CYP450 zymoprotein capable of catalyzing formation of neoandrographolide aglycone, coding gene and application

Abstract

The invention provides a CYP450 zymoprotein capable of catalyzing formation of neoandrographolide aglycone, a coding gene and application, and belongs to the field of biology. According to the present invention, bioinformatics analysis and differential expression analysis are performed on transcriptome data of different tissue parts, the gene ApCYP71BE50 capable of catalyzing (4R, 5S, 9R, 10S)-labda-8 (17), 13-dien-15, 19-diol to form the neoandrographolide aglycone is excavated, and it is verified that the CYP450 enzyme protein encoded by the gene ApCYP71BE50 can perform continuous oxidation on the C16 site of (4R, 5S, 9R, 10S)-labda-8 (17), 13-dien-15, 19-diol to form the neoandrographolide aglycone. The invention provides an important gene element for biosynthesis of andrographolide, and also provides a key gene locus for molecular design breeding of andrographis paniculata.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a CYP450 enzyme protein capable of catalyzing the formation of a new andrographolide aglycone, a coding gene and an application thereof. Background Art

[0002] Cytochrome P450 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. Currently, CYP450 has been reported to be involved in the biosynthesis of a variety of terpenoids.

[0003] Terpenoids are widely present in nature, including oxygen-containing derivatives such as alcohols, aldehydes, ketones, carboxylic acids, and esters. Their diversity mainly depends on the modification of specific chemical groups, the rearrangement of the skeleton structure, and the reaction after modification. Most of the modification reactions are catalyzed by cytochrome P450 monooxygenases, most of which belong to the CYP71 family. Therefore, the discovery and identification of plant CYP450 genes, especially the identification of CYP71 family members, plays a vital role in the exploration of terpenoid biosynthesis.

[0004] Andrographis paniculata is a traditional Chinese medicine and a representative species of southern medicine with important medical value. Its diterpenoid component andrographolide is the main medicinal secondary metabolite. Andrographolide has good effects of removing heat and detoxification, anti-inflammatory and analgesic, and has special effects on bacterial and viral upper respiratory tract infections and dysentery. It is known as a natural antibiotic drug. Its huge clinical usage leads to a tight supply of andrographolide, but the research on its biosynthetic pathway is relatively weak. Therefore, it is urgent to use new technologies and methods to solve the problem of tight supply of andrographolide resources.

[0005] Diterpenoids refer to terpenoids containing four isoprene units. Their synthetic precursors, isoprene pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), are mainly derived from the mevalonate pathway (MVA) and the 2-C-methyl-D-erythritol-4-phosphate pathway (MEP). IPP and DMAPP synthesize geranylgeranyl pyrophosphate (E,E,E)-geranylgeranyl diphosphate (GGPP) through condensation reaction, and then are catalyzed by copalyl / labdadienyl diphosphate synthases (CPS) to form a variety of diterpene skeletons. Although no CYP450 has been reported to catalyze intermediates in the synthesis of andrographolide, a large number of studies have shown that the CYP71 family can oxidatively modify a variety of diterpene skeletons, indicating that the CYP71 family may play an important role in the hydroxylation modification of diterpenoids, and different CYP71 family genes have different substrate specificity and different catalytic activities.

[0006] Moreover, the synthesis pathway of andrographolide does not involve modifications such as glycosylation and acylation, and hydroxylation is the only modification method for the synthesis of andrographolide. Therefore, analyzing the genes that can catalyze 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 andrographolide, and also provides an important reference for the study of hydroxylation modification of diterpenoid compounds. Summary of the invention

[0007] The purpose of the present invention is to provide a CYP450 enzyme protein, encoding gene and application that can catalyze the formation of new andrographolide aglycones, provide important gene elements for the biosynthesis of andrographolide, and also provide key gene sites for the molecular design breeding of andrographolide.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The invention provides a CYP450 enzyme protein capable of catalyzing the formation of a new andrographolide aglycone. The CYP450 enzyme protein can catalyze the continuous oxidation of the C16 position of (4R, 5S, 9R, 10S)-labda-8(17), 13-dien-15, 19-diol to form a new andrographolide aglycone. The amino acid sequence of the CYP450 enzyme protein is shown in SEQ ID NO.1.

[0010] Furthermore, the CAS number of the R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol is 37886-56-9, and its structure is shown in formula (I):

[0011]

[0012] Furthermore, the structure of the new andrographolide aglycone is shown in formula (II):

[0013]

[0014] The present invention also provides a gene ApCYP71BE50 encoding a CYP450 enzyme protein, and the sequence of the gene ApCYP71BE50 is shown in SEQ ID NO.2.

[0015] The present invention also provides an application of the gene ApCYP71BE50 encoding CYP450 enzyme protein in synthesizing new andrographolide aglycones.

[0016] The present invention also provides a new andrographolide aglycone synthesized by the gene ApCYP71BE50 encoding the CYP450 enzyme protein.

[0017] The present invention also provides an application of the new andrographolide aglycone in the preparation of andrographolide.

[0018] The beneficial effects of the present invention compared with the prior art are:

[0019] The present invention conducts bioinformatics analysis and differential expression analysis on transcriptome data of different tissue parts, and finds the CYP450 gene ApCYP71BE50 that catalyzes (4R, 5S, 9R, 10S)-labda-8(17), 13-dien-15, 19-diol to form new andrographolide aglycones, and verifies the formation of the lactone ring using a tobacco transient expression system, indicating that the CYP450 enzyme protein encoded by the ApCYP71BE50 gene can continuously oxidize the C16 position of (4R, 5S, 9R, 10S)-labda-8(17), 13-dien-15, 19-diol and form new andrographolide 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

[0020] 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 labor.

[0021] Figure 1The expression of ApCYP71BE50 gene in different Andrographis paniculata tissues in Example 1;

[0022] Figure 2 The MeJA response of Andrographis paniculata seedlings in Example 1;

[0023] Figure 3 The results of ApCYP71BE50 gene cloning and vector construction in Example 2;

[0024] Figure 4 This is the GC-MS chart for identification of the injected tobacco product in Example 2, wherein A is the standard substance (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol, and B is the new andrographolide aglycone;

[0025] Figure 5 This is the GC-MS image of the identification of the injected tobacco product in Example 2; DETAILED DESCRIPTION

[0026] 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.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments 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. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0028] 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.

[0029] 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 the skilled artisan. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Amino acid sequence of CYP450 enzyme protein (SEQ ID NO.1):

[0032] METTTIQLLILASFLFSILMIFKQAKKSRPNLPPGPPKLPLIGNLHNLIGGGMPHQAIHQLALKYGPLMHLKLGGVSTVVVSSPDVARQVMKTHDLNF ASRPPLLATEILNYGKSGVIFSPYGESWRHLRKICTLELLSNKRVQSFRPVRQKVHLELARMIAAGEGSAVDLSSKLYSSTYDVASRVAVGESNKSRKARLMEIINEAGELSAGFHIAELYPSIKVLGKITGLEKKLQKLHWETDELLEGIIGDHREAPEKEDGKKVDDLVDVLLKLQGEEHQSFLTSNSIKSVIMDILGA GSETSATTLDWAMTELLKNPSVMDRAQREVRAVFDRKGHVNESDVNELKYLNSVIKETLRLHTPGPLLVPRMCIEACQINGCDIPENTRVIVNAWTINRDPDYWQDPLSFKPERFLDSAVDYGGDSFEYLPFGAGRRICPGISFGLANIQSPLAMLLYHFDWKLPDGMKPEDIDMSEKFGVTTRRLINLRAVPVITRPLP;

[0033] ApCYP71BE50 gene nucleotide sequence (SEQ ID NO.2):

[0034] ATGGAGACGACGACGATCCAGTTGCTAATCCTCGCTTCATTCTTATTCTCAATCTTGATGATTTTCAAACAAGCTAAAAAATCACGACCGAATCTCCCTCCAGGGCCGCCGAAGCTGCCGCTGATCGGAAATCTCCACAACCTGATCGGCGGCGGAATGCCCCACCAGGCAATACATCAATTGGCCCTGAAATACGGCCCCCTGATGCACCTGAAGCTCGGCGGGGTTTCCACCGTCGTCGTCTCGTCGCCGGACGTCGCCAGACAAGTCATGAAAACTCACGACCTGAACTTCGCGTCCCGGCCGCCGCTCCTCGCCACCGAGATCCTCAATTACGGCAAATCCGGCGTCATCTTCAGCCCCTACGGCGAATCCTGGAGGCACCTGAGGAAGATCTGCACGCTGGAGCTGCTGAGCAACAAGCGGGTCCAATCATTCCGGCCGGTCCGGCAAAAGGTGCATTTAGAGCTCGCGAGAATGATCGCCGCCGGCGAGGGCTCCGCCGTCGACTTATCGTCGAAGCTTTATTCTTCCACGTACGACGTCGCTTCGCGGGTGGCGGTCGGGGAATCGAACAAATCGAGGAAAGCCAGGTTAATGGAGATTATCAACGAAGCCGGTGAGCTCTCAGCAGGGTTCCATATCGCCGAACTGTATCCCTCCATTAAAGTCCTCGGGAAGATCACCGGCCTGGAGAAGAAGCTGCAGAAACTCCACTGGGAAACCGACGAACTTCTCGAAGGCATCATCGGCGACCACAGAGAAGCTCCTGAGAAAGAAGATGGAAAGAAAGTCGACGATCTCGTCGACGTTCTTCTGAAATTACAGGGTGAAGAGCACCAATCTTTTTTAACCTCCAACAGCATCAAATCCGTGATCATGGACATTCTCGGCGCCGGCAGCGAGACGTCCGCAACGACATTAGATTGGGCGATGACGGAGCTGCTGAAAAATCCAT;

[0035] ApCYP71BE50-F (SEQ ID NO. 3):

[0036] CAAATTCGCGACCGGT ATGGAGACGACGACGATCCA; the underlined part is the vector fragment;

[0037] ApCYP71BE50-R (SEQ ID NO. 4):

[0038] AGTTAAAGGCCTCGAG TTACGGCAATGGCCTCGT; the underlined part is the vector fragment.

[0039] The result of andrographolide in the present invention is shown in formula (III):

[0040]

[0041] Example 1

[0042] In Example 1 of the present invention, the gene ApCYP71BE50 for catalyzing the synthesis of new andrographolide aglycones and the recombinant plasmid containing the gene ApCYP71BE50 were analyzed and obtained, and specifically comprised the following steps:

[0043] (1) Andrographolide has the characteristic of accumulating in large quantities in stems and leaves. The transcriptome sequencing of different tissue parts such as roots, stems, leaves, and flowers of Andrographis paniculata was performed using the Illumina platform. Through bioinformatics analysis and differential expression analysis of transcriptome data from different tissue parts, a total of 57 CYP450 genes belonging to the CYP71 family were screened, and 25 CYP450 genes highly expressed in stems and leaves were selected as candidate genes.

[0044] Since andrographolide can respond to methyl jasmonate (MeJA) stress, we treated seedlings with methyl jasmonate (MeJA) and performed transcriptome sequencing, and found that ApCYP71BE50 was specifically highly expressed in stems and leaves and could respond to MeJA stress (see Figure 1 , 2 ).

[0045] (2) Mature leaves of Andrographis paniculata were collected, and RNA was extracted from the mature leaves of Andrographis paniculata using an RNA extraction kit (Adlai, RN38EASYspinplus). After the RNA quality was tested and qualified, reverse transcription was performed to obtain cDNA of qualified quality.

[0046] AgeI and XhoI were selected as the connection sites, and primer sequences (as shown in SEQ ID NO.3 and SEQ ID NO.4) were designed. Andrographis paniculata cDNA was used as a template and KOD-Plus-Neo high-fidelity enzyme was used to clone the ApCYP71BE50 gene fragment.

[0047] The total volume of the KOD high-fidelity enzyme PCR system was 50 μL: 5 μL 10× Buffer, 3 μL MgSO4, 5 μL dNTP (2 mM), 1.5 μL forward primer (10 μM), 1.5 μL reverse primer (10 μM), 1 μL template, 1 μL KOD enzyme and 32 μL water. The reaction procedure is shown in Table 1.

[0048] Table 1 KOD high fidelity enzyme PCR reaction program

[0049]

[0050] The PEAQ vector backbone was digested with AgeI and XhoI, and the PCR product of ApCYP71BE50 was cut and recovered together. The backbone and fragment were ligated overnight at 16°C using T4 ligase. The total volume of the ligation system was 3 μL: 0.5 μL PEAQ vector and 2.5 μL ApCYP71BE50. The ApCYP71BE50 fragment was successfully ligated to the vector. After ligation, the TransT1 competent cells were directly transformed, and positive clones were screened on 50 mg / mL Kan-resistant LB solid medium and PCR detection was performed (see Figure 3 ), and the recombinant plasmid PEAQ-HT-ApCYP71BE50 was extracted from the successfully sequenced positive clones for subsequent experiments.

[0051] The total volume of the colony PCR system was 12.5 μL: 6.25 μL 2× TaqPCR Mix, 1 μL template, 0.25 μL forward primer, 0.25 μL reverse primer and 4.75 μL water. The reaction procedure is shown in Table 2.

[0052] Table 2 Colony PCR reaction procedure

[0053]

[0054] Example 2

[0055] Example 2 of the present invention uses the recombinant plasmid containing gene ApCYP71BE50 prepared in Example 1 to verify the function of gene ApCYP71BE50, which specifically includes the following steps:

[0056] (1) Preparation of recombinant Agrobacterium EHA105:

[0057] A. Take out the 0.1 cm electric shock cup and cup cover from the storage solution and invert it on clean absorbent paper for 5 minutes to drain the ethanol. Place it upright for 5 minutes to allow the ethanol to evaporate completely. After the ethanol evaporates completely, immediately insert it into ice and compact the ice surface. Keep the top of the electrode cup 0.5 cm away from the ice surface to facilitate the cup cover. Let it stand in the ice for 5 minutes to fully cool down.

[0058] B. Take out the EH105 Agrobacterium competent cells from the -80℃ refrigerator, put them in ice for 5 minutes, wait for them to melt, add 0.01-1μg recombinant plasmid PEAQ-HT-ApCYP71BE50 (volume not more than 6μl), stir the bottom of the tube to mix, immediately put it in ice, use a 200μl pipette tip to quickly transfer the competent cell-plasmid mixture to the electroporation cup, cover the cup, and keep the empty tube for later use.

[0059] C. Start the electroporator, quickly place the electroporation cup into the electroporation tank, quickly insert it into ice after electroporation is completed, add 700 μL of LB without antibiotics and transfer it to the competent empty tube, and culture at 28°C with shaking for 2 to 3 hours.

[0060] D. Centrifuge at 6000 rpm for one minute to collect the bacteria. Take about 100 μL of the supernatant and gently blow to resuspend the bacteria. Apply the plate to the LB plate containing 50 mg / L Kan and 50 mg / L Rif antibiotics. Place the plate upside down in a 28°C incubator and culture for 2-3 days.

[0061] E. Pick a single colony on the LB plate with a sterilized toothpick, and culture it in 500 μL of liquid LB medium containing 50 mg / L Kan and 50 mg / L Rif antibiotics at 28°C with shaking for 8-12 hours. Select the empty pEAQ-HT vector without the target gene and the upstream and downstream primers of the ApCYP71BE50 gene for PCR identification, and the positive strains will be used for the next tobacco transient expression test;

[0062] (2) Tobacco transient expression and product identification:

[0063] A. Injection of recombinant Agrobacterium EHA105 into Nicotiana benthamiana leaves: Inoculate the recombinant Agrobacterium EHA105 containing the recombinant plasmid into 5 ml of LB liquid medium containing 50 mg / L Rif and 50 mg / L Kan antibiotics, and culture at 28°C in a shaking incubator until the OD600 is about 1.0; centrifuge at 4000 g for 5 min, discard the supernatant, collect the bacterial precipitate, and use an equal volume of MMA solution (concentration of 10 mM MES, 10MmMgCl2, 200μM acetosyringone mixed solution) were resuspended to OD600 of 0.8-1.2 and placed at room temperature for 3h; select Nicotiana benthamiana with good growth, and gently pierce the surface of the leaf with a syringe needle; Nicotiana benthamiana injected with only the substrate (4R, 5S, 9R, 10S)-labda-8(17), 13-dien-15, 19-diol was used as the control group, and the Nicotiana benthamiana injected with ApCYP71BE50 and (4R, 5S, 9R, 10S)-labda-8(17), 13-dien-15, 19-diol was used as the experimental group, and 3 tobacco leaves were injected in each combination as a replicate. The injected tobacco was placed in the dark for 1d, then taken out and cultured in the light for 4d, and the leaves injected with Agrobacterium were collected, freeze-dried and tested on the machine.

[0064] B. Analysis and identification of catalytic products: Grind the injected tobacco leaves into powder in liquid nitrogen, take about 0.1g of the powder and put it into 500uL of extractant (extraction reagent: methanol: water: potassium hydroxide = 9:1:1, v:v:w), extract at 65℃ for 2 hours, shake once every half hour, then add 250ul of water and shake to mix, then add 500ul of n-hexane, shake to mix, centrifuge at 12000g for 1min, take 100ul of the upper n-hexane solution, concentrate to dryness using a vacuum centrifugal concentrator (Concentratorplus, Eppendor); add 100uL of ethyl acetate to dissolve it, transfer it to an Agilent sample bottle for GC-MS detection.

[0065] 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°C for 2 min, then heated to 280°C at 15°C / min, and then kept at 280°C for 5 min (40 min in total). The flow rate of carrier gas (He) was 1 mL / min. The injection volume was 1 μL. The vaporization chamber temperature was 250°C. The mass spectrometer was Agilent 8250-Q-Tof. The mass spectrometer scanning range was 50-500u. The GC-MS analysis results are shown in the figure. Figure 4 , 5 shown.

[0066] (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol (CAS: 37886-56-9) is a standard product isolated and purified from Andrographis paniculata in our laboratory.

[0067] When (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol was injected alone into tobacco, it peaked at 24.982.

[0068] When ApCYP71BE50 and (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol were co-injected, (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol was significantly consumed and a product peak with 205 as a characteristic fragment appeared at 27.660.

[0069] The NIST library comparison was neoandrographolide aglycone. To further determine whether the product was neoandrographolide aglycone, the neoandrographolide aglycone standard (CAS: 82209-74-3) was dissolved to 1 μg / mL standard solution and tested on the machine. The peak time and characteristic fragments were consistent with the product. It can be inferred that the CYP450 enzyme protein encoded by the ApCYP71BE50 gene can continuously oxidize the C16 position of (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol and form neoandrographolide aglycone.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A CYP450 enzyme protein that can catalyze the formation of new andrographolide aglycones, It is characterized in that The CYP450 enzyme protein can catalyze the continuous oxidation of the C16 position of (4R, 5S, 9R, 10S)-labda-8(17), 13-dien-15, 19-diol to form new andrographolide aglycones; the amino acid sequence of the CYP450 enzyme protein is shown in SEQ ID NO.

1.

2. The CYP450 enzyme protein capable of catalyzing the formation of new andrographolide aglycones according to claim 1, It is characterized in that The CAS number of the R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol is 37886-56-9, and its structure is shown in formula (I):

3. The CYP450 enzyme protein capable of catalyzing the formation of new andrographolide aglycones according to claim 1, It is characterized in that The structure of the new andrographolide aglycone is shown in formula (II):

4. A gene ApCYP71BE50 encoding the CYP450 enzyme protein as claimed in claim 1, It is characterized in that The nucleotide sequence of the gene ApCYP71BE50 is shown in SEQ ID NO.

2.

5. Use of the gene ApCYP71BE50 encoding CYP450 enzyme protein according to claim 4 in synthesizing new andrographolide aglycones.

6. A new andrographolide aglycone synthesized by using the gene ApCYP71BE50 encoding CYP450 enzyme protein according to claim 4.

7. Use of the new andrographolide aglycone according to claim 6 in the preparation of andrographolide.

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