C-glycosyl transferase and application thereof

Abstract

The invention belongs to the field of gene engineering, and discloses C-glycosyl transferase and application thereof. In particular to a novel C-glycosyl transferase derived from ardisia japonica, a nucleic acid molecule thereof, an expression vector containing the nucleic acid molecule and a host cell containing the vector. The invention also provides a production method of the C-glycosyl transferase, and an application of the C-glycosyl transferase or a host cell in catalytic synthesis of a bergenin precursor, namely gallic acid-2-beta-D-C-glucoside and 4-O-methyl-gallic acid-2-beta-D-C-glucoside, by virtue of position and stereoselectivity. The invention also provides a preparation method of the C-glycosyl transferase and an application of the C-glycosyl transferase or the host cell. According to the C-glycosyl transferase provided by the invention, bergenin precursors, namely gallic acid-2-beta-D-C-glucoside, 4-O-methyl-gallic acid-2-beta-D-C-glucoside and the like, can be subjected to catalytic synthesis in a high-efficiency, position-selective and stereoselective manner; the 4-O-methyl-gallic acid-2-beta-D-C-glucoside can be converted into the bergenin under acid treatment, and a new method is provided for obtaining the bergenin and derivatives thereof.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and specifically relates to a novel C-glycosyltransferase AjCGT derived from Ardisia japonica, its nucleic acid molecule, an expression vector containing the nucleic acid molecule and a host cell containing the vector, a production method of the C-glycosyltransferase, and the application of the C-glycosyltransferase or the host cell in the regioselective and stereoselective catalytic synthesis of the bergenin precursor gallic acid-2-C-β-D-glucoside, 4-O-methyl-gallic acid-2-C-β-D-glucoside and its derivatives, and bergenin. Background Art

[0002] Bergenin ( Figure 1) is a phenolic acid C-glycoside compound, with gallic acid as the parent nucleus, formed through O-methylation, C-glycosylation, and intramolecular esterification reactions. It was named because it was first isolated from the traditional Chinese medicine Bergenia purpurascens. It is an important pharmacodynamic component of traditional Chinese medicinal materials and ethnic medicines such as Bergenia purpurascens, Bergenia purpurascens var. delavayi, Astilbe chinensis, Ardisia crenata, and Ardisia japonica. Pharmacological studies have shown that bergenin has significant antitussive and expectorant effects, can effectively treat respiratory diseases such as chronic bronchitis, emphysema, cor pulmonale, and bronchial asthma, and is safe and non-toxic. Currently, it has been developed into compound bergenin tablets and is clinically used to treat chronic bronchitis (Wang Meng, Niu Youhong, Wu Yanfen. Research progress on the anti-tumor activity of bergenin and its derivatives. Pharmaceutical Research, 2018, 37, 408-412). At the same time, this compound also has a variety of good pharmacological activities such as antifungal, antiviral, anti-arrhythmic, anti-type 2 diabetes, anti-tumor, anti-anxiety, lipid-lowering, neuroprotection, liver protection, anti-inflammatory, and alleviating pulmonary interstitial fibrosis, and has low side effects and small toxicity, showing the potential to be developed into other new drugs (Bajracharya G.B. Diversity, pharmacology and synthesis of bergenin and its derivatives: Potential materials for therapeutic usages. Fitoterapia, 2015, 101, 133–152; Cui Shuang, Zhang Mingqian, Liang Wulin, et al. Research progress on the pharmacological effects of Bergenia purpurascens. Western Journal of Traditional Chinese Medicine, 2022, 35, 123-128; Baraia P., Ravala N., Acharyab S., et al. Neuroprotective effects of bergenin in Alzheimer’s disease: investigation through molecular docking, in vitro and in vivo studies. Behav. Brain Res., 2019, 356, 18-40; Bomgning C.L.K., Sinda P.V.K., Ponou B.K. Hepatoprotective effects of extracts, fractions and compounds from the stem bark of Pentaclethra macrophylla Benth: Evidence from in vitro and in vivo studies. Biomed. Pharmacother., 2021, 136, 111242; Tang Q., Wang Q., Sun Z.Bergenin monohydrate attenuates inflammatory response via MAPK and NF-κB pathways against Klebsiella pneumonia infection. Front. Pharmacol., 2021, 12, 651664; Li X., Wang Y., Liang J., et al. Bergenin attenuates bleomycin-induced pulmonary fibrosis in mice via inhibiting TGF-β1 signaling pathway. Phytother. Res., 2021, 35, 5808–5822.).

[0003] Due to the diverse pharmacological activities and low toxicity of bergenin, structural modification of bergenin as a lead compound to obtain derivatives with higher activity has also become a research hotspot among scholars at home and abroad ( Figure 1)。Compared with bergenin, triacetylbergenin has enhanced anti-inflammatory activity and anti-ulcer effects (Jung J.-C., Lim E., Kim S.H., et al. Practical synthesis and biological evaluation of bergenin analogs. Chem. Biol. Drug Res., 2011, 78, 725–729); 6′-O-galloylbergenin has good activities such as anti-inflammatory and analgesic, antioxidant, liver protection, and selective inhibition of α-glucosidase (Qiu J., Chen X., Liang P., et al. Integrating approach to discover novel bergenin derivatives and phenolics with antioxidant and anti-inflammatory activities from bio-active fraction of Syzygium brachythyrsum. Arab. J. Chem., 2022, 15, 103507; Uddin G., Sadat A., Siddiqui B.S. Comparative antioxidant and antiplasmodial activities of 11-O-galloylbergenin and bergenin isolated from Bergenia ligulata. World Appl. Sci. J., 2013, 27, 977–981; Habtemariam S., Cowley R.A. Antioxidant and anti-α-glucosidase compounds from the rhizome of Peltiphyllum peltatum (Torr.) Engl. Phytother. Res., 2012, 26, 1656–1660; Kumar T.V., Tiwari A.K., Robinson A., et al. Synthesis and antiglycation potentials of bergenin derivatives. Bioorg. Med. Chem. Lett., 2011, 21, 4928–4931.).In vivo activity evaluation showed that bergenin derivative 6′-O-p-cyanocoumaroylbergenin had stronger anti-hepatocellular carcinoma activity and lower toxicity than the control 5-fluorouracil (Liang C., Pei S., Ju W., et al. Synthesis and in vitro and in vivo antitumor activity study of 11-hydroxyl esterified bergenin / cinnamic acid hybrids. Eur. J. Med. Chem., 2017, 133, 319-328.), and it has the potential to be developed into a new type of antitumor drug.

[0004] The good pharmacological activities and clinical application values of bergenin and its derivatives have led to an increasing demand for bergenin. Currently, bergenin used clinically is mainly extracted from medicinal source plants such as Bergenia purpurascens, B. crassifolia, Ardisia creanata, and A. japonica (Bajracharya G.B. Diversity, pharmacology and synthesis of bergenin and its derivatives: Potential materials for therapeutic usages. Fitoterapia, 2015, 101, 133–152.), but the contents are all relatively low. Moreover, most of the medicinal source plants of bergenin grow in high-altitude areas (for example, Bergenia purpurascens is distributed in areas with an altitude of 2700-4800 meters), the plant growth rate is slow, the growth cycle is long, and the yield is low. At the same time, long-term over-exploitation has seriously damaged the wild medicinal source plant resources of bergenin, further exacerbating the tense situation of the medicinal source of bergenin; according to statistics, the resources of Bergenia purpurascens were on the verge of exhaustion in the 1990s, and the purchase prices of the raw materials and related medicinal materials of bergenin have been rising year by year (Li P.P., Yang S.C., Zeng Y.H. Research progress on the medicinal source plant resources of bergenin. Chinese Traditional and Herbal Drugs, 2009, 40, 1500-1505.). Chemical synthesis has many difficulties, especially in this structure with multiple hydroxyl groups, and there are problems such as insufficient regioselectivity in glycosylation using chemical methods. Synthetic biology based on biosynthesis developed in recent years provides a new strategy to solve this problem.

[0005] Based on the structure of bergenin, its biosynthetic pathway can be preliminarily speculated: gallic acid passes through Figure 2One of the metabolic pathways shown is that 2-C-glycosyl-transferase (2-CGT) or / and 4-O-methyltransferase (4-OMT) catalyze 2-C-glycosylation or / and 4-O-methylation in sequence with position and stereoselectivity, and bergenin is formed through intramolecular esterification. However, there is currently no report on 2-C-glycosyltransferase that can catalyze the selective glycosylation of gallic acid and 4-O-methylgallic acid by key enzymes.

[0006] Therefore, discovering and functionally identifying the key enzyme 2-C-glycosyltransferase in the bergenin biosynthetic pathway can not only (1) reveal its biosynthetic pathway, but also (2) use this enzyme to catalyze the position and stereoselective synthesis of bergenin precursors such as gallic acid-2-β-D-C-glucoside and 4-O-methyl-gallic acid-2-β-D-C-glucoside from gallic acid and 4-O-methylgallic acid; furthermore, (3) it can also combine it with other key enzyme genes such as 4-O-methyltransferase in microorganisms with the gallic acid biosynthetic pathway to construct a microbial "cell factory", so as to produce bergenin using synthetic biology technology, which has potential application value. Summary of the Invention

[0007] In order to overcome the deficiencies in the prior art, the technical problem solved by the present invention is to provide a new C-glycosyltransferase derived from Ardisia japonica, its nucleic acid molecule, an expression vector containing the nucleic acid molecule, and a host cell containing the vector. The present invention also provides a production method of the C-glycosyltransferase, and the application of the C-glycosyltransferase or host cell in the position and stereoselective catalytic synthesis of bergenin precursors such as gallic acid-2-C-β-D-glucoside, 4-O-methyl-gallic acid-2-C-β-D-glucoside and their derivatives, as well as bergenin.

[0008] To solve the technical problems of the present invention, the following technical solutions are provided:

[0009] In the first aspect, the present invention provides a new C-glycosyltransferase.

[0010] The C-glycosyltransferase has the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence is named AjCGT; or has an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO.1 and has the same function; or has an amino acid sequence obtained by conservative variation of the amino acid sequence shown in SEQ ID NO.1, that is, an amino acid sequence with the same function formed by substitution, deletion or addition of amino acids.

[0011] The C-glycosyltransferase provided by the present invention can be isolated from plants such as Ardisia japonica, Bergenia purpurascens, Bergenia crassifolia, and Astilbe chinensis.

[0012] Conventional modifications can be performed on the C-glycosyltransferase provided by the present invention. The conventional modifications may include one or more of acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, and immobilization modification.

[0013] A tag for detection or purification can also be linked to the C-glycosyltransferase provided by the present invention. The tag can be selected from conventional tags known in the art that can be used for detecting or purifying proteins. Specifically, the tag can be selected from one or more of His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact.

[0014] In a second aspect, the present invention provides a nucleic acid molecule encoding the C-glycosyltransferase described in the first aspect of the present invention.

[0015] As a preferred embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO.2 and is named AjCGT.

[0016] The nucleic acid molecule provided by the present invention can be cloned from plants such as Ardisia japonica, Bergenia purpurascens, Bergenia crassifolia, and Astilbe chinensis.

[0017] In a third aspect, the present invention provides a recombinant expression vector containing the nucleic acid molecule described in the second aspect of the present invention. Specifically, the nucleic acid molecule described in the second aspect of the present invention is ligated to an expression vector by enzymatic digestion.

[0018] As a preferred embodiment, the vector can be an expression vector for expressing a target protein in bacteria, yeast, plant cells, and animal cells. More preferably, the vector is an expression vector capable of expressing a target protein in Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae cells.

[0019] As a specific embodiment, the vector is the pET28a(+) vector.

[0020] In a fourth aspect, the present invention provides a host cell containing the recombinant expression vector described in the third aspect of the present invention.

[0021] As a preferred embodiment, the host cell is selected from bacteria, yeast, plant cells and animal cells, and more preferably from Escherichia coli, Pichia pastoris and Saccharomyces cerevisiae host cells.

[0022] In a fifth aspect, the present invention provides a method for producing the C-glycosyltransferase described in the first aspect of the present invention. The method includes: culturing the host cell described in the fourth aspect of the present invention, collecting the culture, and obtaining a crude enzyme containing the C-glycosyltransferase through extraction.

[0023] The method provided by the present invention may further include: purifying the crude enzyme to obtain a pure enzyme of the C-glycosyltransferase.

[0024] The culturing in the present invention is carried out under a culture medium and culture conditions suitable for the host cell.

[0025] In a sixth aspect, the present invention provides the application of the C-glycosyltransferase described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, or the crude enzyme or pure enzyme produced by the method described in the fifth aspect in a C-glycosylation reaction. The C-glycosylation reaction described in the present invention can achieve specific position and stereoselectivity.

[0026] As a preferred embodiment, the C-glycosylation reaction refers to the reaction of a glycosyl acceptor and a glycosyl donor under the action of a C-glycosyltransferase to obtain a C-glycosylation product.

[0027] As a specific embodiment, the glycosyl acceptor compound in the present invention is gallic acid or its derivative. Using uridine diphosphate-glucose (UDP-glucose) as the glycosyl donor, gallic acid or its derivative can generate gallic acid-2-C-β-D-glucoside or its derivative under the action of the C-glycosyltransferase. The C-glycosyltransferase provided by the present invention can catalyze the 2-C-glycosylation reaction of gallic acid with position and stereoselectivity.

[0028] As a specific embodiment, the glycosyl acceptor compound in the present invention is 4-O-methylgallic acid or its derivative. Using uridine diphosphate-glucose (UDP-glucose) as the glycosyl donor, 4-O-methylgallic acid can generate 4-O-methyl-gallic acid-2-C-β-D-glucoside or its derivative under the action of the C-glycosyltransferase. The C-glycosyltransferase provided by the present invention can catalyze the 2-C-glycosylation reaction of 4-O-methylgallic acid with position and stereoselectivity.

[0029] Seventh aspect, the present invention provides a method for synthesizing bergenin. The method uses the C-glycosyltransferase described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, or the crude enzyme or pure enzyme obtained by the method described in the fifth aspect, to catalyze 4-O-methylgallic acid to perform C-glycosylation to generate 4-O-methyl-gallic acid-2-C-β-D-glucoside, and then adjust the pH value of the reaction system to acidic to obtain bergenin.

[0030] In the method, the pH value of the reaction system is preferably adjusted to 0.1-2.0, more preferably 0.1-1.0. In the method, the pH value of the reaction system is preferably adjusted using an inorganic acid, more preferably hydrochloric acid.

[0031] Supplementary description

[0032] Unless otherwise specified, the scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein can also be used to implement the present invention. Specific embodiments, preferred methods and materials are described herein, but the present invention is not limited in any way.

[0033] Beneficial technical effects

[0034] Bergenin has various pharmacological activities and is clinically used to treat chronic tracheitis. However, its natural resources are scarce and chemical synthesis is difficult, especially the position-selective 2-C-glycosylation. The C-glycosyltransferase described in the present invention, including recombinant enzymes and recombinant engineering cells, can efficiently, position- and stereoselectively catalyze the 2-C-glycosylation reaction of gallic acid, 4-O-methylgallic acid, etc. to synthesize the bergenin precursors such as gallic acid-2-C-β-D-glucoside, 4-O-methylgallic acid-2-C-β-D-glucoside, etc., and synthesize bergenin under acid treatment, overcoming the above deficiencies. Furthermore, it can also be combined with other key enzyme genes such as 2-carbon glycosyltransferase in microorganisms with the endogenous gallic acid biosynthetic pathway to construct a microbial "cell factory", thereby producing bergenin using synthetic biology technology, which has potential application value. Description of the drawings

[0035] Figure 1 . Structural formulas of bergenin and some of its derivatives;

[0036] Figure 2 . Possible biosynthetic pathway from gallic acid to bergenin; 2-CGT represents 2-C-glycosyltransferase; 4-OMT represents 4-O-methyltransferase;

[0037] Figure 3. The recombinant AjCGT protein catalyzes the C-glycosylation reaction of gallic acid and UDP-glucose; among them, Figure 3 A is the reaction formula and the HPLC detection spectrum of the reaction product; Figure 3 B is the first-order and second-order mass spectrometry spectra of the product gallic acid-2-C-β-D-glucoside;

[0038] Figure 4 . The recombinant AjCGT protein catalyzes the C-glycosylation reaction of 4-O-methyl gallic acid and UDP-glucose; among them, Figure 4 A is the reaction formula and the HPLC detection spectrum of the reaction product; Figure 4 B is the first-order and second-order mass spectrometry spectra of the product 4-O-methyl gallic acid-2-C-β-D-glucoside. Specific implementation manners

[0039] The following examples are provided to further illustrate various aspects of the present invention. These examples are non-limiting and should not be construed as limiting any aspect of the present invention. The protection scope of the present invention is only limited by the claims. Without departing from the scope of the claims, those skilled in the art can make various modifications and improvements to various aspects of the present invention, and these modifications and improvements also fall within the protection scope of the present invention. For example, replacing the promoters and expression vectors used in the examples with other commonly used promoters and expression vectors in the art can be understood and implemented by those of ordinary skill in the art.

[0040] In addition, it should be noted that unless otherwise specified, various materials and reagents used in the following examples are commonly used materials and reagents in the art and can be obtained through conventional commercial channels; the methods used are all conventional methods well known to those skilled in the art.

[0041] Example 1: Cloning of AjCGT gene

[0042] 1. Extraction of total RNA and synthesis of the first strand of cDNA

[0043] Using fresh young leaves of Ardisia japonica as materials according to E.Z.N.A TMExtract total RNA according to the operation instructions of the Plant RNA Kit. The specific operations are as follows: Weigh an appropriate amount of the sample and quickly transfer it to a mortar pre-cooled with liquid nitrogen. Grind it thoroughly into a powder; transfer the powdered sample to a centrifuge tube, add 500 μL of Buffer RCL per 100 mg of tissue, homogenize it with a vortex mixer and pipette it repeatedly to mix evenly; incubate at 55 °C for 3 min; centrifuge at 15,000×g for 5 min, take the supernatant and transfer it to a filter column, and centrifuge at 14,000×g for 2 min. Add an equal volume of Buffer RCB to the filtered liquid and invert it 5 - 10 times. Transfer the liquid to an RNA adsorption column and centrifuge at 10,000×g for 1 min; discard the filtered liquid, add 400 μL of RWC wash Buffer to the adsorption column and centrifuge at 10,000×g for 1 min; after the adsorption column is air-dried at room temperature, elute the total RNA with 50 μL of ddH2O. Detect the integrity of the total RNA by 1.0% non-denaturing agarose gel electrophoresis and measure the OD 260 / OD 280 ratio and the RNA concentration. Use SMARTer TM RACE cDNA Amplification Kit (Clontech, USA) to synthesize the first strand of cDNA.

[0044] 2. RT-PCR amplification of the target gene fragment

[0045] According to the information of the Ardisia japonica transcriptome, determine the candidate amino acid sequence (named AjCGT, as shown in SEQ ID NO.1) and the corresponding nucleotide sequence (named AjCGT, as shown in SEQ ID NO.2), design specific primers for the candidate gene AjCGT (SEQ ID NO.3, SEQ ID NO.4), use KOD DNA Polymerase for PCR amplification to obtain the full length of the AjCGT1 gene. Gel-purify the PCR product (target gene fragment), ligate it to the pEASY-blunt vector, transform Trans1-T1 competent cells, perform blue-white screening and colony PCR screening, and send the positive clones to a sequencing company for sequencing to confirm the sequence information of the candidate gene.

[0046] 2.1. PCR reaction system (50 μL):

[0047]

[0048] 2.2. PCR reaction conditions:

[0049] Denature at 94°C for 2 min; denature at 98°C for 10 sec, anneal at a temperature gradient of 55°C for 30 sec, extend at 68°C for 1 min, and perform 35 cycles; after the last cycle ends, incubate at 68°C for 10 min. Store at 4°C.

[0050] Example 2: Expression of AjCGT gene, separation, purification of recombinant protein and study on catalytic function

[0051] 1. Construction, induced expression and detection of recombinant expression strain Rosetta-pET28a-AjCGT

[0052] The pET-28a(+) vector contains a "T7" strong promoter and a "T7" translation initiation signal. The vector contains His-tag protein sequences at both the N-terminal and C-terminal. In addition, the vector contains multiple common multiple cloning sites, and His tags can be added to the N-terminal or C-terminal of the target protein for fusion expression according to needs.

[0053] According to the gene sequences on both sides of the multiple cloning site of the pET28a(+) vector, primers (SEQ ID NO.5, SEQ ID NO.6) containing 17 bp vector homologous arms were designed. Homologous arms were introduced at the N-terminal and C-terminal of the coding sequence (CDS) of the target gene by PCR technology, and the PCR products were recovered by gel extraction. The target gene was ligated to the linearized pET28a(+) vector using a homologous recombinase to construct the recombinant plasmid pET28a-AjCGT. Escherichia coli (Trans1 T1) was used as the cloning host for transformation. Positive transformants were screened by PCR and sent to a sequencing company for sequencing to verify the correctness of the inserted target gene. The correctly sequenced transformants were cultured overnight for amplification, plasmids were extracted, and Escherichia coli Rosetta competent cells were transformed. Positive clones were picked and verified by PCR for the correctness of the expression system. The positive transformants were stored at -80°C in 15% glycerol.

[0054] The steps for induced expression of recombinant protein, extraction of crude enzyme solution and detection are as follows: Inoculate the positive transformants with correct sequences identified by sequencing into LB medium containing both kanamycin and chloramphenicol, and culture at 37°C with shaking at 200 rpm for 12 h; inoculate the activated seed solution into LB medium containing both kanamycin and chloramphenicol at a ratio of 1:100. Use 250 mL Erlenmeyer flasks, with a liquid volume of 50 mL per flask; culture at 37°C with shaking at 200 rpm until OD 600The value is approximately 0.6. Add IPTG with a final concentration of 0.5 mM to each bottle of culture; induce the expression of the target protein at 18 °C and 200 rpm; collect the bacterial cells after 18 h of induction culture, centrifuge at 6,000×g for 5 min to collect the bacterial cells, and wash the bacterial cells 3 times with double-distilled water; add protein extraction buffer (50 mM Tris-HCl, 0.5 mg / mL lysozyme, 1 mM PMSF, pH 7.4) with a volume 5 times the mass of the bacterial cells to the bacterial cells, shake and mix well; place the centrifuge tube in an ice-water mixture and ultrasonically disrupt it for 15 min (130 w, 3 s / 3 s), centrifuge at 4 °C and 15,000×g for 30 min, and the supernatant is the crude enzyme solution.

[0055] 2. Isolation and purification of His-AjCGT recombinant protein

[0056] Use the Rosetta-pET28a-AjCGT recombinant expression strain for protein isolation and purification. The target gene is fused with 6×His·tag for expression. Therefore, use Ni Sepharose TM 6Fast Flow resin (GE Healthcare) affinity chromatography column and PD-10 Desalting Columns (GE Healthcare) to isolate and purify the fusion protein containing His tag. The specific steps are as follows:

[0057] 2.1. Preparation of buffers:

[0058] 1) binding buffer: 20 mM phosphate buffer, 500 mM NaCl, 20 mM imidazole, pH 7.4;

[0059] 2) elution buffer: 20 mM phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 7.4;

[0060] 3) desalting buffer: 50 mM Tris-HCl, 1 mM DTT, 1% glycerol, 50 mM NaCl, pH 7.4.

[0061] 2.2. Pretreatment of Ni Sepharose TM 6Fast Flow resin affinity chromatography column:

[0062] According to every 1 mL of Ni Sepharose TMFor the 6Fast Flow resin column bed, select an appropriate-sized affinity chromatography column for packing according to the proportion of the protein expressed in 2 L of the recombinant strain culture solution. Wash it successively with 10 column volumes of ddH2O, binding buffer, and elution buffer, and finally equilibrate the chromatography column with the binding buffer.

[0063] 2.3 Preparation and loading of the crude extract of AjCGT recombinant protein

[0064] The preparation steps of the crude extract of AjCGT recombinant protein are the same as those in Example 1 of this embodiment. The obtained crude protein extract is filtered through a 0.45 μm filter membrane and loaded onto the affinity chromatography column at a flow rate of 1 mL / min.

[0065] 2.4 Elution of AjCGT recombinant protein

[0066] After the loading is completed, wash the impurities with 10 column volumes of binding buffer at a flow rate of 1 mL / min to elute the impurities that are not tightly bound to the column bed. When the UV absorption value of the eluate at 280 nm tends to 0, elute the target protein. Prepare elution buffers with different imidazole concentrations to perform gradient elution on the target protein. The imidazole concentration gradients are as follows: 50 mM, 100 mM, 200 mM, 300 mM. Elute 5 column volumes for each concentration gradient and perform SDS-PAGE detection on each fraction. Transfer the high-purity fraction containing the target protein to an ultrafiltration centrifugal tube (specification 30 kDa, Millipore) for desalting and concentration. During this period, desalting buffer can be supplemented and centrifuged continuously until the volume of the pure enzyme solution is about 1 mL, and then measure the protein content. Store it at -80 °C for later use.

[0067] Example 3: Positional and stereoselective catalysis of recombinant AjCGT to generate gallic acid-2-C-β-D-glu coside from gallic acid

[0068] Using UDP-glucose as the glycosyl donor and gallic acid as the glycosyl acceptor substrate, study the function of AjCGT through the recombinant protease-catalyzed reaction. The reaction system is as follows: 0.2 mM glycosyl acceptor, 0.4 mM glycosyl donor, 50 μg recombinant AjCGT protein, total reaction volume 100 μL, reaction buffer (50 mM Tris-HCl, pH 7.0), react in a 40 °C water bath for 1 h, add 200 μL of ice methanol to terminate the reaction, centrifuge at 15,000×g for 30 min, and take the supernatant for HPLC-MS / MS analysis ( Figure 3 )

[0069] Example 4: Positional and stereoselective catalysis of recombinant AjCGT to generate 4-O-methylgallate-2-C-β-D-glu coside from 4-O-methylgallate

[0070] Using UDP-glucose as the glycosyl donor and 4-O-methyl gallic acid as the glycosyl acceptor substrate, the function of AjCGT was studied through a recombinant protease-catalyzed reaction. The reaction system was as follows: 0.2 mM glycosyl acceptor, 0.4 mM glycosyl donor, 50 μg recombinant AjCGT protein, total reaction volume of 100 μL, reaction buffer (50 mM Tris-HCl, pH 7.0), reaction in a water bath at 40 °C for 1 h, add 200 μL of ice-cold methanol to terminate the reaction, centrifuge at 15,000×g for 30 min, and take the supernatant for HPLC-MS / MS analysis( Figure 4 ).

[0071] Example 5: Conversion of 4-O-methylgallate-2-C-β-D-glucoside to bergenin

[0072] The pH value of the fermentation broth in Example 4 was adjusted to pH 0.1 with concentrated hydrochloric acid, and all of the 4-O-methyl gallic acid-2-C-β-D-glucoside could be converted into bergenin.

Claims

1. A C-glycosyltransferase, characterized in that, Having the amino acid sequence shown in SEQ ID NO.1; or having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO.1 and having the same function; or having an amino acid sequence obtained by conservative variation of the amino acid sequence shown in SEQ ID NO.1 and having the same function.

2. The C-glycosyltransferase according to claim 1, wherein Conventional modifications have been made to the C-glycosyltransferase; the conventional modifications are selected from one or more of acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, or immobilization modification.

3. The C-glycosyltransferase according to any one of claims 1 or 2, characterized in that, A tag for detection or purification is attached to the C-glycosyltransferase; Preferably, the tag is selected from one or more of His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or Profinity eXact.

4. A nucleic acid molecule, characterized in that, Encoding the C-glycosyltransferase according to any one of claims 1 to 3, preferably having the nucleotide sequence shown in SEQ ID NO.

2.

5. A recombinant expression vector comprising the nucleic acid molecule according to claim 4.

6. A host cell containing the nucleic acid molecule according to claim 4 or the recombinant expression vector according to claim 5; Preferably, the host cell is selected from bacteria, yeast, plant cells, and animal cells; More preferably, the host cell is selected from Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris.

7. A method for producing the C-glycosyltransferase according to any one of claims 1 to 3, characterized in that, Culturing the host cell according to claim 6, collecting the culture, and obtaining a crude enzyme containing the C-glycosyltransferase by extraction; preferably, purifying the crude enzyme to obtain a pure enzyme of the C-glycosyltransferase.

8. Use of the C-glycosyltransferase according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, or the crude enzyme or pure enzyme produced by the method according to claim 7, in a C-glycosylation reaction.

9. The application according to claim 8, characterized in that, The C-glycosylation reaction refers to reacting a glycosyl acceptor and a glycosyl donor under the action of the C-glycosyltransferase to obtain a C-glycoside product; Preferably, the glycosyl acceptor is selected from gallic acid or its derivatives, 4-O-methylgallic acid or its derivatives.

10. The application according to claim 9, characterized in that, The glycosyl acceptor is gallic acid or its derivative, the glycosyl donor is uridine diphosphate-glucose, and the product is gallic acid-2-C-β-D-glucoside or its derivative; Or, the glycosyl acceptor is 4-O-methylgallic acid or its derivative, the glycosyl donor is uridine diphosphate-glucose, and the product is 4-O-methyl-gallic acid-2-C-β-D-glucoside or its derivative.

11. A method for synthesizing bergenin, characterized in that, The crude enzyme or pure enzyme produced by using the C-glycosyltransferase according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, or the method according to claim 7 is used to catalyze 4-O-methylgallic acid to carry out C-glycosylation to generate 4-O-methyl-gallic acid-2-C-β-D-glucoside, and then the pH value of the reaction system is adjusted to acidic to obtain bergenin; Preferably, the pH value of the reaction system is adjusted to 0.1 to 2.0, preferably 0.1 to 1.0; Preferably, an inorganic acid is used to adjust the pH value of the reaction system, and more preferably hydrochloric acid.

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