O-methyltransferase and application thereof

Abstract

The invention belongs to the field of gene engineering, and discloses O-methyltransferase and application thereof. In particular to a novel O-methyltransferase 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 O-methyltransferase, and an application of the O-methyltransferase or the host cell in position selective catalytic synthesis of bergenin precursor 4-O-methylgallic acid-2-C-beta-D-glucoside and a derivative thereof, and bergenin. According to the O-methyltransferase provided by the invention, bergenin precursor 4-O-methylgallic acid-2-C-beta-D-glucoside, a derivative thereof and bergenin can be efficiently and selectively catalyzed and synthesized in a position manner, and a new method is provided for obtaining the bergenin and the derivative thereof.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a novel O-methyltransferase AjOMT2 derived from the plant 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 O-methyltransferase, and an application of the O-methyltransferase or the host cell in the regioselective catalytic synthesis of the bergenin precursor 4-O-methylgallate-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 active ingredient in traditional Chinese medicinal materials and ethnic medicines such as Bergenia purpurascens, Bergenia ciliata, 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, pulmonary 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 antitumor activities of bergenin and its derivatives. Pharmaceutical Research, 2018, 37, 408-412). At the same time, this compound also has various good pharmacological activities such as antifungal, antiviral, anti-arrhythmic, anti-type 2 diabetes, antitumor, anti-anxiety, lipid-lowering, neuroprotective, liver-protective, anti-inflammatory, and alleviating pulmonary interstitial fibrosis, and has low side effects and small toxicity, showing the potential for development 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 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 anti-inflammatory, analgesic, antioxidant, liver-protective, selective α-glucosidase inhibitory activities, etc. (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 was possible to develop it into a new type of anti-tumor drug.

[0004] The good pharmacological activities and clinical application values of bergenin and its derivatives have led to an increasing demand for bergenin. At present, 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 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, the long-term overexploitation has seriously damaged the wild medicinal source plant resources of bergenin, further intensifying the shortage of bergenin medicinal sources; 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 Pingping, Yang Shengchao, Zeng Yunheng. Research progress on the resources of medicinal source plants 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 methylation using chemical methods. Synthetic biology based on biosynthesis developed in recent years provides a new strategy to solve this problem.

[0005] From the structure of bergenin, its biosynthetic pathway can be preliminarily speculated: gallic acid passes through Figure 2One of the metabolic pathways shown is that 4-O-methyltransferase (4-OMT) and 2-C-glycosyl-transferase (2-CGT) sequentially catalyze 4-O-methylation and 2-C-glycosylation in a position-selective manner, and bergenin is formed through intramolecular esterification. However, there is currently no report on 4-O-methyltransferase that can catalyze the selective methylation of gallic acid-2-C-β-D-glucoside, isolithospermic acid, etc.

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

[0007] In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a new O-methyltransferase, 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 O-methyltransferase, and the application of the O-methyltransferase or the host cell in the position-selective catalytic synthesis of the bergenin precursor 4-O-methyl gallic acid-2-C-β-D-glucoside and its 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 O-methyltransferase.

[0010] The O-methyltransferase has the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence is named AjOMT2; 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 O-methyltransferase provided by the present invention can be isolated from plants such as Ardisia japonica, Bergenia purpurascens, B. crassifolia, and Astilbe chinensis.

[0012] Conventional modifications can be carried out on the O-methyltransferase provided by the present invention. The conventional modifications can 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 O-methyltransferase provided by the present invention.

[0014] The tag can be selected from the 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.

[0015] In a second aspect, the present invention provides a nucleic acid molecule encoding the O-methyltransferase described in the first aspect of the present invention.

[0016] As a preferred embodiment, the nucleic acid molecule has the nucleic acid sequence shown in SEQ ID NO.2 and is named AjOMT2.

[0017] The nucleic acid molecule provided by the present invention can be cloned from plants such as Ardisia japonica, Bergenia purpurascens, B. crassifolia, and Astilbe chinensis.

[0018] 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 the expression vector by restriction enzyme digestion.

[0019] As a preferred embodiment, the vector can be an expression vector for expressing the target protein in bacteria, yeast, plant cells, and animal cells. More preferably, the vector is an expression vector capable of expressing the target protein in Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae cells.

[0020] As a specific embodiment, the vector is the pET28a(+) vector.

[0021] As a specific embodiment, the vector is the pCDFDuet-1 vector.

[0022] Fourthly, the present invention provides a host cell containing the recombinant expression vector described in the third aspect of the present invention.

[0023] As a preferred option, the host cell is selected from bacteria, yeast, plant cells and animal cells, and more preferably from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae host cells.

[0024] Fifthly, the present invention provides a method for producing the O-methyltransferase 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 O-methyltransferase through extraction.

[0025] The method provided by the present invention may further include: purifying the crude enzyme to obtain a pure enzyme of the O-methyltransferase.

[0026] Sixthly, the present invention provides the application of the O-methyltransferase 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 the O-methylation reaction. The O-methylation reaction described in the present invention can achieve specific regioselectivity.

[0027] As a specific embodiment, the O-methylation reaction refers to the reaction of a methyl acceptor and a methyl donor under the action of an O-methyltransferase to obtain an O-methylated product.

[0028] As a specific embodiment, the methyl acceptor compound of the present invention is gallic acid-2-C-β-D-glucoside. Using S-adenosylmethionine as the methyl donor, gallic acid-2-C-β-D-glucoside can generate the direct precursor of bergenin, 4-O-methylgallic acid-2-C-β-D-glucoside under the action of the O-methyltransferase. The O-methyltransferase provided by the present invention can regioselectively catalyze the 4-O-methylation reaction of gallic acid-2-C-β-D-glucoside.

[0029] As a specific embodiment, the methyl acceptor compound of the present invention is isolithospermic acid. Using S-adenosylmethionine as the methyl donor, isolithospermic acid can generate bergenin under the action of the O-methyltransferase. The O-methyltransferase provided by the present invention can regioselectively catalyze the 4-O-methylation reaction of isolithospermic acid.

[0030] In a seventh aspect, the present invention provides a method for synthesizing bergenin using a crude enzyme or a pure enzyme obtained by using the O-methyltransferase 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 method described in the fifth aspect of the present invention.

[0031] As a specific embodiment, the method uses the crude enzyme or pure enzyme obtained by using the O-methyltransferase 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 method described in the fifth aspect of the present invention to catalyze the O-methylation of isobergenin to produce bergenin.

[0032] As a specific embodiment, the method uses the crude enzyme or pure enzyme obtained by using the O-methyltransferase 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 method described in the fifth aspect of the present invention to catalyze the O-methylation of gallic acid-2-C-β-D-glucoside to produce 4-O-methyl-gallic acid-2-C-β-D-glucoside, and then adjusts the pH value of the reaction system to acidic to obtain bergenin.

[0033] In the method, the pH value of the reaction system is preferably adjusted to 0.1 - 2.0, more preferably 0.1 - 1.0.

[0034] In the method, the pH value of the reaction system is preferably adjusted using an inorganic acid, more preferably hydrochloric acid.

[0035] Supplementary note

[0036] 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. Beneficial technical effects:

[0037] Bergenin has a variety of 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 4-O-methylation. The O-methyltransferase described in the present invention, including recombinant enzymes and recombinant engineering cells, can efficiently and position-selectively catalyze the 4-O-methylation reaction of isobergenin, gallic acid-2-C-β-D-glucoside, etc. to synthesize products such as bergenin and its precursor 4-O-methylgallic acid-2-C-β-D-glucoside. 4-O-methylgallic acid-2-C-β-D-glucoside can be converted into 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 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

[0038] Figure 1 .Structural formulas of bergenin and some of its derivatives;

[0039] Figure 2 .Possible biosynthetic pathway from gallic acid to bergenin; 2-CGT represents 2-C-glycosyltransferase; 4-OMT represents 4-O-methyltransferase;

[0040] Figure 3 .Recombinant AjOMT2 protein catalyzes the O-methylation reaction of isobergenin and SAM; among them, Figure 3 A is the reaction formula, Figure 3 B is the HPLC analysis spectrum of the reaction product, Figure 4 C is the mass spectrum of the product bergenin;

[0041] Figure 4 .Recombinant engineering bacteria E.coli-AjCGT1-AjOMT2 whole cells catalyze the reaction of gallic acid; among them, Figure 4 A is the reaction formula, Figure 4 B is the HPLC analysis spectrum of the reaction product, Figure 4 C is the mass spectrum of the product 1a. Detailed Embodiments

[0042] 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 scope of protection 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 scope of protection 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.

[0043] In addition, it should be noted that unless otherwise specified, all kinds of 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.

[0044] Example 1: Cloning of AjOMT2 Gene

[0045] 1. Extraction of total RNA and synthesis of the first strand of cDNA

[0046] Using fresh young leaves of Ardisia japonica as materials, total RNA was extracted according to the operation instructions of the E.Z.N.A TM Plant RNAKit. 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, and grind it thoroughly until it becomes powdery; Transfer the ground powdery sample to a centrifuge tube, add 500 μL of Buffer RCL per 100 mg of tissue, homogenize it with a vortex mixer and aspirate it repeatedly with a pipette until it is well mixed; 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 up and down. Transfer the liquid to an RNA adsorption column and centrifuge at 10,000×g for 1 min; Discard the filtrate, 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 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 using a UV spectrophotometer. Use the SMARTer TM RACE cDNA amplification kit (Clontech, USA) to synthesize the first strand of cDNA.

[0047] 2. RT-PCR amplification of the target gene fragment

[0048] According to the information of the Ardisia japonica transcriptome, a candidate amino acid sequence (named AjOMT2, as shown in SEQ ID NO.1) was determined corresponding to the nucleotide sequence (named AjOMT2, as shown in SEQ ID NO.2). Specific primers for the candidate gene AjOMT2 (SEQ ID NO.3, SEQ ID NO.4) were designed, and PCR amplification was performed using KOD DNA Polymerase to obtain the full-length AjOMT2 gene. The PCR product (target gene fragment) was recovered by gel electrophoresis, ligated to the pEASY-blunt vector, transformed into Trans1-T1 competent cells, and blue-white screening and colony PCR screening were carried out. The positive clones were sent to a sequencing company for sequencing to confirm the sequence information of the candidate gene.

[0049] 2.1. PCR reaction system (50 μL):

[0050]

[0051] 2.2. PCR reaction conditions:

[0052] Denaturation at 94 °C for 2 min; denaturation at 98 °C for 10 sec, annealing at a temperature gradient of 55 °C for 30 sec, extension at 68 °C for 1 min, for 35 cycles; after the last cycle, incubate at 68 °C for 10 min. Store at 4 °C.

[0053] Example 2: Expression of AjOMT2 Gene, Isolation and Purification of Recombinant Protein

[0054] 1. Construction, recombinant protein induction expression and detection of Rosetta-pET28a-AjOMT2 recombinant expression strain

[0055] The pET-28a(+) vector contains a "T7" strong promoter and a "T7" start translation signal. The vector contains His-tagged protein sequences at both the N-terminal and C-terminal. In addition, the vector contains multiple common polylinker sites, and His tags can be added for fusion expression at the N-terminal or C-terminal of the target protein as needed.

[0056] According to the gene sequences on both sides of the multiple cloning site of the pET28a(+) vector, primers containing 17bp vector homologous arms (SEQ ID NO.5, SEQ ID NO.6) were designed. Homologous arms were introduced at the N-terminus and C-terminus of the target gene coding sequence (CDS) respectively by PCR technology, and the PCR products were recovered by gel extraction. The target gene was ligated to the linearized pET28a(+) vector using homologous recombinase to construct the recombinant plasmid pET28a-AjOMT2. Escherichia coli (Trans1 T1) cloning host was transformed, positive transformants were screened by PCR, and sent to a sequencing company for sequencing to verify the correctness of the inserted target gene. The transformants with correct sequencing were cultured overnight for amplification, plasmids were extracted, and then transformed into Escherichia coli Rosetta competent cells. 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.

[0057] Steps for induced expression, crude enzyme solution extraction and detection of the recombinant protein are as follows: The positive transformants with correct sequence identified by sequencing were inoculated into LB medium containing both kanamycin and chloramphenicol, and cultured at 37 °C with shaking at 200 rpm for 12 h; The activated seed solution was inoculated into LB medium containing both kanamycin and chloramphenicol at a ratio of 1:100. A 250 mL Erlenmeyer flask was used, with a liquid volume of 50 mL per flask (for scale-up experiments, a 500 mL Erlenmeyer flask was used, with a liquid volume of 180 mL per flask); Cultured at 37 °C with shaking at 200 rpm until the OD 600 value was approximately 0.6, and IPTG with a final concentration of 0.5 mM was added to each flask of the culture; The target protein was induced to express at 18 °C with shaking at 200 rpm; After 18 h of induced culture, the cells were collected, and the cells were collected by centrifugation at 6,000×g for 5 min. The cells were washed 3 times with double-distilled water; 5 times the mass-volume of protein extraction buffer (50 mM Tris-HCl, 0.5 mg / mL lysozyme, 1 mM PMSF, pH 7.4) was added to the cells, and mixed well by shaking; The centrifuge tube was placed in an ice-water mixture and sonicated for 15 min (130 w, 3 s / 3 s), and centrifuged at 4 °C and 15,000×g for 30 min. The supernatant was the crude enzyme solution.

[0058] 2. Isolation and purification of His-AjOMT2 recombinant protein

[0059] The Rosetta-pET28a-AjOMT2 recombinant expression strain was used for protein isolation and purification. The target gene was fused with 6×His·tag for expression, so Ni Sepharose TM6 Fast Flow resin (GE Healthcare) affinity chromatography column and PD-10 Desalting Columns (GE Healthcare) were used to separate and purify the His-tagged fusion protein. The specific steps are as follows:

[0060] 2.1 Preparation of buffers:

[0061] 1) Binding buffer: 20 mM phosphate buffer, 500 mM NaCl, 20 mM imidazole, pH 7.4;

[0062] 2) Elution buffer: 20 mM phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 7.4;

[0063] 3) Desalting buffer: 50 mM Tris-HCl, 1 mM DTT, 1% glycerol, 50 mM NaCl, pH 7.4.

[0064] 2.2 Pretreatment of Ni Sepharose TM Pretreatment of 6 Fast Flow resin affinity chromatography column:

[0065] According to the ratio that every 1 mL of Ni Sepharose TM The 6 Fast Flow resin column bed can bind the protein expressed in 2 L of recombinant strain culture solution, select an appropriate size of affinity chromatography column for packing, and wash it successively with 10 column volumes of ddH2O, binding buffer, and elution buffer, and finally equilibrate the chromatography column with binding buffer.

[0066] 2.3 Preparation and loading of crude AjOMT2 recombinant protein extract

[0067] The preparation steps of the crude AjOMT2 recombinant protein extract were the same as those in Example 1 of this embodiment. The obtained crude protein extract was filtered through a 0.45 μm filter membrane and loaded onto the affinity chromatography column at a flow rate of 1 mL / min.

[0068] 2.4 Elution of crude AjOMT2 recombinant protein

[0069] After sample loading, wash the miscellaneous proteins with 10 column volumes of binding buffer at a flow rate of 1 mL / min to elute the miscellaneous proteins 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: 20 mM, 50 mM, 100 mM, 250 mM, 500 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 perform protein content determination. Store at -80 °C for later use.

[0070] Example 3: Selective Catalysis of Recombinant AjOMT2 for 4-O-Methylation of Isobergenin to Generate Bergenin

[0071] Using SAM as the methyl donor and isobetulinic acid as the methyl acceptor substrate, the function of AjOMT2 was studied through recombinant protease-catalyzed reaction. The reaction system is as follows: methyl acceptor (40 mmol·L -1 ) 1 μL, SAM (40 mmol·L -1 ) 2 μL, MgCl2 (40 mmol·L -1 ) 2.5 μL, purified protein 200 μg, Tris-HCl buffer (pH 7.4, 50 mmol·L -1 ) make up to 100 μL. React at 37 °C for 6 h, terminate the reaction with 100 μL of cold methanol, centrifuge at 15 000×g for 30 min, and the supernatant is used for HPLC and LC / MS detection ( Figure 3 ). HPLC-UV / MS spectral analysis of the recombinant AjOMT2-catalyzed isobetulinic acid showed that compared with the blank control group, there was a new product peak in this catalytic reaction. The molecular weight shown by the first-order mass spectrum of this product was 328, which was 14 higher than the molecular weight of the substrate. This product was a single methylation product of the substrate isobetulinic acid. Under the same HPLC detection conditions, the retention time of the new product peak was consistent with that of the standard betulinic acid, indicating that AjOMT2 could catalyze isobetulinic acid to generate its corresponding methylation product betulinic acid.

[0072] Example 4: Whole-Cell Catalysis of Gallic Acid by Recombinant E.coli-AjCGT-AjOMT2 to Generate 4-O-Methylgallic Acid-2-C-β-D-Glucoside Figure 4

[0073] The C - glycosyltransferase gene AjCGT1 (shown in SEQ ID NO.7) and the O - methyltransferase gene AjOMT2 (shown in SEQ ID NO.2) in the bergenin biosynthesis pathway discovered from Ardisia japonica were respectively ligated to the MCS2 and MCS1 sites of the vector pCDFDuet - 1 and transformed into BL21(DE3) competent cells to obtain the recombinant engineering bacterium E.coli - AjCGT - AjOMT2. Take out the already constructed recombinant engineering bacterium stored in an ultra - low - temperature refrigerator and culture it overnight at 37°C and 200 rpm. Then add 34 mg / mL streptomycin to the culture medium, and at the same time add fresh LB medium for scale - up culture in a ratio of 1:100. Culture the bacteria at 37°C and 200 rpm. When the cell density OD600 is 0.6, add IPTG with a final concentration of 0.2 mM to the bacterial solution to induce protein expression, and perform induction treatment at 16°C and 200 rpm for 16 - 20 hours.

[0074] Centrifuge and collect the bacteria after induction. The collection conditions are centrifugation at 3,400 g for 5 min, and use 1×M9 medium to remove the residual bacterial solution. Suspend the bacteria with 1×M9 medium, gently mix well, measure the value of the cell density OD600, and then adjust the OD600 value to 6.0 with M9 medium. Add gallic acid (substrate concentration is 1.2 mM), and react at 30°C and 200 rpm for 24 hours. Take samples of the reaction solution in a laminar flow hood, and then add twice the volume of cold methanol to terminate the reaction, and perform HPLC and LC / MS detection on the whole - cell reaction solution ( Example 5: Conversion of 4-O-Methylgallic Acid-2-C-β-D-Glucoside to Bergenin ).

[0075] From the HPLC - MS spectrum of the whole - cell catalyzed reaction of gallic acid, it can be seen that compared with the control group, a new product peak appears at the retention time of 13.1 min. The retention time of this product is the same as that of the standard product and 4 - O - methylgallic acid C - glucoside. Mass spectrometry analysis shows that the substance with a retention time of 13.1 min has a mass - to - charge ratio of [M - H] - = 345.25 in the negative ion mode, further confirming that the substance corresponding to the newly generated product peak is 4 - O - methylgallic acid C - glucoside. The above analysis shows that: the whole - cell of the recombinant engineering bacterium E.coli - AjCGT1 - AjOMT2 catalyzes the reaction of gallic acid to generate the direct precursor of bergenin, 4 - O - methylgallic acid - 2 - C - β - D - glucoside.

[0076] ​

[0077] Adjust the pH value of the fermentation broth in Example 4 to pH 0.1 with concentrated hydrochloric acid, and 4 - O - methylgallic acid - 2 - C - β - D - glucoside can be completely converted into bergenin.

Claims

1. An O-methyltransferase, 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 equivalent functions; or having an amino acid sequence with equivalent functions obtained by conservative variation of the amino acid sequence shown in SEQ ID NO.

1.

2. The O-methyltransferase according to claim 1, wherein The O-methyltransferase has undergone conventional modifications; 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 O-methyltransferase according to any one of claims 1 or 2, characterized in that, A tag for detection or purification is attached to the O-methyltransferase; 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, It encodes the O-methyltransferase 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 O-methyltransferase 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 O-methyltransferase by extraction; preferably, purifying the crude enzyme to obtain a pure enzyme of the O-methyltransferase.

8. Use of the O-methyltransferase 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 catalytic O-methylation reaction.

9. The application according to claim 8, characterized in that, The O-methylation reaction refers to reacting a methyl acceptor and a methyl donor under the action of the O-methyltransferase to obtain an O-methylated product; Preferably, the methyl acceptor is selected from isolithospermic acid and gallic acid-2-C-β-D-glucoside.

10. The application according to claim 9, characterized in that, The methyl acceptor compound is isolithospermic acid, the methyl donor is S-adenosylmethionine, and the product is bergenin. Or, the methyl acceptor compound is gallic acid-2-C-β-D-glucoside, the methyl donor is S-adenosylmethionine, and the product is 4-O-methylgallic acid-2-C-β-D-glucoside.

11. A method for synthesizing bergenin, characterized in that, Using the O-methyltransferase 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 to catalyze the O-methylation of isolithospermic acid to generate bergenin.

12. A method for synthesizing bergenin, characterized in that, The crude enzyme or pure enzyme produced by using the O-methyltransferase 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 catalyzes the O-methylation of gallic acid-2-C-β-D-glucoside to generate 4-O-methylgallic acid-2-C-β-D-glucoside, and then adjusts the pH value of the reaction system 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, preferably hydrochloric acid.

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