A CYP450 enzyme protein capable of catalyzing the formation of new andrographolide aglycones, its encoding gene, and its applications.

Abstract

This invention provides a CYP450 enzyme protein, its encoding gene, and its applications that can catalyze the formation of new andrographolide aglycones, belonging to the field of biology. This invention utilizes bioinformatics analysis and differential expression analysis of transcriptome data from different tissue sites to identify the gene ApCYP71BE50, which catalyzes the formation of new andrographolide aglycones from (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol. It also verifies 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 to form new andrographolide aglycones. This invention provides an important gene element for the biosynthesis of andrographolide and also provides a key gene locus for the molecular design breeding of andrographis paniculata.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a CYP450 enzyme protein, encoding gene, and application of a catalytically formed andrographolide aglycone. Background Technology

[0002] Cytochrome P450 is an ancient superfamily of enzymes found in almost all prokaryotes and eukaryotes. It was also the first group of enzymes classified as a "superfamily," comprising over 1000 families and 2500 subfamilies. Currently, CYP450 has been reported to participate in the biosynthesis of various terpenoids.

[0003] Terpenes are widely found 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 skeletal structure, and the reactions following the modification. Most of these post-modification reactions are catalyzed by cytochrome P450 monooxygenases, the majority 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 crucial role in the exploration of terpenoid biosynthesis.

[0004] Andrographis paniculata is a traditional Chinese medicine and a representative species of southern Chinese medicinal herbs, possessing significant medicinal value. Its diterpenoid component, andrographolide, is its main medicinal secondary metabolite. Andrographolide has excellent antipyretic, detoxifying, anti-inflammatory, and analgesic effects, showing special efficacy against bacterial and viral upper respiratory tract infections and dysentery. It is hailed as a natural antibiotic, and its clinical use is enormous, leading to a shortage of andrographolide supply. However, research on its biosynthetic pathway is relatively weak. Therefore, there is an urgent need to utilize new technologies and methods to solve the resource shortage problem of andrographolide.

[0005] Diterpenoids are terpenoid compounds 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 diphosphate (GGPP) via a condensation reaction, which is then catalyzed by copalyl / labdadienyl diphosphate synthases (CPS) to form a variety of diterpenoid skeletons. Although no CYP450 has yet been reported to catalyze the intermediates of andrographolide synthesis, numerous studies have shown that the CYP71 family can oxidatively modify various diterpenoid skeletons. This suggests that the CYP71 family may play an important role in the hydroxylation modification of diterpenoids, and different CYP71 family genes exhibit different substrate specificities and catalytic activities.

[0006] Furthermore, the andrographolide synthesis pathway does not involve modifications such as glycosylation or acylation; hydroxylation is the only modification involved in andrographolide synthesis. Therefore, analyzing genes that catalyze intermediates in the andrographolide synthesis pathway not only provides key gene modules for the synthetic biology research of andrographolide but also provides key sites for the breeding of new varieties of andrographis paniculata, and offers important references for the study of hydroxylation modifications of diterpenoid compounds. Summary of the Invention

[0007] The purpose of this invention is to provide a CYP450 enzyme protein, encoding gene, and application that can catalyze the formation of new andrographolide aglycones, providing an important gene element for the biosynthesis of andrographolide and a key gene locus for the molecular design breeding of andrographis paniculata.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a CYP450 enzyme protein capable of catalyzing the formation of a new andrographolide aglycone, wherein the CYP450 enzyme protein catalyzes the continuous oxidation of (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol at position C16 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 novel andrographolide aglycone is shown in formula (II):

[0013]

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

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

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

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

[0018] The beneficial effects of this invention compared to the prior art are as follows:

[0019] This invention, through bioinformatics analysis and differential expression analysis of transcriptome data from different tissue sites, identified the CYP450 gene – ApCYP71BE50 – which catalyzes the formation of a new andrographolide aglycone from (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol. The formation of the lactone ring was verified 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, forming a new andrographolide aglycone. This invention not only provides an important gene element for the biosynthesis of andrographolide but also provides a key gene locus for the molecular design breeding of andrographis paniculata. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1The expression of the 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 The GC-MS diagram for identifying the injected tobacco product in Example 2 is shown in Figure 2. In Figure 2, A is the standard (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol, and B is the neoandrographolide.

[0025] Figure 5 Here is a GC-MS image of the injected tobacco product identification in Example 2; Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0031] The amino acid sequence of the 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 carrier fragment;

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

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

[0039] The results of andrographolide in this invention are shown in formula (III):

[0040]

[0041] Example 1

[0042] Example 1 of this invention analyzed and obtained the gene ApCYP71BE50, which catalyzes the synthesis of new andrographolide aglycones, and a recombinant plasmid containing the gene ApCYP71BE50, specifically including the following steps:

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

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

[0045] (2) Collect mature leaves of Andrographis paniculata and extract RNA from the mature leaves using an RNA extraction kit (Adley, RN38EASYspinplus). After the RNA quality is qualified, reverse transcription is performed to obtain qualified cDNA.

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

[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 procedure

[0049]

[0050] The PEAQ vector backbone was digested with AgeI and XhoI enzymes, and then gel-extracted along with the PCR product of the amplified ApCYP71BE50. The backbone and fragment were ligated overnight at 16°C using T4 ligase in a 3 μL ligation system: 0.5 μL PEAQ vector and 2.5 μL ApCYP71BE50. The ApCYP71BE50 fragment was successfully ligated into the vector. After ligation, the cells were directly transformed into TransT1 competent cells, and positive clones were screened on 50 mg / mL Kan resistance LB agar and detected by PCR (see [link to relevant documentation]). Figure 3 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×TaqPCRMix, 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 this invention uses the recombinant plasmid containing the gene ApCYP71BE50 prepared in Example 1 to verify the function of the gene ApCYP71BE50, specifically including the following steps:

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

[0057] A. Remove the 0.1cm electrode cup and its lid from the storage solution and place them upside down on clean absorbent paper for 5 minutes to allow the ethanol to drain. Then, place them upright for 5 minutes to allow the ethanol to evaporate completely. Once the ethanol has evaporated, immediately insert them into ice and press the ice surface firmly. The top of the electrode cup should be 0.5cm away from the ice surface to facilitate closing the lid. Let them stand in the ice for 5 minutes to cool down completely.

[0058] B. Remove EH105 Agrobacterium competent cells from the -80℃ freezer, insert them into ice for 5 minutes, and add 0.01-1 μg of recombinant plasmid PEAQ-HT-ApCYP71BE50 (volume not exceeding 6 μl) after they thaw. Mix well by hand by stirring the bottom of the tube, immediately insert it into ice, and quickly transfer the competent cell-plasmid mixture into an electroporation cup using a 200 μl pipette tip. Cover the cup and keep the empty tube for later use.

[0059] C. Start the electroporation apparatus, quickly place the electroporation cup into the electroporation tank, and after electroporation is complete, quickly insert it into ice, add 700 μL of antibiotic-free LB and transfer it to a competent empty tube, and incubate at 28°C with shaking for 2-3 hours.

[0060] D. Centrifuge at 6000 rpm for one minute to collect the bacteria. Take about 100 μL of supernatant, gently pipette and resuspend the bacterial block, and spread it on LB agar plates containing 50 mg / L Kan and 50 mg / L Rif antibiotics. Invert the plates and incubate them at 28°C for 2-3 days.

[0061] E. Use a sterile toothpick to pick a single colony from an LB plate and incubate 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 upstream and downstream primers of the empty pEAQ-HT vector without the target gene and the ApCYP71BE50 gene without the target gene for PCR identification. Positive strains will be used for the next step of transient tobacco expression experiment.

[0062] (2) Transient expression and product identification in tobacco:

[0063] A. Injection of recombinant Agrobacterium EHA105 into Tobacco Benzoentae leaves: Recombinant Agrobacterium EHA105 containing the recombinant plasmid was inoculated into 5 ml of LB liquid medium containing 50 mg / L Rif and 50 mg / L Kan antibiotics, and cultured at 28°C on a shaker until the OD600 reached approximately 1.0; centrifuged at 4000g for 5 min, the supernatant was discarded, the bacterial pellet was collected, and an equal volume of 10 mM MMA solution was used. Resuspend the tobacco leaves in a mixture of MES, 10MmMgCl2, and 200μM acetylsyleugenol until the OD600 is 0.8–1.2, and leave them at room temperature for 3 hours. Select healthy *Nicotiana benthamiana* leaves and gently puncture small holes on the leaf surface with a syringe needle. Use *Nicotiana benthamiana* leaves injected only with the substrate (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol as the control group, and use leaves injected with both ApCYP71BE50 and (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol as the experimental group. Each group is injected with 3 tobacco leaves as replicates. After injection, the tobacco leaves are placed in the dark for 1 day, then taken out and cultured under light for 4 days. Collect the leaves injected with *Agrobacterium*, freeze-dry them, and then perform instrumental analysis.

[0064] B. Analysis and identification of catalytic products: The injected tobacco leaves were ground into powder in liquid nitrogen. Approximately 0.1 g of the powder was placed in 500 μL of extraction solvent (extraction reagent: methanol:water:potassium hydroxide = 9:1:1, v:v:w) and extracted at 65°C for 2 hours, shaking once every half hour. Then, 250 μL of water was added and the mixture was shaken to mix. Next, 500 μL of n-hexane was added and shaken to mix. The mixture was centrifuged at 12000 g for 1 min. 100 μL of the upper n-hexane solution was collected and concentrated to dryness using a vacuum centrifuge concentrator (Concentratorplus, Eppendor). After redissolving in 100 μL of ethyl acetate, the solution was transferred to an Agilent sample vial for GC-MS detection.

[0065] GC-MS analysis was performed using an Agilent 8250 gas chromatograph. The gas chromatographic column was HB-5 (30m × 0.25mm × 0.25um, Agilent). The temperature program was: 50℃ for 2 min, increased to 280℃ at 15℃ / min, and held at 280℃ for 5 min (total 40 min). The carrier gas (He) flow rate was 1 mL / min; the injection volume was 1 μL; the vaporization chamber temperature was 250℃; the mass spectrometer detector was an Agilent 8250-Q-Tof; and the mass spectrometry scan range was 50-500 u. The GC-MS analysis results are as follows: Figure 4 , 5 As shown.

[0066] (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol (CAS:37886-56-9) is a standard obtained by our laboratory from Andrographis paniculata after isolation and purification.

[0067] When (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol was injected alone into tobacco, it eluted 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 were significantly consumed, and a product peak with 205 as the characteristic fragment appeared at 27.660.

[0069] NIST library comparison identified it as neoandrographolide. To further confirm whether the product was indeed neoandrographolide, a neoandrographolide standard (CAS: 82209-74-3) was dissolved in a 1 μg / mL standard solution and analyzed. 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 to form neoandrographolide.

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A gene encoding the CYP450 enzyme protein ApCYP71BE50 Application in the synthesis of novel andrographolide aglycones; characterized in that... The amino acid sequence of the CYP450 enzyme protein is shown in SEQ ID NO.1; the gene ApCYP71BE50 The nucleotide sequence is shown in SEQ ID NO.2; The CYP450 enzyme protein catalyzes the continuous oxidation of (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol at the C16 position to form a new andrographolide aglycone. The CAS number of (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol is 37886-56-9, and its structure is shown in Formula I: Equation I; The structure of the new andrographolide aglycone is shown in Formula II: Formula II.

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