Rhizoma coptidis dioxygenase gene, application, expression vector and host bacterium

Abstract

The invention discloses a coptis dioxygenase gene, an application, an expression vector and a host bacterium, and relates to a catalytic method for carrying out dihydroxylation modification by taking benzylisoquinoline alkaloids (including berberine, epiberberine, coptisine, columbine and palmatine) as substrates. The dioxygenase gene sequence provided by the invention comes from the traditional Chinese medicine coptis chinensis, and is one of key genes on a biological synthesis path of benzylisoquinoline alkaloids in the coptis chinensis. Based on the significant substrate and reaction heterogeneity of the gene, an important theoretical basis is provided for coptis chinensis breeding and industrial production of coptis chinensis medicinal components, and an important reference is provided for modifying complex organic compounds or natural products.

Description

Technical Field

[0001] The invention relates to a coptis chinensis dioxygenase gene, application, expression vector and host bacteria, and belongs to the technical field of genetic engineering and synthetic biology. Background Art

[0002] The medicinal value of Coptis chinensis was first recorded in Shennong's Herbal Classic. The rhizome of Coptis chinensis is a traditional Chinese medicinal material and has been widely used in China for more than 2000 years. Modern pharmacological studies have shown that Coptis chinensis has a wide range of pharmacological activities, including antibacterial, antiviral, hypoglycemic and lipid-lowering, anti-tumor, anti-gastric ulcer, and cardiovascular and cerebrovascular protection. According to studies, benzylisoquinoline alkaloids (BIAs) are the main active ingredients of Coptis chinensis.

[0003] It is known that coptis chinensis oxygen methyltransferase and multiple cytochrome P450 enzymes are involved in the biosynthesis of coptis chinensis, and both types of enzymes have the characteristics of catalyzing substrate promiscuity. However, the biosynthetic pathway of benzylisoquinoline alkaloids has not been fully elucidated, and more functional enzymes have not yet been discovered.

[0004] Therefore, how to further reveal the biosynthetic pathway of benzylisoquinoline alkaloids and find the key genes for modifying benzylisoquinoline alkaloids is an urgent problem to be solved in the field of synthetic biology. Summary of the invention

[0005] In view of this, the present invention provides a Coptis chinensis dioxygenase gene, application, expression vector and host bacteria to solve the above problems existing in the prior art.

[0006] One of the solutions of the present invention is a Coptis chinensis dioxygenase gene, the gene nucleic acid sequence of which is shown in SEQ ID NO.1: It can catalyze the cis-dihydroxylation reaction of various benzylisoquinoline alkaloids and modify the aromatic ring structure of benzylisoquinoline alkaloids.

[0007] Another embodiment of the present invention is the use of the above gene in modifying benzylisoquinoline alkaloid compounds.

[0008] The protein encoded by the gene catalyzes the dihydroxylation reaction of benzylisoquinoline alkaloids. The 1 mL reaction system contains 100 mM Tris-HCl buffer (pH 7.4), 10 mM ascorbic acid, 1 mM α-ketoglutarate, 0.5 mM ferrous sulfate, 14 mM β-mercaptoethanol, 0.5 mM substrate, and 10 μM purified enzyme. The reaction mixture is incubated at 30°C for 4 hours; The protein encoded by the gene catalyzes a variety of benzylisoquinoline alkaloids and has substrate promiscuity. It uses a variety of benzylisoquinoline alkaloids as substrates, including berberine, coptisine, epiberberine, palmatine, and tertium palmatine, to achieve the cis-dihydroxylation reaction of the substrates.

[0009] Another embodiment of the present invention is a recombinant expression vector and a host bacteria containing the above gene.

[0010] The recombinant expression vector uses pET-28a as the vector, and the cloning site of the gene is BamHI / XhoI; The host bacteria is BL21 (DE3) strain, and the recombinant expression vector plasmid is transferred into the host bacteria by heat shock method.

[0011] The beneficial effects of the present invention are: The present invention explores new uses for enzymes, structurally modifies benzylisoquinoline alkaloids through a green biosynthetic approach, and the enzyme has significant substrate and reaction promiscuity, providing an important theoretical basis for subsequent research on the biosynthetic pathway of Coptis chinensis and this type of dioxygenase. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results of SDS-polyacrylamide gel electrophoresis of the CcCODM genetically engineered bacteria of the present invention; Figure 2 It is the product result detected by HPLC of the catalytic reaction in the prokaryotic expression system of CcCODM of the present invention; Figure 3 It is the product result of mass spectrometry detection of the catalytic reaction in the CcCODM prokaryotic expression system of the present invention. DETAILED DESCRIPTION

[0013] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0014] In the following examples, the coptis chinensis dioxygenase gene, the gene nucleic acid sequence is shown in SEQ ID NO.1: Example 1 Construction of prokaryotic expression vector of CcCODM from Coptis chinensis The CcCODM gene sequence was cloned to obtain the pET28a-CcCODM recombinant plasmid, which was transferred into the competent BL21 (DE3) strain by heat shock method. The specific implementation method is as follows: take a tube containing 100 microliters of competent cells from the -80℃ refrigerator, place it in an ice-water mixture for thawing for about 3-5 minutes; take 3 microliters of plasmid and add it to the thawed competent cells, flick the centrifuge tube to mix; then place the centrifuge tube in the ice-water mixture for 10 minutes; immerse 2 / 3 of the above centrifuge tube in a 42℃ water bath, and heat shock for 90 seconds; after the heat shock, place the centrifuge tube in an ice bath for 3 minutes; then add 600 microliters of LB medium, and resuscitate and culture at 37℃ shaker (200rpm) for 45 minutes; after the recovery, culture overnight on a plate to obtain a pET28a-CcCODM single clone.

[0015] Example 2 Inducible expression of recombinant strains The above single clones and the BL21 strain with empty pET28a as a blank control were selected and cultured in LB medium (kanamycin, 50 μg / mL) on a shaker (37°C, 200 rpm) until the OD600 value was about 0.6. Then, 3 mL of bacterial solution was taken from the experimental group and the control group, and cultured on a shaker (37°C, 200 rpm) until the OD600 value was about 0.6, and IPTG solution was added to a final concentration of 1 mM, and placed in a shaker at 28°C and 100 rpm to induce expression overnight.

[0016] Example 3 Purification of recombinant proteins After the bacterial solution for induced protein expression was divided into aliquots, it was centrifuged at 4°C and 5000 rpm for 15 min, the bacteria were collected, and resuspended with 15 mL of binding solution (10 mM Tris-HCl, pH 7.9, 500 mM NaCl, 20 mM imidazole). The resuspended solution was then placed on ice and placed in an ultrasonic cell disruptor for disruption. The program conditions of the disruptor were 30% power, 3s ultrasound, 3s stop, and 30min disruption. The resuspended bacterial solution was then centrifuged at 4°C and 12000 rpm for 15 min. The supernatant and precipitate were collected separately, and samples of the supernatant and precipitate were retained for 12% polyacrylamide gel electrophoresis detection. The supernatant obtained by centrifugation was transferred to Ni +Chromatographic affinity column, bind for 3 hours, wash impurities with 10mL binding solution, elute recombinant protein with 3mL elution solution of 200mM imidazole concentration. The collected eluted recombinant protein was loaded into a dialysis bag with a molecular weight cutoff of 8000-14000, and then the dialysis bag containing the protein was placed in a dialysis solution (10% glycerol, 250mM NaCl, 2 mM Tris-HCl, pH7.4) for 4 hours at 4℃, and then placed in a new dialysis solution (10% glycerol, 150 mM NaCl, 2 mM Tris-HCl, pH7.4) for dialysis at 4℃. The dialyzed protein was collected the next day, which was the purified recombinant protein.

[0017] The above samples were subjected to 12% polyacrylamide gel electrophoresis. The results were as follows: Figure 1 As shown, Figure 1 In the figure, lane 1: protein marker (10-180KDa); lane 2: BL21 (DE3) carrying pET-28a empty plasmid; lane 3: BL21 (DE3) carrying pET-28a recombinant plasmid carrying dioxygenase gene; lane 4: precipitate after ultrasonic disruption of bacterial solution; lane 5: supernatant after ultrasonic disruption of bacterial solution; lane 6: supernatant after passing through nickel column; lane 7: impurity protein after binding solution passed through nickel column; lane 8: target protein after eluent 1 (200mM imidazole concentration) passed through nickel column; lane 9: target protein after eluent 2 (400mM imidazole concentration) passed through nickel column. The electrophoresis results showed that an obvious specific protein expression band appeared between the molecular weight of 43kDa and 55kDa, which was consistent with the theoretical value of about 44.63k Da.

[0018] Example 4 In vitro enzymatic reactions and assays The protein encoded by the above gene uses a variety of benzylisoquinoline alkaloids as substrates, including berberine, coptisine, epiberberine, palmatine, and tertiapine, to achieve cis-dihydroxylation of the substrate. The 1mL reaction system contains 100 mMTris-HCl buffer (pH 7.4), 10 mM ascorbic acid, 1 mM α-ketoglutaric acid, 0.5 mM ferrous sulfate, 14 mMβ-mercaptoethanol, 0.5 mM substrate, and 10 μM purified enzyme. The reaction mixture was incubated at 30°C for 4 hours.

[0019] The reaction solution was filtered through a 0.22 μm membrane and then analyzed using an Agilent 1260 high-performance liquid chromatography system. The chromatographic column used was an Agilent Extend C18 column (size 250 mm × 4.6 mm, particle size 5 μm), and the mobile phase was acetonitrile-water solution containing 0.3% phosphoric acid and 0.2% triethylamine (volume ratio 70:30). The flow rate of the mobile phase was set to 0.7 mL / min, and 10 μL of sample was used for each analysis. The column temperature was controlled at 20 °C, and the detection wavelength was 345 nm.

[0020] The product results of HPLC detection of catalytic reaction in CcCODM prokaryotic expression system are as follows Figure 2 As shown, A is the product result of HPLC detection of berberine catalyzed in the CcCODM prokaryotic expression system; B is the product result of HPLC detection of epiberberine catalyzed in the CcCODM prokaryotic expression system; C is the product result of HPLC detection of coptisine catalyzed in the CcCODM prokaryotic expression system; D is the product result of HPLC detection of bamipine catalyzed in the CcCODM prokaryotic expression system; E is the product result of HPLC detection of fangquercetin catalyzed in the CcCODM prokaryotic expression system; CK is the blank control.

[0021] After the reaction, the solution was filtered through a 0.22 μm membrane and then analyzed by mass spectrometry using an Agilent LC-MS. The mobile phase was acetonitrile-water solution containing 0.1% formic acid (volume ratio 90:10). Independent ESI ion source; mass range: m / z 2-1450; maximum scan rate: 10,400 amu / s; dynamic range: > 6×106; sensitivity: ESI+ mode: 1 pg of reserpine was injected on the LC-MS column, and the ion m / z 609 was selected for monitoring, with a signal-to-noise ratio of ≥40:1; ESI- mode: 1 pg of chloramphenicol was injected on the LC-MS column, and the ion m / z 321 was selected for monitoring, with a signal-to-noise ratio of ≥40:1; positive and negative mode switching time: ≤25 ms; mass axis stability: ±0.1 amu / 12 hours; measurement accuracy: 0.13 amu; minimum SIM dwell time: 1 ms.

[0022] The product results of mass spectrometry detection of catalytic reactions in the CcCODM prokaryotic expression system are as follows: Figure 3As shown, A is the product result of mass spectrometry detection of berberine catalyzed in the CcCODM prokaryotic expression system; B is the product result of mass spectrometry detection of epiberberine catalyzed in the CcCODM prokaryotic expression system; C is the product result of mass spectrometry detection of coptisine catalyzed in the CcCODM prokaryotic expression system; D is the product result of mass spectrometry detection of bamipine catalyzed in the CcCODM prokaryotic expression system; E is the product result of mass spectrometry detection of fangdeline catalyzed in the CcCODM prokaryotic expression system.

Claims

1. A Coptis chinensis dioxygenase gene, characterized in that: The gene nucleic acid sequence is shown in SEQ ID NO.

1.

2. Use of the gene according to claim 1 in modifying benzylisoquinoline alkaloid compounds.

3. The use according to claim 2, characterized in that: The protein encoded by the gene catalyzes the dihydroxylation reaction of benzylisoquinoline alkaloids.

4. The use according to claim 2, characterized in that: The protein encoded by the gene catalyzes the synthesis of multiple benzylisoquinoline alkaloids with substrate promiscuity.

5. A recombinant expression vector and a host bacteria containing the gene according to claim 1.

6. The recombinant expression vector and host bacteria according to claim 5, characterized in that: The recombinant expression vector uses pET-28a as the vector, and the cloning site of the gene is BamHI / XhoI.

7. The recombinant expression vector and host bacteria according to claim 5, wherein the host bacteria is a BL21 (DE3) strain.

Images