Demethylase ODM gene for synthesizing benzylisoquinoline alkaloid and application of demethylase ODM gene

Abstract

The invention discloses a demethylase ODM gene for synthesizing benzylisoquinoline alkaloids and application thereof, the demethylase ODM gene is an ODM1 gene or an ODM2 gene, the nucleotide sequence of the CDS region of the ODM1 gene is shown as SEQ ID NO: 1, and the nucleotide sequence of the CDS region of the ODM2 gene is shown as SEQ ID NO: 2. Aiming at the current situation that the research basis of a key gene for synthesizing corydalmine in rhizoma corydalis is weak, the ODM1 gene for catalyzing tetrahydropalmatine demethylation to specifically generate corydalmine and tetrahydrojatrorrhizine and the ODM2 gene for specifically generating tetrahydrojatrorrhizine are cloned from rhizoma corydalis for the first time. An important theoretical basis is provided for producing the corydalmine by utilizing a synthetic biological method in the future, and the corydalmine mutant has a wide application prospect and a great economic value.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a demethylase ODM gene for synthesizing benzylisoquinoline alkaloids and an application thereof. Background Art

[0002] Yanhusuo is the dried tuber of the poppy family plant Yanhusuo. It tastes bitter, pungent and warm, and enters the liver, spleen and lung meridians. It has the effects of promoting blood circulation, promoting qi and relieving pain. In clinical practice, Yanhusuo is mainly used for chest and flank pain, abdominal pain, chest pain, heart pain, amenorrhea, dysmenorrhea, postpartum blood stasis, swelling and pain caused by falls, etc. Modern pharmacological studies have shown that Yanhusuo has analgesic, anti-anxiety, sedative and hypnotic effects, anti-myocardial ischemia, cerebral ischemia, anti-gastrointestinal ulcer effects, anti-tumor, and drug addiction treatment effects.

[0003] The main chemical components of Corydalis yanhusuo are alkaloids, followed by anthraquinones, organic acids, amino acids, inorganic acids and other compounds. At present, a variety of active alkaloid components have been isolated from Corydalis yanhusuo, mainly benzylisoquinoline alkaloids, including protoberberine alkaloids, aporphine alkaloids, opioid alkaloids, etc., which are potential drugs for the treatment of various diseases. According to phytochemical analysis, a benzylisoquinoline alkaloid corydaline was found in the tubers of Corydalis yanhusuo, which can relieve neuropathic pain by inhibiting the ASK1-p38 MAPK / NF-κB pathway, upregulating the Nrf2 / HO-1 / CO signaling pathway or inhibiting the nf-κb-dependent CXCL1 / CXCR2 signaling pathway, and can effectively treat drug addiction and has good biological activity. However, the key factor that has not been able to become a drug is that it exists in trace amounts in nature.

[0004] In recent years, the rise of molecular biology and metabolic engineering has brought new ideas to people. The use of metabolic engineering methods to produce medicinal ingredients at the level of organisms or organelles has the advantages of less time consumption, higher yields, and lower costs than chemical synthesis. Therefore, the use of molecular biology and metabolic engineering technology to produce active ingredients has broad application prospects.

[0005] The application of metabolic engineering is limited by the lack of key enzymes and coding genes that control the biosynthesis of active ingredients. Structurally, corydaline is C-10 monohydroxytetrahydrobamatine, that is, corydaline can be obtained by removing the methyl group of the methoxy group at position 10 of tetrahydrobamatine to form a hydroxyl group. Tetrahydrobamatine is also a type of benzylisoquinoline alkaloid that exists in large quantities in Corydalis yanhusuo. It is cheap and easy to obtain. If metabolic engineering can be used to convert tetrahydrobamatine into trace amounts of corydaline in microorganisms, it will provide a basis for the subsequent development of corydaline medicine.

[0006] It has been found that a cytochrome P450 produced by Streptomyces griseus can selectively demethylate the hydroxyl group on the D-ring of tetrahydropalmatine to produce corydaline. However, due to its low conversion efficiency, long reaction time, and huge differences between enzymes in bacteria and plants, it is necessary to find this specific ODM enzyme in Corydalis yanhusuo to provide a basis for the production of benzylisoquinoline alkaloids using synthetic biology methods. Summary of the invention

[0007] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a demethylase ODM gene for synthesizing benzylisoquinoline alkaloids and its application.

[0008] Technical solution: To achieve the above-mentioned invention object, the present invention adopts the following technical solution:

[0009] In the first aspect, the present invention provides a demethylase ODM gene for synthesizing benzylisoquinoline alkaloids, wherein the demethylase ODM gene is an ODM1 gene or an ODM2 gene, the nucleotide sequence of the CDS region of the ODM1 gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the CDS region of the ODM2 gene is shown in SEQ ID NO: 2.

[0010] The invention uses bioinformatics technology to analyze the results of genome sequencing and transcriptome sequencing of Corydalis yanhusuo, and excavates an ODM gene ODM1 that specifically generates corydaline and tetrahydrojatrorrhizine and an ODM gene ODM2 that specifically generates tetrahydrojatrorrhizine. PCR amplification is performed on the cDNA of Corydalis yanhusuo tuber samples, and the CDS sequence (full length 1074bp) of the functional gene ODM1 that catalyzes the demethylation of tetrahydropalmatine to generate corydaline and tetrahydrojatrorrhizine and the CDS sequence (full length 1080bp) of the functional gene ODM2 that catalyzes the demethylation of tetrahydropalmatine to generate tetrahydrojatrorrhizine in Corydalis yanhusuo are obtained.

[0011] In a second aspect, the present invention provides a protein encoded by the demethylase ODM gene, wherein the amino acid sequence of the protein encoded by the ODM1 gene is shown in SEQ ID NO:3; and the amino acid sequence of the protein encoded by the ODM2 gene is shown in SEQ ID NO:4. The amino acid sequence of the protein also includes an amino acid sequence with equivalent functions formed by replacing, deleting or adding one or more amino acids in the above sequence. The proteins are demethylases ODM1 and ODM2.

[0012] In a third aspect, the present invention provides a recombinant expression vector, which contains the demethylase ODM gene.

[0013] As a specific embodiment, the expression vector is pET28a.

[0014] Preferably, the recombinant expression vector is pET28a-ODM1 or pET28a-ODM2.

[0015] In a fourth aspect, the present invention provides a recombinant expression engineered bacterium, wherein the recombinant expression engineered bacterium expresses the demethylase ODM gene, or contains the recombinant expression vector.

[0016] As a specific implementation scheme, the recombinant expression engineered bacteria is the Escherichia coli strain BL21 (DE3).

[0017] In addition, the present invention provides not only the recombinant expression vector, the recombinant expression engineering bacteria and the demethylases ODM1 and ODM2, but also the expression cassettes of ODM1 and ODM2 including the demethylase genes.

[0018] In a fifth aspect, the present invention provides the use of the demethylase ODM gene, the protein, the recombinant expression vector, and the recombinant expression engineering bacteria in the synthesis of benzylisoquinoline alkaloids.

[0019] As a specific embodiment, the benzylisoquinoline alkaloids are corydaline and / or tetrahydrojatrorrhizine, the ODM1 gene is used to catalyze the demethylation of tetrahydrobamatine to produce corydaline and tetrahydrojatrorrhizine, and the ODM2 gene catalyzes the demethylation of tetrahydrobamatine to produce tetrahydrojatrorrhizine.

[0020] In a sixth aspect, the present invention provides a method for synthesizing benzylisoquinoline alkaloids, the method comprising utilizing the demethylase ODM gene to synthesize the benzylisoquinoline alkaloids by a genetic engineering method.

[0021] As a specific embodiment, the benzylisoquinoline alkaloids are corydaline and / or tetrahydrojatrorrhizine, the ODM1 gene is used to catalyze the demethylation of tetrahydrobamatine to produce corydaline and tetrahydrojatrorrhizine, and the ODM2 gene catalyzes the demethylation of tetrahydrobamatine to produce tetrahydrojatrorrhizine.

[0022] As a specific implementation scheme, the method comprises constructing a recombinant expression vector using the demethylase ODM gene, then introducing the recombinant expression vector into an engineered bacterium to obtain a recombinant expression engineered bacterium, fermenting, purifying to obtain a purified protein (demethylase ODM1 or ODM2), and finally using tetrahydrobamipine as a raw material and the purified protein as a catalyst to carry out an enzymatic reaction to obtain the benzylisoquinoline alkaloid.

[0023] More specifically, the recombinant expression engineered bacteria is a recombinant Escherichia coli, which is inoculated into a fermentation medium and cultured at 35-37°C until OD 600When the value is 0.6-0.8, add inducing agent isopropyl-β-D-thiogalactoside and ferment for 18 hours at 18°C. The temperature of the enzymatic reaction is 20-30°C, the time is 0.5-2.5 hours, and the reaction medium is Tris-HCl buffer or phosphate buffer.

[0024] In one embodiment, the final concentration of the inducing agent isopropyl-β-D-thiogalactoside is 0.3 mM;

[0025] In one embodiment, the fermentation medium includes but is not limited to LB or TB medium.

[0026] In one embodiment, when the ODM1 gene is used to catalyze the demethylation of tetrahydropalmatine to produce corydaline and tetrahydrojatrorrhizine, the optimal enzymatic reaction conditions are: using a Tris-HCl buffer with a pH of 8.0 and reacting at 20°C for 120 minutes. When the ODM2 gene is used to catalyze the demethylation of tetrahydropalmatine to produce tetrahydrojatrorrhizine, the optimal enzymatic reaction conditions are: using a phosphate buffer with a pH of 6.5 and reacting at 30°C for 50 minutes.

[0027] In a seventh aspect, the present invention provides the use of the demethylase ODM gene, the recombinant expression vector, and the recombinant expression engineering bacteria in increasing the content of corydaline and / or tetrahydrojatrorrhizine in plants.

[0028] Beneficial effects: In view of the weak research foundation of key genes for synthesizing corydaline in Corydalis yanhusuo, the present invention cloned the ODM1 gene that catalyzes the demethylation of tetrahydropalmatine to specifically generate corydaline and tetrahydrojatrorrhizine and the ODM2 gene that specifically generates tetrahydrojatrorrhizine from Corydalis yanhusuo for the first time. This provides an important theoretical basis for the future production of corydaline using synthetic biology methods, and has broad application prospects and great economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the demethylation reaction of tetrahydrobamatin catalyzed by ODM1 protein in Example 2 to synthesize corydaline and tetrahydrojatrorrhizine;

[0030] Figure 2 is a schematic diagram of the synthesis of tetrahydrojatrorrhizine by the demethylation reaction of tetrahydrobamipine catalyzed by the ODM2 protein in Example 2;

[0031] Figure 3 The mass spectra of the ODM1 protease catalysis products detected by LC-MS in Example 2, wherein (A) is the MS1 and MS2 analysis of the standard substance Corydalis. (B) is the MS1 and MS2 analysis of the standard substance Tetrahydrojatrorrhizine. (CD) Mass spectrometry detection diagram of ODM1 protein catalysis samples;

[0032] Figure 4 The mass spectra of the ODM2 protease catalytic products detected by LC-MS in Example 2, wherein (A) MS1 and MS2 analysis of the standard tetrahydrojatrorrhizine. (B) Mass spectrometry detection of the ODM2 protein catalytic sample;

[0033] Figure 5 The enzymatic investigation of ODM1 and ODM2 in Example 2 was used to improve the catalytic efficiency of demethylase, wherein (A) is the enzymatic investigation result of ODM1, and (B) is the enzymatic investigation result of ODM2. DETAILED DESCRIPTION

[0034] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001, or the conditions recommended by the manufacturer's instructions.

[0035] Example 1 ODM1 and ODM2 gene cloning

[0036] According to the analysis of the previous genome sequencing and transcriptome sequencing results of Corydalis yanhusuo, two pairs of specific primers were designed:

[0037] ODM1-F:5'-ATGGGTCGCGGATCCGAATTCGGGCCCATGAAGAATGAGATCAACC-3'ODM1-R:5'-GGTGGTGGTGGTGCTCGAGCTAATCCAATTTCATGAATTCC-3'

[0038] ODM2-F:5'-ATGGGTCGCGGATCCGAATTCGGGCCCATGAACAATGCGATTATCAATAG-3'

[0039] ODM2-R:5'-GTGGTGGTGGTGGTGCTCGAGTTAATCCAATTTCATGAATTCCAAT-3'

[0040] Total RNA was extracted from tubers of Corydalis yanhusuo using a total RNA extraction kit, and reverse transcribed to synthesize cDNA. The CDS sequences of the ODM1 gene (full length 1074 bp) and ODM2 (full length 1080 bp) genes of tetrahydropalmatine demethylation in Corydalis yanhusuo as shown in SEQ ID NO: 1 and SEQ ID NO: 2 were amplified from the cDNA obtained by reverse transcription using the above primers.

[0041] (1) Grind fresh Corydalis tubers with liquid nitrogen cooling, accurately weigh 100 mg of powder and add it to a 2 mL centrifuge tube pre-cooled with liquid nitrogen, and add the EASYspin Universal Plant RNA Kit total RNA kit lysis buffer, vortex mix and let stand for 10 minutes;

[0042] (2) Extracting total RNA from Corydalis tubers according to the instructions of the EASYspin Universal Plant RNA Kit;

[0043] (3) Total RNA extracted from Corydalis tubers was used as a template and reverse transcriptase kit PrimeScript was used to TM It was reverse transcribed and synthesized into the first strand of cDNA using RTMaster Mix (purchased from Takara Biotech Dalian Co., Ltd.), and the reaction conditions were carried out according to the instructions of the kit;

[0044] (4) Using the above primers ODM1-F and ODM1-R, ODM2-F and ODM2-R, the CDS sequences of the functional gene ODM1 that catalyzes the demethylation of tetrahydropalmatine to produce corydaline and tetrahydrojatrorrhizine and the functional gene ODM2 that produces tetrahydrojatrorrhizine in Corydalis yanhusuo were amplified from the cDNA obtained by reverse transcription of RNA.

[0045] (5) Reaction conditions: 95℃ pre-denaturation for 3min; 95℃ for 15sec, 56℃ for 90sec, 72℃ for 1.5min, 30 cycles; 72℃ extension for 15min. The reaction product was purified using a DNA purification kit from Vazyme. The purified PCR product was inserted between the EcoRI and XhoI restriction sites of the expression vector pET28a using a ClonExpress MultiS one-step cloning kit (purchased from Vazyme, Nanjing, China), transformed into Escherichia coli DH5α competent cells, screened positive clones and sequenced to obtain the required full-length gene. Plasmids carrying the ODM1 and ODM2 gene sequences were extracted from the positive clones and named pET28a-ODM1 and pET28a-ODM2 plasmids.

[0046] Example 2 Verification of recombinant expression of ODM1 and ODM2 genes in Escherichia coli

[0047] Materials and Methods

[0048] LB medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, sterilized at 121°C for 15 min.

[0049] TB medium: 12 g / L peptone, 5 g / L glycerol, 24 g / L yeast extract, 17 mM potassium dihydrogen phosphate, 72 mM potassium dihydrogen phosphate, sterilized at 121°C for 15 min.

[0050] Fermentation conditions:

[0051] (1) The pET28a-ODM1 and pET28a-ODM2 plasmids obtained in Example 1 were transformed into Escherichia coli BL21 (DE3) competent cells, positive clones were screened and subcultured in LB liquid medium containing Kana antibiotics, and the recombinant Escherichia coli was cultured at 35-37° C. until the OD600 value was 0.6-0.8, and isopropyl-β-D-thiogalactoside was added and continued to be cultured at 16-20° C. for 12-20 h. The concentration of isopropyl-β-D-thiogalactoside was 0.3 mM.

[0052] (2) The fermentation broth obtained in step (1) was centrifuged at 4°C and 8000 rpm for 10-20 min to collect the bacterial cells, and the bacterial cells were washed with Tris-HCl buffer at pH 8.0. After washing, the cells were used for crude enzyme solution reaction or whole cell catalysis.

[0053] Enzymatic reaction:

[0054] (3) The washed bacteria in step (2) were resuspended in 20 mM imidazole so that the OD value of the different recombinant bacterial suspensions was 15, and the crude enzyme solution was obtained by ultrasonic disruption. The suspension was centrifuged at 14000 rpm at 4°C for 30-60 min, and the supernatant was purified by a nickel column after passing through a membrane. The detailed steps are described in the instructions.

[0055] (4) The purified protein obtained in step (3) was used for in vitro functional verification. The experimental steps were as follows: reaction volume 200 μL, 400 μM tetrahydropalmatine, 500 μM α-ketoglutarate, 10 mM ascorbic acid, 2.5 mM Fe 2+ , 36 μg of ODM purified protein, 100 mM Tris-HCl Buffer (PH8.5) to make up to 200 μL; negative control: 400 μM tetrahydropalmatine, 500 μM α-ketoglutaric acid, 10 mM ascorbic acid, 2.5 mM Fe 2+ , ODM purified protein was inactivated by boiling. The reaction was carried out at 30°C for 2 hours. After the reaction was completed, 1 / 2 volume of acetonitrile was added for mixing. The reaction mixture was centrifuged at 14000 rpm for 10-20 minutes, and the supernatant was passed through a membrane for high performance liquid chromatography analysis.

[0056] The ODM1 protein catalyzed reaction is described in detail in Figure 1 , ODM2 protein catalyzed reactions are described in Figure 2 .

[0057] HPLC analysis:

[0058] (5) Mobile phase: Phase A is ultrapure water containing 0.1% formic acid, and phase B is acetonitrile

[0059] (6) Chromatographic column: Shimadzu LC-2010C AT high performance liquid chromatograph, Hedera ODS-2C18 chromatographic column (4.0 mm×250 mm, 5 μm), mobile phase acetonitrile: 0.1% formic acid aqueous solution, gradient elution, elution flow rate 1 mL / min, injection volume 20 μL, column temperature 25°C; elution program: 0 min:10:90 (v / v), 5 min:30:70 (v / v), 10 min:95:5 (v / v), 14 min:45:55 (v / v), 12 min:50:50 (v / v). Use ultraviolet detector, detection wavelength 280 nm.

[0060] (7) Mass spectrometry parameters: Agilent Poroshell 120SB-Aq (3.0*150mm, 2.7μm) was used. Acetonitrile: 0.1% formic acid water was used as the mobile phase. The linear elution conditions were 0min:10:90 (v / v), 5min:30:70 (v / v), 10min:95:5 (v / v), 14min:45:55 (v / v), 12min:50:50 (v / v). The ESI source conditions were: dry gas (N2) flow rate 8.0L / min; collision energy, 35eV; spray voltage 3.5kV; capillary temperature: 320℃; auxiliary gas heater temperature, 300℃; sheath gas flow rate: 35arb; auxiliary gas flow rate: 15arb; sweep gas flow rate: 5arb; auxiliary gas, sheath gas, and sweep gas were all high-purity nitrogen. All operations and data analysis were performed in positive ion mode.

[0061] The results of ODM1 protein function verification were obtained by LC-MS detection. Figure 3 , Figure A is the mass spectrum of the standard substance Corydalis, with a molecular weight of 343.1757[H+1] + Figure B is the mass spectrum of the standard tetrahydrojatrorrhizine, with a molecular weight of 342.1698. Figure C is the mass spectrum of the sample of ODM1 protein catalyzing tetrahydrobamipine to produce corydaline, and the catalytic product with a molecular weight of 342.1703 can be detected. Figure D is the mass spectrum of the sample of ODM1 protein catalyzing tetrahydrobamipine to produce tetrahydrojatrorrhizine, and the catalytic product with a molecular weight of 342.1704 can be detected. The functional verification results of ODM2 protein were obtained by LC-MS detection, and the results are detailed in Figure 4, where Figure A is the mass spectrum of the standard tetrahydrochalcone, with a molecular weight of 342.1698, and Figure B is the mass spectrum of the ODM2 protein catalytic sample, in which the catalytic product with a molecular weight of 342.1798 can be detected;

[0062] (8) The above enzymatic reaction conditions were optimized, and the effects of different reaction pH (100mM phosphate buffer at pH 6.0-6.5, 100mM Tris-HCl buffer at pH 7.0-8.5, and 100mM Na2CO3-NaHCO3 buffer at pH 9.0-11.0), temperature (15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C), time (15min, 45min, 60min, 90min, 120min, 150min, 180min, 240min) and different ions (Cu2+, Mg2+, Ag+, Ca2+, Zn2+, Mn2+, Fe3+, Co2+, Ni+ in the presence or absence of Fe2+) were investigated. Sampling and HPLC analysis were performed according to the above method. The results are shown in Figure 1. Figure 5 As shown, the optimal reaction conditions for ODM1 are: Tris-HCl buffer with a pH of 8.0, and reaction at 20°C for 120 min. The optimal reaction conditions for ODM2 are: phosphate buffer with a pH of 6.5, and reaction at 30°C for 50 min.

[0063] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A demethylase ODM gene for synthesizing benzylisoquinoline alkaloids, characterized in that: The demethylase ODM gene is an ODM1 gene or an ODM2 gene. The nucleotide sequence of the CDS region of the ODM1 gene is shown in SEQ ID NO:1, and the nucleotide sequence of the CDS region of the ODM2 gene is shown in SEQ ID NO:

2.

2. The protein encoded by the demethylase ODM gene according to claim 1, characterized in that The amino acid sequence of the protein encoded by the ODM1 gene is shown in SEQ ID NO:3; the amino acid sequence of the protein encoded by the ODM2 gene is shown in SEQ ID NO:

4.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the demethylase ODM gene according to claim 1.

4. A recombinant expression engineering bacterium, characterized in that: The recombinant expression engineering bacteria expresses the demethylase ODM gene described in claim 1, or contains the recombinant expression vector described in claim 3.

5. Use of the demethylase ODM gene according to claim 1, the protein according to claim 2, the recombinant expression vector according to claim 3, and the recombinant expression engineering bacteria according to claim 4 in the synthesis of benzylisoquinoline alkaloids.

6. The use according to claim 5, characterized in that: The benzylisoquinoline alkaloids are corydaline and / or tetrahydrojatrorrhizine, the ODM1 gene is used to catalyze the demethylation of tetrahydrobamatine to generate corydaline and tetrahydrojatrorrhizine, and the ODM2 gene is used to catalyze the demethylation of tetrahydrobamatine to generate tetrahydrojatrorrhizine.

7. A method for synthesizing benzylisoquinoline alkaloids, characterized in that: The method comprises utilizing the demethylase ODM gene of claim 1 to synthesize the benzylisoquinoline alkaloids by genetic engineering method.

8. The method for synthesizing benzylisoquinoline alkaloids according to claim 7, characterized in that: The benzylisoquinoline alkaloids are corydaline and / or tetrahydrojatrorrhizine, the ODM1 gene is used to catalyze the demethylation of tetrahydrobamatine to generate corydaline and tetrahydrojatrorrhizine, and the ODM2 gene is used to catalyze the demethylation of tetrahydrobamatine to generate tetrahydrojatrorrhizine.

9. The method for synthesizing benzylisoquinoline alkaloids according to claim 7, characterized in that: The method comprises constructing a recombinant expression vector using the demethylase ODM gene according to claim 1, then introducing the recombinant expression vector into an engineering bacterium to obtain a recombinant expression engineering bacterium, fermenting, purifying to obtain a purified protein, and finally using tetrahydrobamipine as a raw material and the purified protein as a catalyst to perform an enzymatic reaction to obtain the benzylisoquinoline alkaloid.

10. Use of the demethylase ODM gene according to claim 1, the recombinant expression vector according to claim 3, and the recombinant expression engineering bacteria according to claim 4 in increasing the content of corydaline and / or tetrahydrojatrorrhizine in plants.

Images