CYP450 zymoprotein as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a CYP450 zymoprotein as well as a preparation method and application thereof. The nucleotide sequence of the CYP450 zymoprotein is as shown in SEQ ID NO.1, and the amino acid sequence of the CYP450 zymoprotein is as shown in SEQ ID NO.2. The CYP450 gene for catalyzing neoandrographolide aglycone to form the 14-deoxidized andrographolide aglycone is analyzed and excavated by utilizing transcriptomics, and the formation of the 14-deoxidized andrographolide aglycone is verified by utilizing a tobacco transient expression system. The invention not only provides an important gene element for biosynthesis of andrographolide, but also provides a key gene locus for molecular design and breeding of andrographis paniculata.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a CYP450 enzyme protein and a preparation method and application thereof. Background Art

[0002] Cytochrome P450 (CYP450) is an ancient supergene family that exists in almost all prokaryotes and eukaryotes. It is also the first group of enzymes classified as a "superfamily", which includes more than 1,000 families and 2,500 subfamilies. Existing literature shows that CYP450 participates in the secondary metabolism of plants and is an important regulatory substance in the biosynthesis of terpenoids. There are many kinds of terpenoids in plants, among which diterpenoids refer to terpenoids containing four isoprene units. Its synthetic precursors IPP and DMAPP are mainly derived from the MVA and MEP pathways. IPP and DMAPP synthesize GGPP through condensation reactions, and then are catalyzed by CPS to form a variety of diterpene skeletons, and then modified by specific functional groups under the action of modifying enzymes (such as P450).

[0003] As a traditional Chinese medicine, Andrographis paniculata is a representative species of southern medicine . . Andrographolide is a diterpene lactone component derived from the traditional Chinese medicine Andrographis paniculata. Andrographolide has been proven to be the main material basis for the pharmacological effects of Andrographis paniculata, and has multiple pharmacological activities such as anti-infection, anti-inflammatory and anti-tumor. Due to the relatively weak research on the analysis of the biosynthetic pathway of andrographolide, the production of andrographolide mainly relies on plant extraction. It is worth mentioning that in the synthesis pathway of andrographolide, hydroxylation is the only modification method for the synthesis of andrographolide. In the face of a large number of medicinal needs, the study of CYP450 is crucial for the analysis of the biosynthesis of andrographolide. In the face of huge market demand, it is urgent to use new technologies and new methods to analyze the biosynthetic pathway of andrographolide. Analyzing the function of CYP450 catalyzing the intermediates in the synthesis pathway of andrographolide not only provides key gene modules for the synthetic biology research of andrographolide, but also provides key sites for the cultivation of new varieties of Andrographis paniculata, and also provides important references for the study of hydroxylation modification of diterpene compounds. Summary of the invention

[0004] The purpose of the present invention is to provide a CYP450 enzyme protein and a preparation method and application thereof. The inventors subjected Andrographis paniculata to methyl jasmonate (MeJA) stress and then performed transcriptome sequencing, and used differential expression analysis to mine out the CYP450 enzyme that catalyzes the formation of new andrographolide aglycones to 14-deoxyandrographolide, which not only provides a key gene module for the synthetic biology research of andrographolide, but also provides a key gene site for the molecular design breeding of Andrographis paniculata.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a CYP450 enzyme protein capable of catalyzing the formation of 14-deoxyandrographolide from a new andrographolide aglycone. The nucleotide sequence of the CYP450 enzyme protein is shown in SEQ ID NO.1, and the amino acid sequence of the CYP450 enzyme protein is shown in SEQ ID NO.2.

[0007] The present invention also provides a recombinant vector, which comprises the nucleotide sequence of the CYP450 enzyme protein.

[0008] Furthermore, the nucleotide sequence of the CYP450 enzyme protein is inserted between the SalI and XhoI sites of the vector.

[0009] Furthermore, the primer sequences used to construct the recombinant vector are shown in SEQ ID NO.5-6.

[0010] The present invention also provides a recombinant strain, which comprises the CYP450 enzyme protein or the recombinant vector.

[0011] The present invention also provides an application of the CYP450 enzyme protein, the recombinant vector or the recombinant strain in catalyzing the formation of 14-deoxyandrographolide from neoandrographolide aglycone.

[0012] The present invention discloses the following technical effects:

[0013] Because andrographolide has good effects of removing heat and detoxification, anti-inflammatory and analgesic, it is praised as a natural antibiotic drug. However, the source of andrographolide currently relies solely on plant extraction, and the amount used in clinical practice is huge, resulting in a tight supply of andrographolide. Therefore, it is urgent to use new technologies and new methods to analyze the synthesis pathway of andrographolide and solve the supply problem of andrographolide. The present invention uses transcriptomics analysis to dig out the CYP450 gene that catalyzes the formation of 14-deoxyandrographolide aglycones from new andrographolide aglycones, and uses a tobacco transient expression system to verify the formation of 14-deoxyandrographolide aglycones. The present invention not only provides important gene elements for the biosynthesis of andrographolide, but also provides key gene sites for the molecular design breeding of andrographolide. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 This is a diagram showing the MeJA response of the ApCYP706U5 gene involved in the present invention;

[0016] Figure 2 The molecular structural formula diagram of the new andrographolide aglycone, 14-deoxyandrographolide and andrographolide involved in the present invention;

[0017] Figure 3 The diagrams are for the verification of the ApCYP706U5 gene cloning and vector construction in Example 1 of the present invention, wherein the left diagram is a diagram of the amplification results using the designed primers during the extraction and verification of the ApCYP706U5 gene fragment, the middle diagram is a diagram of the amplification results using the designed primers during the construction of the tobacco transient expression vector, and the right diagram is a diagram of the results of screening pEAQ-HT-ApCYP706U5 positive clones and PCR detection;

[0018] Figure 4 The GC-MS images of the identification of the injected tobacco products in Example 2 of the present invention, wherein the left image is the effect of the injection of ApCYP706U5 on the neoandrographolide aglycone, and the right image is a comparison of the products obtained by the co-injection of ApCYP706U5 and the neoandrographolide aglycone with the 14-deoxyandrographolide standard;

[0019] Figure 5 This is the GC-MS fragmentation diagram of the product in Example 2 of the present invention. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] Based on the property that andrographolide can respond to methyl jasmonate (MeJA), the transcriptome sequencing of Andrographis paniculata seedlings treated with MeJA was performed using the Illumina platform, and it was found that ApCYP706U5 can respond to MeJA stress, specifically Figure 1 As shown, ApCYP706U5 contains 1521 nucleotides, encodes a protein of 506 amino acids, and is officially named ApCYP71BE50, whose nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO.2.

[0026] In the following examples, the standard products of neoandrographolide aglycone (CAS: 82209-74-3) and 14-deoxyandrographolide (CAS: 4176-97-0) were obtained from Xili Company, and the other conventional reagents and equipment were commercially available unless otherwise specified. The molecular structures of neoandrographolide aglycone, 14-deoxyandrographolide and andrographolide are shown in Figure 2 shown.

[0027] Example 1

[0028] 1. Extraction and verification of ApCYP706U5 gene fragment

[0029] Mature leaves of Andrographis paniculata were collected, and RNA was extracted from the mature leaves of Andrographis paniculata using an RNA extraction kit (Quanshijin, polysaccharide and polyphenol RNA extraction kit). After the test was qualified, cDNA was obtained by reverse transcription. Primer sequences were designed (specifically shown in Table 1).

[0030] Table 1 Primer sequences used for cloning genes

[0031]

[0032] Using cDNA as a template, the ApCYP706U5 gene fragment was cloned using 2*Keypo high-fidelity enzyme (2*Keypo high-fidelity enzyme; the total volume of the PCR system was 50 μL: 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 2 μL template, 25 μL 2*Keypo Mix and 19 μL water, the procedure is shown in Table 2).

[0033] Table 22*Keypo high fidelity enzyme PCR reaction program

[0034]

[0035] After confirming that the size of the amplified band was correct, the ApCYP706U5 fragment was connected to the pEASY-Blunt vector (the total volume of the connection system was 2.5 μL: 0.5 μL pEASY-Blunt vector and 2.0 μL cDNA template, and the connection reaction was carried out at 25°C for 1 hour). The connection system was directly transformed into DH5α competent cells, and positive clones were selected for sequencing (the total volume of the colony PCR system was 25 μL: 13 μL 2×Taq PCR Mix, 1 μL template, 1 μL forward primer, 1 μL reverse primer and 9 μL water, and the program was shown in Table 3). After alignment with the reference sequence, the nucleotide sequence had a 100% similarity with the original data.

[0036] Table 3 Colony PCR reaction procedure

[0037]

[0038] 2. Construction of tobacco transient expression vector

[0039] (1) Design primer sequences. Design primers using SalI and XhoI as the ligation sites (specific primers are shown in Table 4).

[0040] Table 4 Primer sequences used for vector construction

[0041]

[0042] The sequenced pEASY-Blunt-ApCYP706U5 was used as a template and amplified using 2*Keypo high-fidelity enzyme (the total volume of the system was 50 μL: 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 2 μL template, 25 μL 2*Keypo Mix and 19 μL water, the procedure is shown in Table 5).

[0043] Table 52*Keypo high fidelity enzyme PCR reaction program

[0044]

[0045] (2) Ligation reaction: SalI and XhoI were used to digest the PEAQ backbone, and the PCR product of ApCYP706U5 obtained by amplification was cut and recovered together. The backbone and fragments were ligated overnight at 16°C using T4 ligase, and transferred into DH5α competent medium. Positive clones of pEAQ-HT-ApCYP706U5 were screened on 50 mg / mL Kan-resistant LB solid medium and PCR tested. The recombinant plasmids of the positive clones that were successfully sequenced were extracted for subsequent experiments. The specific results of ApCYP706U5 gene cloning and vector construction are shown in the figure. Figure 3 .

[0046] Example 2

[0047] This example uses the Agrobacterium transformation method of GV3101 of Weidi Biotechnology to perform Agrobacterium transformation:

[0048] (1) GV3101 Agrobacterium competent cells were taken out from -80℃, placed in ice for 5 minutes, and after melting, 1 μL of pEAQ-HT-ApCYP706U5 positive plasmid DNA was added. The cells were placed in an ice bath for 5 minutes. After taking out the centrifuge tube, the cells were placed in liquid nitrogen for 5 minutes, and then placed in a 37℃ water bath for 5 minutes. Finally, 700 μL of non-resistant LB liquid culture medium was added. The cells were shaken at 200 rpm for 3 hours in a 28℃ shaker, and then taken out. The cells were centrifuged at 6000 rpm for one minute to collect the bacteria. 100 μL of the supernatant was retained and gently blown to resuspend the bacterial mass and spread on an LB plate containing 25 mg / L Kan and 25 mg / L Rif antibiotics. The plates were placed upside down in a 28℃ incubator and cultured for 2-3 days.

[0049] (2) A single colony was picked from the plate with a sterilized toothpick and cultured in 500 μL of liquid LB medium containing 25 mg / L Kan and 25 mg / L Rif antibiotics in a 28°C incubator at 200 rpm for 8-12 h. When the bacterial solution became turbid, the upstream universal primers of pEAQ-HT-DEST and the downstream primers of ApCYP706U5 were selected for PCR identification. The positive strains were subjected to the next tobacco transient expression test.

[0050] The specific methods for tobacco transient expression and product identification are as follows:

[0051] (1) Injection of Agrobacterium tumefaciens GV3101 into Nicotiana benthamiana leaves

[0052] The recombinant plasmid pEAQ-HT-ApCYP706U5 was inoculated into 5 ml LB liquid medium containing 25 mg / L Rif and 25 mg / L Kan antibiotics, and cultured at 28 °C in a shaking incubator until the OD 600 The bacterial precipitate was collected by centrifugation at 6000 g for 5 min and resuspended in an equal volume of MMA solution (a mixed solution of 10 mM MES, 10 Mm MgCl2, and 200 μM acetosyringone) to an OD of about 1.0. 600 0.2, placed at room temperature for 2-3 hours before use; select Nicotiana benthamiana with good growth, use a syringe needle to gently puncture a small hole on the back of the leaf, and then inject the resuspended bacterial solution into the leaf; only the substrate neoandrographolide aglycone (CAS: 82209-74-3) was injected as the control group, and pEAQ-HT-ApCYP706U5 and neoandrographolide aglycone were injected as the experimental group, and 3 tobacco leaves were injected in each combination as a repeat. The injected tobacco was placed in dark culture for about 24 hours, then taken out and cultured in light for 5 days, and the leaves injected with Agrobacterium were collected and freeze-dried for 36 hours before being tested on the machine.

[0053] (2) Analysis and identification of catalytic products

[0054] The injected tobacco leaves were ground into powder using a grinder, and about 0.2 g of the powder was taken in 1000 uL of ethyl acetate, and the product was extracted after ultrasonication for 30 min. After centrifugation at 12000 rpm for 20 min, 400 μL of the supernatant was transferred to an Agilent sample bottle, and the reaction substrate (neoandrographolide aglycone) was detected using GC-MS. At the same time, 400 μL of the supernatant was transferred to a 2 mL centrifuge tube for rotary evaporation, and after complete drying, 100 μL of BSTFA derivatization reagent was added, and it was kept at 80 ° C for 30 min and then transferred to a liquid phase vial for detection. Agilent 8250 was used for GC-MS detection. The gas chromatography column was hb-5 (30m×0.25mm×0.25um, Aglient). The temperature program was: 50℃ for 2min, then heated to 280℃ at 15℃ / min, and then kept at 280℃ for 5min (40min in total). The flow rate of carrier gas (He) was 1mL / min. The injection volume was 1μL. The vaporization chamber temperature was 250℃. The mass spectrometer was Agilent 8250-Q-Tof, and the mass spectrometer scanning range was 50-500u. The GC-MS analysis results are shown in the figure. Figure 2-Figure 4 As shown, the standards of neoandrographolide aglycone (CAS: 82209-74-3) and 14-deoxyandrographolide (CAS: 4176-97-0) were both from Xili Company.

[0055] After the above analysis and identification, it was found that when the new andrographolide aglycone was injected into tobacco alone, it peaked at 27.677 (specifically, Figure 4When ApCYP706U5 and neoandrographolide aglycone were co-injected, neoandrographolide aglycone was consumed and a product peak with 173 as a characteristic fragment appeared at 28.611 (as shown in Figure 4 The new product has the same peak time and characteristic fragments as the 14-deoxyandrographolide standard (CAS: 4176-97-0) (as shown in Figure 5 It can be speculated that ApCYP706U5 can oxidize the neoandrographolide aglycone to form 14-deoxyandrographolide.

[0056] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the scope of protection determined by the claims of the present invention. In addition, the examples described are only one application of the present invention, so the use of the relevant repair mode in the present invention for site-directed insertion of other DNA fragments (not limited to the DNA sequence of the fusion tag) should also fall within the scope of protection determined by the claims of the present invention.

Claims

1. A CYP450 enzyme protein that can catalyze the formation of 14-deoxyandrographolide from a new andrographolide aglycone, characterized in that: The nucleotide sequence of the CYP450 enzyme protein is shown in SEQ ID NO.1, and the amino acid sequence of the CYP450 enzyme protein is shown in SEQ ID NO.

2.

2. A recombinant vector, characterized in that: The recombinant vector comprises the nucleotide sequence of the CYP450 enzyme protein according to claim 1.

3. The recombinant vector according to claim 2, characterized in that The nucleotide sequence of the CYP450 enzyme protein is inserted between the SalI and XhoI sites of the vector.

4. The recombinant vector according to claim 2, characterized in that The primer sequences used to construct the recombinant vector are shown in SEQ ID NO.5-6.

5. A recombinant strain, characterized in that: The recombinant strain comprises the CYP450 enzyme protein according to claim 1 or the recombinant vector according to any one of claims 2-4.

6. Use of the CYP450 enzyme protein according to claim 1, the recombinant vector according to any one of claims 2 to 4, or the recombinant strain according to claim 5 in catalyzing the formation of 14-deoxyandrographolide from neoandrographolide aglycone.

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