Rodgersia podophylla carbon glycosyl transferase RsCGT1 gene and application thereof in preparation of bergenin

Abstract

The invention relates to a Rodgersia aesculifolia carbon glycosyl transferase RsCGT1 gene and application thereof in preparation of bergenin, and belongs to the technical field of biology. The nucleotide sequence of the Rodgersia podophylla carbon glycosyl transferase RsCGT1 gene is as shown in SEQ ID NO.1, and the full length of the sequence is 1458bp; the amino acid sequence of the encoded protein is shown as SEQ ID NO.2, and 485 amino acid residues are encoded. The rodgersia podophylla carbon glycosyl transferase RsCGT1 gene can be used as a biosynthesis regulation gene of bergenin, is applied to preparation of bergenin, and is remarkable in application prospect and easy to popularize and apply.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a Coleus fasciatus carbonyl transferase RsCGT1 gene and an application thereof in preparing bergenin. Background Art

[0002] Rodgersiasambucifolia Hemsl is a perennial herbaceous plant of the genus RodgersiaA.Gray of the family Saxifragaceae. It is also known as the feathery-leaved Rodgersia. There are 5 species in this genus, distributed in East Asia and the Himalayas. There are 4 species in my country, produced in Northeast China, Northwest China, Central China and Southwest China, mainly in Southwest China. There are many medicinal ingredients in the plants of Rodgersiasambucifolia Hemsl, and bergenin is one of the main active ingredients. Bergenin has attracted wide attention for its various pharmacological activities, such as antitussive, anti-inflammatory, antianxiety, antioxidant, antimalarial, anticancer, treatment of diabetes, anti-hepatotoxicity, immunomodulation and neuroprotection. Unlike morphine-like cough suppressants that inhibit the central nervous system, bergenin has a selective inhibitory effect on the cough center and has no inhibitory effect on other nerve centers. It has the characteristics of small toxic and side effects, few adverse reactions, and no drug resistance after continuous use. Bergenin has been made into various dosage forms and is widely used in China to treat respiratory diseases, such as compound bergenin tablets, bergenin capsules, etc.

[0003] The precursor of bergenin biosynthesis is 2-glucose-4-methoxygallic acid, which is generated by intramolecular dehydration, rearrangement and ring closure; 2-glucose-4-methoxygallic acid is generated by 4-methoxygallic acid connecting glucose at the 2-position with uridine diphosphate glucose (UDP-glucose) as the glycosyl donor under the catalysis of carbon glycosyltransferase (CGT); 4-methoxygallic acid is catalyzed by gallic acid oxygen methyltransferase (OMT) with gallic acid as substrate and S-adenosyl-methionine (SAM) as the methyl donor; gallic acid is catalyzed by shikimate dehydrogenases (SDH) with NADP+ as the proton donor.

[0004] With the increasing number of chronic bronchitis diseases, the gap in raw materials for bergenin is increasing. At present, the raw materials for bergenin on the market are mainly obtained by extraction, and the annual demand for dry medicinal materials for extracting bergenin has reached more than 2,000 tons. The extensive mining in recent decades has caused a sharp decline in wild resource reserves and is on the verge of exhaustion. At the same time, the high altitude, many mountainous areas and cold climate conditions in the origin make it difficult to cultivate artificially on a large scale, further expanding the gap in the market for bergenin raw materials and causing prices to rise year by year. In recent years, with the rapid development of the field of synthetic biology, the use of synthetic biology technology to produce natural drug monomers can effectively solve the above problems. However, to understand the biosynthetic pathways of these active ingredients, it is necessary to identify the key genes related to these pathways, and discovering these catalytic enzyme genes has become a key link in studying the biosynthetic pathways of plant metabolites. Currently, the synthesis pathway of 4-methoxygallic acid catalyzing the glycosylation reaction at the C-2 position to form 2-glucose-4-methoxygallic acid has been clarified, but the function of the carbon glycosyltransferase responsible for glycosylation has been less verified, which has affected the advancement of the biosynthesis of bergenin. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a C. corymbifera carbonyl transferase RsCGT1 gene, which can be used as a biosynthesis regulatory gene of bergenin and can be used in the preparation of bergenin.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a southwestern Coleus corydalis carbonyl transferase RsCGT1 gene, the nucleic acid sequence of the southwestern Coleus corydalis carbonyl transferase RsCGT1 gene is shown in SEQ ID NO.1 below, and the full length of the sequence is 1458 bp.

[0008]

[0009] The second aspect of the present invention provides a protein encoded by the above-mentioned Coleus corymbifera carbonyl transferase RsCGT1 gene, the amino acid sequence of the protein is as follows SEQ ID NO.2, encoding 485 amino acid residues.

[0010] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMSNSGNQEPRPHIALLPSAGMGHLNPLLRLTATLAARGCHVTVITPIPTFTAAESEHMDEFFSTYPNISRLKFQIVGDEPPSEFDPPI FLQWQSINRSGHILRPLLTALSPPITALFSDFVAGLCICPIADDLKIPNYFVVTSSARFFSLWALLPYINKSHDDEILIPGLSPFHISTIPPPFFNPNHLFTKLATSNAPLLSKAKGILLNTF DYFEPDTITAIRKGTVLSDLPQVFPIGPLKDYKFGKGNYPTWLDDQEEGSVVYVSFGSRTTLSRDQIRELGDGLERSGFPFLWVLKTSKEDEGELRDLLGDTFFEKTGKKGIVVKGWADQEA ILAHPAIGGFVSHCGWNSVTETTRFGVPILAWPQHGDQRLNAEVVEKAGMGVWERDWGWQRERLIKGEEIGEKIEKWMTDEKSKTQAKKIRVEAKKALEANGSSEKALSDLITTLKKN;(SEQ ID NO.2)

[0011] The third aspect of the present invention provides a recombinant plasmid containing the above-mentioned Coleus corydalis carbonyl transferase RsCGT1 gene.

[0012] Preferably, the recombinant plasmid is obtained by homologous recombination of the above-mentioned Coleus corymbifera carbonyl transferase RsCGT1 gene with the pET28a vector and is named pET28a-RsCGT1.

[0013] The fourth aspect of the present invention provides a transgenic engineering bacterium, which contains the recombinant plasmid, or the genome of the genetically engineered bacterium is integrated with the exogenous carbonyl transferase RsCGT1 gene of Coleus corymbifera.

[0014] Preferably, the genetically modified bacteria is Escherichia coli BL21 (DE3) strain.

[0015] The fifth aspect of the present invention provides an application of a C. corymbifera carbonyl transferase RsCGT1 gene in the preparation of bergenin.

[0016] Preferably, 4-methoxygallic acid and glycosyl donor uridine diphosphate glucose (UDP-glucose) are used as raw materials, and a glycosylation reaction is carried out at the C-2 position of 4-methoxygallic acid under the catalysis of the southwestern Coleus carbonyl transfer encoded by the above-mentioned Coleus carbonyl transferase RsCGT1 gene to produce bergenin.

[0017] The present invention obtains the target protein after in vitro expression through the recombinant plasmid, and directly generates bergenin by further catalyzing the substrate 4-methoxygallic acid.

[0018] The southwestern Coleus affine carbonyl transferase RsCGT1 gene of the present invention is obtained by extracting RNA from the rhizome of the southwestern Coleus affine plant using an RNA reagent, and then reversely transcribing it into cDNA and performing PCR amplification. The amplification primers of the southwestern Coleus affine carbonyl transferase RsCGT1 gene are as follows:

[0019] 5'F: ATGGGCAGCAGCCATCA; (SEQ ID NO.3)

[0020] 3'R: CTAATTTTTCTTCAACGTAGTAATTAAATCAGATAAAGC; (SEQ ID NO.4)

[0021] In addition, when homologous recombination is performed with the vector pET28a, the RsCGT1 gene needs to be amplified and recovered using primers with homologous walls. The primers with homologous walls are as follows:

[0022] 5'F: ggtggacagcaaatgggtcgcggatccATGGGCAGCAGCCATCA; (SEQ ID NO.5)

[0023] 3'R: gagctcgaattcggatccCTAATTTTTCTTCAACGTAGTAATTAAATCAGATAAAGC; (SEQ IDNO.6)

[0024] The glycosyltransferase RsCGT1 gene isolated and identified from the southwestern pollen lamprey can be used as an important marker gene for molecular assisted breeding of the southwestern pollen lamprey, and can also be used as an important candidate gene for the production of bergenin in the construction of yeast chassis cells.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention provides a gene for the carbon glycosyltransferase RsCGT1 of Coleus fasciatus, which can be used as a biosynthesis regulatory gene of bergenin and can be used in the preparation of bergenin.

[0027] (2) With the rapid development of bioinformatics technology, the mining of key enzyme genes in the biosynthetic pathway of bergenin has been greatly promoted. The biosynthetic regulatory gene of bergenin in the present invention, namely the southwestern Coleus carbonyl transferase RsCGT1 gene, was identified and successfully verified for the first time, opening up a new biosynthetic method for producing bergenin. The present invention obtains the target product by enzyme catalysis in vitro through heterologous expression of proteins in Escherichia coli, adopts in vitro biosynthesis, and carries out directional production, which has the advantages of less by-products, etc.

[0028] (3) The present invention also provides a recombinant plasmid, a genetically engineered bacterium and a recombinant protein containing the carbon glycosyltransferase RsCGT1 gene, which lays a foundation for synthesizing a large amount of bergenin by bioengineering methods and further for constructing a cell factory producing bergenin.

[0029] (4) The in vitro biosynthesis of bergenin is highly controllable, which can reduce the demand for raw material planting, produce a single product, facilitate the separation and purification of bergenin in the later stage, and reduce the difficulties of chemical synthesis and the complexity of the synthesis path. The RsCGT1 gene of the southwestern Corydalis scabra is a key gene for the biosynthesis of bergenin, and can also be used for the breeding research of plants rich in bergenin such as Ardisia japonica. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the derived synthetic route of bergenin.

[0031] Figure 2 Schematic diagram of the construction of the recombinant expression plasmid Pet28a-RsCGT1.

[0032] Figure 3 The electrophoresis test results of the recombinant gene of C. corymbifera carbonyl transferase RsCGT1 are shown in Figure 1. M is a nucleic acid marker, and 1 and 2 are the positive single colony test results.

[0033] Figure 4 This is the SDS-PAGE protein electrophoresis detection diagram of the carbonyl transferase RsCGT1 of Coleus fasciatus. Among them, M is the protein molecular weight standard; lanes 1, 2, 3, and 4 are the flow-through eluent, precipitation eluent, 20mmol / L imidazole eluent, and 50mmol / L imidazole eluent, respectively; 5-11 are all proteins in 250mmol / L imidazole eluent.

[0034] Figure 5HPLC was used to detect the glycosylation of 4-methoxygallic acid by RsCGT1, a carbon glycosyltransferase of Coleus corymbosa. The horizontal axis is time, in min, and the vertical axis is the response value, in mAU; among them, CK: the enzyme activity reaction result of the control group (4-methoxygallic acid + UDP-glucose + inactivated RsCGT1, a carbon glycosyltransferase of Coleus corymbosa); standard product: 4-methoxygallic acid standard product + bergenin standard product; RsCGT1: the enzyme activity reaction result of the experimental group (4-methoxygallic acid + UDP-glucose + RsCGT1, a carbon glycosyltransferase of Coleus corymbosa).

[0035] Figure 6 It is the mass spectrometry analysis (LC / MS / MS) spectrum of the standard, wherein A is the retention time of the standard 4-methoxygallic acid, which is 19.46 minutes; B is the retention time of the standard bergenin, which is 20.40 minutes.

[0036] Figure 7 The mass spectrometry analysis (LC / MS / MS) spectrum of the reaction product for enzyme activity verification, wherein A is the retention time of the substrate 4-methoxygallic acid, 19.40 minutes; and B is the retention time of the reaction product bergenin, 20.39 minutes.

[0037] Figure 8 This is the fragment ion pattern of the standard substance bergenin (theoretical molecular weight 328) (LC / MS / MS).

[0038] Fig. 9 The fragment ion pattern of the reaction product bergenin (theoretical molecular weight 328) (LC / MS / MS). DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with embodiments.

[0040] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product specifications are used. If the manufacturer of the materials or equipment used is not specified, they are all conventional products that can be purchased.

[0041] Example 1

[0042] After a series of work including preparation of cDNA of P. sylvatica, amplification and recovery of candidate genes, homologous recombination, protein expression, in vitro enzyme activity reaction, and HPLC and LC / MS detection, the target candidate carbonyl glycosyltransferase RsCGT1 gene ( Figure 1The operation steps of each stage of the synthesis of bergenin are as follows (the reagents, raw materials, instruments and equipment used in the following implementation are all commercially available):

[0043] (1) Preparation of cDNA template

[0044] Fresh samples of Psoralea corylifolia were taken, sliced, and then quickly frozen in liquid nitrogen for RNA extraction. Total RNA was extracted using the HiPure Plant RNA Mini Kit from Magen (Guangzhou Meiji Biotechnology Co., Ltd.). RNA was extracted according to the procedure of the kit. After passing the test, the RNA was reverse transcribed into cDNA using the TAKARA reverse transcription kit and stored at -20°C for later use.

[0045] (2) Gene amplification and recovery

[0046] Primers for amplifying the RsCGT1 gene of the southwestern Coleus syltransferase were designed using primer design software (CE Design) v1.04. The primers consisted of primer F (SEQ ID NO.3) and primer R (SEQ ID NO.4). The KOD high-fidelity enzyme was used according to the instructions for use of the high-fidelity KOD enzyme, and the gene was amplified using the Bergenia cDNA as a template. The PCR reaction program was: 94°C, 5min; 94°C, 30S, 58°C, 90S, 72°C, 1min, 35 cycles; 72°C, 7min. After the PCR was completed, the gel was run, and the target band was recovered after confirming that the amplification was successful. The EasyPure Quick Gel Extraction Kit of Beijing Quanshijin Biotechnology Co., Ltd. was used for gene gel extraction and recovery to recover the target gene. After recovery, the recovery concentration was measured on a NanoReady ultra-micro UV-visible spectrophotometer, and finally stored in a -20°C refrigerator for later use, to obtain a gene fragment of the southwestern Coleus scabra carbonyl transferase RsCGT1. After sequencing, its nucleic acid sequence was shown in SEQ ID NO.1, and the amino acid sequence of the protein was shown in SEQ ID NO.2.

[0047] 5'F: ATGGGCAGCAGCCATCA; (SEQ ID NO.3)

[0048] 3'R: CTAATTTTTCTTCAACGTAGTAATTAAATCAGATAAAGC; (SEQ ID NO.4)

[0049] In addition, when the southwestern Coleus corydalis carbon glycosyltransferase RsCGT1 gene fragment with vector homology arms is homologously recombined with the vector pET28a (the homology arms are Escherichia coli pET28a), the southwestern Coleus corydalis carbon glycosyltransferase RsCGT1 gene needs to be amplified and recovered using primers with homology walls (i.e., homology arm primers). The homology arm primers are composed of an upstream homology arm primer (shown in SEQ ID NO.5 in the sequence table) and a downstream homology arm primer (shown in SEQ ID NO.6 in the sequence table). Using the southwestern Coleus corydalis carbon glycosyltransferase RsCGT1 gene as a template, the high-fidelity KOD enzyme instruction manual is used to perform PCR amplification again to obtain the southwestern Coleus corydalis carbon glycosyltransferase RsCGT1 gene fragment with vector homology arms.

[0050] Upstream homology arm primer:

[0051] 5'F: ggtggacagcaaatgggtcgcggatccATGGGCAGCAGCCATCA. (SEQ ID NO.5)

[0052] Downstream homology arm primer:

[0053] 3'R:gagctcgaattcggatccCTAATTTTTCTTCAACGTAGTAATTAAATCAGATAAAGC. (shown in SEQ IDNO.6).

[0054] The lowercase letters in the above upstream homology arm primer (SEQ ID NO.5) and downstream homology arm primer (shown in SEQ ID NO.6) represent the pET28a homology arms, and the uppercase letters represent the primer sequences for amplifying the southwestern Coleus carbonyl transferase RsCGT1 gene.

[0055] (3) Construction and identification of gene recombination vectors

[0056] A schematic diagram of homologous recombination is shown in Figure 2 . First, the vector pET28a was linearized, and the linearized vector was obtained by single enzyme digestion with BamH I enzyme. During homologous recombination, the assembly was carried out according to the operating instructions of the homologous recombination enzyme, and then the concentrations of the pET28a homologous arms inserted with the vector homologous arms, the capital letters represent the amplified southwestern Coleus carbonyltransferase RsCGT1 gene fragment and the pET28a vector, and the amount of each component was calculated according to the recombination instructions; finally, each component was added to the PCR reaction tube on ice, and the pET28a homologous arms, the capital letters represent the amplified southwestern Coleus carbonyltransferase RsCGT1 gene, and the pET28a vector were homologously recombined to obtain the recombinant plasmid, named pET28a-AcCGT64. After assembly, the results were tested and sent to the company for sequencing. The electrophoresis test results after assembly are shown in Figure 3 , indicating that the assembly is successful. Reassemble the operation according to the following process:

[0057] Table 1 Candidate gene recombination reaction system

[0058]

[0059] Among them, X = (0.02×pET28a base pair number) ng / linearized pET28a concentration ng / μL; Y = (0.02×pET28a base pair number) ng / Southwestern Coleus carbonyl transferase RsCGT1 recovery concentration ng / μL, and the inserted gene fragment is the Southwestern Coleus carbonyl transferase RsCGT1 gene fragment inserted with the vector homology arm.

[0060] (4) SDS-PAGE protein electrophoresis detection

[0061] After a small protein expression test, the protein induction conditions of RsCGT1 were determined to be: 18°C, 0.1mM IPTG, 220r / min, induction for 12h; then shake vigorously, collect bacteria, break the cell wall, obtain protein supernatant after high-speed centrifugation (12000r / min), and then use SDS-PAGE protein electrophoresis and detection. The test results are shown in Figure 4 , Figure 4 It shows that RsCGT1 protein can be eluted and purified in 250mmol / L imidazole elution buffer.

[0062] (5) Enzyme activity reaction

[0063] The enzyme activity of the RsCGT1 gene of the southwestern Coleus was determined by the glycosylation reaction to synthesize bergenin in a 1.5 mL centrifuge tube. The mixture in the experimental sample reaction system contained: 2 microliters of 100mM uridine diphosphate glucose, 2 microliters of 100mM 4-methoxygallic acid, 40μg of purified southwestern Coleus carbonyl transferase RsCGT1 protein, and 50mM Tris-HCl buffer (pH 8.0) was added to a total volume of 100μL, and the total volume of the reaction system was 100μL. After incubation at 32°C for 2 hours, an equal volume of 1M hydrochloric acid was terminated, and the supernatant was taken after brief centrifugation (12000r / min). Finally, the reaction product was detected by HPLC and LC-MS / MS analysis.

[0064] The reaction system of the control group (CK) was as follows: 2 μL of 100 mM 4-methoxygallic acid, 2 μL of 100 mM UDP-glucose, 40 μg of inactivated purified C. corydalis carbonyl transferase RsCGT1 protein, and 50 mM Tris-HCl buffer (pH 8.0) was added to a total volume of 100 μL, and the total volume of the reaction system was 100 μL.

[0065] Standard products: 50 μl of 10 mM 4-methoxygallic acid, 50 μl of 10 mM bergenin.

[0066] (6) Product testing

[0067] HPLC detection conditions are as follows:

[0068] The instrument used for HPLC detection is Agilent 1290 ultra-high performance liquid chromatograph. The chromatographic column is XBridge ShieldRP18 (4.6mm×250mm, 5μm), column temperature: 30°C; the mobile phase for the determination of bergenin is 0.01% v / v formic acid aqueous solution (A)-acetonitrile (B), gradient elution: 0-8min, 1%-5% B; 8-13min, 5%-10% B; 13-20min, 10%-20% B; 20-25min, 20%-45% B; 25-35min, 45%-90% B; 35-38min, 90%-90% B; 38-45min, 90%-100% B; elution time: 45min; injection volume: 10 microliters; flow rate: 0.6ml / min; detection wavelength 270nm. The test results are shown in Figure 5 , indicating that the experimental samples produced bergenin under the catalysis of the southwestern Codonopsis pilosula carbonyl transferase RsCGT1.

[0069] LC-MS / MS detection conditions are as follows:

[0070] In order to further confirm the reaction products detected by HPLC, Agilent 1290UPLC / 6540Q-TOF liquid chromatography-mass spectrometry (LC / MS / MS) was used for detection: Mass spectrometry conditions: the ion source used was negative ion mode, voltage: 3500 V; fragmentation voltage: 135 V; cone voltage: 60 V; radio frequency voltage: 750 V, scanning range: 100-1000 m / z, scanning mode: SRM. Chromatographic conditions: the chromatographic column is XBridge Shield RP18 (4.6mm, × 250mm, 5μm), the column temperature is 32°C, the mobile phase for determining bergenin is 0.01% v / v formic acid aqueous solution (A)-acetonitrile (B), gradient elution: 0-8min, 1%-5% B; 8-13min, 5%-10% B; 13-20min, 10%-20% B; 20-25min, 20%-45% B; 25-35min, 45%-90% B; 35-38min, 90%-90% B; 38-45min, 90%-100% B; elution time: 45min; injection volume: 10 μL; flow rate: 0.6ml / min; detection wavelength is 270nm, and the detector is a diode array detector.

[0071] Test results are shown in Figure 6 to Figure 9 From the results, it can be seen that the peak time and characteristic fragmentation ions of the product are consistent with those of the standard substance bergenin, confirming that the reaction product is bergenin. Finally, it was concluded that the RsCGT1 gene of the southwestern Coleus syltransferase has the ability to catalyze the glycosylation of 4-oxymethyl gallic acid to produce bergenin.

[0072] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A gene for carbonyl transferase RsCGT1 of Coleus chinensis, characterized in that: Coleus chinensis carbonyl transferase RsCGT1 The nucleic acid sequence of the gene is shown in SEQ ID NO.

1.

2. The protein encoded by the gene of Coleus corymbiferum carbonyl transferase RsCGT1 according to claim 1, characterized in that: The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. A recombinant plasmid containing the C. corydalis carbonyl transferase RsCGT1 gene according to claim 1.

4. The recombinant plasmid containing the C. corydalis carbonyl transferase RsCGT1 gene according to claim 3, characterized in that: The RsCGT1 gene of Coleus corymbifera was homologously recombined with the pET28a vector to obtain the pET28a-RsCGT1 recombinant plasmid.

5. A genetically modified bacteria, characterized in that: The recombinant plasmid of claim 3 or 4, or the genome of the genetically engineered bacteria is integrated with the exogenous C. coleus carbonyl transferase RsCGT1 gene of claim 1.

6. The genetically modified bacteria according to claim 5, characterized in that: The transgenic engineering bacteria are Escherichia coli BL21 (DE3) strains.

7. Use of the Corydalis scabra carbonyl transferase RsCGT1 gene described in claim 1 in the preparation of bergenin.

8. The use of the C. corydalis carbonyl transferase RsCGT1 gene in the preparation of bergenin according to claim 7, characterized in that: Using 4-methoxygallic acid as a substrate and uridine diphosphate glucose (UDP-glucose) as a glycosyl donor, a glycosylation reaction is carried out at the C-2 position of 4-methoxygallic acid under the catalysis of the southwestern Coleus edulis carbonyl glycosyltransferase encoded by the above-mentioned Coleus edulis carbonyl glycosyltransferase RsCGT1 gene to produce bergenin.

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