8.0.
[0024] In one embodiment, the concentration (OD) of the genetically engineered bacteria in the reaction system 600nm ≥40 (whole cells).
[0025] In one embodiment, the reaction is carried out at 30-40°C for 20-24 hours.
[0026] In one embodiment, the substrate concentration in the reaction system is ≥3 mM.
[0027] The present invention also provides the application of the glycosyltransferase CaGT83, or the gene, or the recombinant vector, or the host cell, or the genetically engineered bacteria, or the biocatalyst, or the method in the preparation of resorcinol O-glycosides or products containing resorcinol O-glycosides.
[0028] The technical effects of this invention are as follows: (1) This invention utilizes industrial hemp ( Cannabis sativa A previously unstudied glycosyltransferase, CaGT83, was screened from the genome sequence of a cell, and a cell catalyst expressing the glycosyltransferase CaGT83 was prepared.
[0029] (2) The glycosyltransferase CaGT83 screened in this invention can catalyze the synthesis of O-glycoside products from substrates such as 3,5-dihydroxypropylbenzene, 3,5-dihydroxytoluene, resorcinol, 3,5-dihydroxybenzonitrile, methyl 3,5-dihydroxybenzoate, and 6-hydroxyindole. It has high selectivity and a yield of over 97%, and has important application value. Attached Figure Description
[0030] Figure 1 The HPLC chromatogram is shown for the reaction mixture of 3,5-dihydroxypropylbenzene and CaGT83.
[0031] Figure 2 The first-order and second-order mass spectra of the product generated by the reaction of 3,5-dihydroxypropylbenzene with CaGT83 are shown.
[0032] Figure 3 The HPLC chromatogram is of the reaction mixture of 3,5-dihydroxytoluene and CaGT83.
[0033] Figure 4 The first-order and second-order mass spectra of the product generated by the reaction of 3,5-dihydroxytoluene with CaGT83 are shown.
[0034] Figure 5 The image shows the HPLC chromatogram of the reaction mixture of resorcinol and CaGT83.
[0035] Figure 6 These are the primary and secondary mass spectra of the product generated from the reaction of resorcinol with CaGT83.
[0036] Figure 7 The HPLC chromatogram is shown for the reaction mixture of 3,5-dihydroxybenzonitrile and CaGT83.
[0037] Figure 8 The first-order and second-order mass spectra of the product generated by the reaction of 3,5-dihydroxybenzonitrile with CaGT83 are shown.
[0038] Figure 9 The HPLC chromatogram is of the reaction mixture of methyl 3,5-dihydroxybenzoate and CaGT83.
[0039] Figure 10 The first-order and second-order mass spectra of the product generated by the reaction of methyl 3,5-dihydroxybenzoate with CaGT83 are shown.
[0040] Figure 11 The HPLC chromatogram is shown for the reaction mixture of 6-hydroxyindole and CaGT83.
[0041] Figure 12 The first-order and second-order mass spectra of the product generated by the reaction of 6-hydroxyindole with CaGT83 are shown. Detailed Implementation
[0042] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0043] Detection method: 1. Glycosyltransferase activity assay: Glycosyltransferase activity was assayed in 500 μL reaction buffer, which included 3 mM aglycone, 2% glucose (w / v), and 100 mM KH2PO4. K2HPO4 (pH 8.0) and mutant whole cells (OD) 600 nm = 40). Incubate at 30 °C for 24 h, then terminate the reaction by adding 1 volume of methanol, and analyze by HPLC.
[0044] 2. Product Analysis Methods: High-performance liquid chromatography (HPLC) and mass spectrometry (MS / MS) were used. The corresponding HPLC detection method employed a 5µm C18 column with a PDA detector. The mobile phase consisted of phase A (H₂O containing 0.1% formic acid) and phase B (acetonitrile containing 0.1% formic acid). The gradient elution program was: 33% B for 3 min, 33%-60% B for 3 min, 60% B for 3 min, and 60%-33% B for 1 min. The flow rate was 1 mL / min. Yield calculation: Yield = Peak area of product / Sum of peak areas of product and remaining substrate × 100%.
[0045] Culture media involved in the examples: Solid culture medium (1 L): 5 g yeast extract, 10 g peptone, 10 g sodium chloride, 15 g agar, diluted with deionized water and autoclaved.
[0046] LB medium (1 L): 5 g yeast extract, 10 g peptone, 10 g sodium chloride, bring to volume with deionized water, and autoclave.
[0047] TB medium (1 L): 24 g yeast extract, 12 g peptone, 4 mL glycerol. Add 900 mL deionized water to dissolve, then autoclave. Add 100 mL of a 0.17 M KH₂PO₄ / 0.72 M K₂HPO₄ 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.) Example 1: Synthesis of glycosyltransferase CaGT83 The inventors used an enzyme from a laboratory enzyme library that catalyzes the production of O-glucosinolates from resorcinol substrates as a template for gene sequence analysis, and selected 100 sequences from industrial hemp (…) from the database. Cannabis sativa The glycosyltransferase was identified, and CaGT83 with the amino acid sequence shown in SEQ ID NO.1 was screened by fixing key residues. The gene with the nucleotide sequence shown in SEQ ID NO.2 was synthesized by Sangon Biotech Co., Ltd., and ligated into plasmid pET28a to construct the recombinant plasmid pET28a-CaGT83.
[0048] Example 2: Expression of glycosyltransferase CaGT83 The recombinant plasmid CaGT83 from Example 1 was chemically transformed into competent BL21 DE3 cells. An appropriate amount of bacterial culture was plated onto a solid medium containing kanamycin and incubated at 37°C for 12-15 h. A single colony containing the transformed recombinant plasmid was then transferred to LB medium (containing 50 μg / mL kanamycin). -1 The strain was cultured overnight at 37 °C with kanamycin and the correct strain was named the genetically engineered strain CaGT83.
[0049] Cell cultures prepared in 1% LB medium were inoculated into TB medium (containing 50 μg / mL). -1 The protein was incubated with kanamycin and sterilized with 0.05% glucose (after membrane sterilization), then cultured at 37 °C to the logarithmic growth phase (OD value 0.6–0.8). α-lactose monohydrate at a final concentration of 5 g / L was added as an inducer, and the culture was transferred to 18 °C and 220 rpm for 18–20 h. Protein expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
[0050] The expression cells were collected using a centrifuge pre-cooled to 18 °C and then centrifuged with 100 mM KH2PO4. Wash twice with K2HPO4 (pH 8.0) buffer solution, add a small amount of buffer, freeze quickly in liquid nitrogen, and thaw in an ice-water bath before use in the reaction.
[0051] Example 3: Synthesis of O-glucoside from 3,5-dihydroxypropylbenzene catalyzed by glycosyltransferase CaGT83 Genetically engineered bacterial cells were prepared using the method described in Example 2, with 100 mM KH2PO4. Wash twice with K2HPO4 (pH 8.0) buffer solution as a biocatalyst.
[0052] In a 500 μL reaction system containing, by final concentration: 3 mM substrate 3,5-dihydroxypropylbenzene, 20 g / L glucose, and 2% DMSO (v / v), a biocatalyst was added until its OD value in the reaction system was reached. 600 nm The concentration was 40. The reaction was carried out at 30 °C for 24 h to obtain the corresponding O-glucosinolate product. The reaction was terminated by adding 1 volume of ice-cold methanol, and the results were analyzed by high performance liquid chromatography and mass spectrometry.
[0053] Depend on Figure 1 and Figure 2 It can be seen that CaGT83 catalysis can yield O-glucoside product from 3,5-dihydroxypropylbenzene with a yield as high as 97%. Example 4: Synthesis of O-glucoside from 3,5-dihydroxytoluene catalyzed by glycosyltransferase CaGT83 Genetically engineered bacterial cells were prepared using the method described in Example 2, with 100 mM KH2PO4. Wash twice with K2HPO4 (pH 8.0) buffer solution as a biocatalyst.
[0054] In a 500 μL reaction system containing, by final concentration: 3 mM substrate 3,5-dihydroxytoluene, 20 g / L glucose, and 2% DMSO (v / v), a biocatalyst was added until its OD value in the reaction system was reached. 600 nm = 40, reacted at 30 °C for 24 h to obtain the corresponding O-glucosinolate product. The reaction was terminated by adding 1 volume of ice-cold methanol, and analyzed by high performance liquid chromatography and mass spectrometry.
[0055] Depend on Figure 3 and Figure 4 It can be seen that CaGT83 catalysis can yield O-glucoside product from 3,5-dihydroxytoluene with a yield as high as 99%.
[0056] Example 5: Glycosyltransferase CaGT83 catalyzes the synthesis of O-glucoside from resorcinol Genetically engineered bacterial cells were prepared using the method described in Example 2, with 100 mM KH2PO4. Wash twice with K2HPO4 (pH 8.0) buffer solution as a biocatalyst.
[0057] In a 500 μL reaction system, 3 mM resorcinol, 20 g / L glucose, and 2% DMSO (v / v) were added, and the biocatalyst was added until its OD value in the reaction system was reached. 600 nm = 40, reacted at 30 °C for 24 h to obtain the corresponding O-glucosinolate product. The reaction was terminated by adding 1 volume of ice-cold methanol, and analyzed by high performance liquid chromatography and mass spectrometry.
[0058] Depend on Figure 5 and Figure 6 It can be seen that CaGT83 catalyzes resorcinol to yield O-glucoside products with a yield as high as 97%.
[0059] Example 6: Glycosyltransferase CaGT83 catalyzes the synthesis of O-glucoside from 3,5-dihydroxybenzonitrile. Genetically engineered bacterial cells were prepared using the method described in Example 2, with 100 mM KH2PO4. Wash twice with K2HPO4 (pH 8.0) buffer solution as a biocatalyst.
[0060] In a 500 μL reaction system, 3 mM substrate 3,5-dihydroxybenzonitrile, 20 g / L glucose, and 2% DMSO (v / v) were added, and the biocatalyst was added until its OD value in the reaction system was reduced. 600 nm = 40, reacted at 30 °C for 24 h to obtain the corresponding O-glucosinolate product. The reaction was terminated by adding 1 volume of ice-cold methanol, and analyzed by high performance liquid chromatography and mass spectrometry.
[0061] Depend on Figure 7 and Figure 8 It can be seen that CaGT83 catalysis can yield O-glucoside product from 3,5-dihydroxybenzonitrile with a yield as high as 99%.
[0062] Example 7: Glycosyltransferase CaGT83 catalyzes the synthesis of O-glucoside from methyl 3,5-dihydroxybenzoate. Genetically engineered bacterial cells were prepared using the method described in Example 2, with 100 mM KH2PO4. Wash twice with K2HPO4 (pH 8.0) buffer solution as a biocatalyst.
[0063] In a 500 μL reaction system, 3 mM methyl 3,5-dihydroxybenzoate, 20 g / L glucose, and 2% DMSO (v / v) were added, and the biocatalyst was added until its OD value in the reaction system was reached. 600 nm= 40, reacted at 30 °C for 24 h to obtain the corresponding O-glucosinolate product. The reaction was terminated by adding 1 volume of ice-cold methanol, and analyzed by high performance liquid chromatography and mass spectrometry.
[0064] Depend on Figure 9 and Figure 10 It can be seen that CaGT83 catalyzes the production of O-glucoside from methyl 3,5-dihydroxybenzoate with a yield as high as 98%.
[0065] Example 8: Glycosyltransferase CaGT83 catalyzes the synthesis of O-glucoside from 6-hydroxyindole. Genetically engineered bacterial cells were prepared using the method described in Example 2, with 100 mM KH2PO4. Wash twice with K2HPO4 (pH 8.0) buffer solution as a biocatalyst.
[0066] In a 500 μL reaction system, the amounts of 3 mM substrate 6-hydroxyindole, 20 g / L glucose, 2% DMSO (v / v), and biocatalyst (the genetically engineered bacteria obtained in Example 2) were approximately OD. 600 nm = 40, reacted at 30 °C for 24 h to obtain the corresponding O-glucosinolate product. The reaction was terminated by adding 1 volume of ice-cold methanol, and analyzed by high performance liquid chromatography and mass spectrometry.
[0067] Depend on Figure 11 and Figure 12 It can be seen that CaGT83 catalyzes 6-hydroxyindole to yield O-glucoside products with a yield as high as 98%.
[0068] Comparative Example 1: The specific implementation method is the same as in Examples 1 and 2, except that a glycosyltransferase with the sequence shown in SEQ ID NO.3 was also prepared and a whole-cell catalytic reaction was carried out according to the method in Example 3. The results showed that the product yield was only 2%, while the yield of CaGT83 was as high as 97% under the same conditions.
[0069] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person 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. Glycosyltransferase CaGT83, characterized in that, Contains the amino acid sequence shown in SEQ ID NO:
1.
2. The gene encoding the glycosyltransferase CaGT83 of claim 1.
3. A recombinant vector carrying the gene of claim 2.
4. A host cell expressing the glycosyltransferase CaGT83 of claim 1, or containing the gene of claim 2, or containing the recombinant vector of claim 3.
5. A genetically engineered bacterium, characterized in that, The glycosyltransferase CaGT83 of claim 1 was expressed using Escherichia coli as the host and pET series plasmids as vectors.
6. A biocatalyst, characterized in that, Contains the glycosyltransferase CaGT83 as described in claim 1, the host cell as described in claim 4, or the genetically engineered bacteria as described in claim 5.
7. A method for synthesizing resorcinol O-glycosides, characterized in that, Resorcinol-based O-glycosides were prepared using resorcinol compounds as acceptors and the biocatalyst described in claim 6.
8. The method according to claim 7, characterized in that, The resorcinol compounds include 3,5-dihydroxypropylbenzene, 3,5-dihydroxytoluene, resorcinol, 3,5-dihydroxybenzonitrile, methyl 3,5-dihydroxybenzoate, or 6-hydroxyindole.
9. The method according to claim 7 or 8, characterized in that, The reaction system contains glucose; the concentration of glucose is 15~25 g / L; the substrate concentration in the reaction system is ≥3 mM; and the reaction temperature is 30~40℃.
10. The use of the glycosyltransferase CaGT83 of claim 1, or the gene of claim 2, or the recombinant vector of claim 3, or the host cell of claim 4, or the genetically engineered bacteria of claim 5, or the biocatalyst of claim 6, or the method of any one of claims 7 to 9 in the preparation of resorcinol O-glycosides or products containing resorcinol O-glycosides.