Biosynthesis pathway process for andrographolide
Through transcriptomic analysis and verification of tobacco transient expression system, the CYP450 gene catalyzing the synthesis of punctate lactide was excavated, which solved the problem of unresolved biosynthetic pathways, provided key gene elements and breeding sites, and promoted the synthesis and supply of punctate lactide.
Patent Information
- Application Number
- CN202510130060.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-05
- Publication Date
- 2025-05-09
AI Technical Summary
The biosynthesis pathway of punctate has not been analyzed, which has resulted in the acquisition method that can only rely on plant extraction, resulting in the problem of tight supply.
CYP450 gene that catalyzes (4R, 5S, 9R, 10S)-labda-8(17), 13-dien-15, 19-diol continuous oxidation and formation of new azolidol aglycones was excavated through transcriptomic association analysis, and the formation of lactone ring was verified using the tobacco transient expression system.
It provides important genetic elements for the biosynthesis of punctyl lactone and provides key genetic loci for the molecular design breeding of punctyl lactone, solving the problem of unresolved biosynthetic pathways and promoting its synthesis and supply.
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Figure CN119955748A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of andrographolide biosynthesis, in particular to a process for andrographolide biosynthesis pathway. Background Art
[0002] Cytochrome P450 enzymes (P450s) are one of the most widely used oxidases in nature and the first group of enzymes to be classified as a "super family", which includes more than 1,000 families and 2,500 subfamilies. Different types of P450 oxidases catalyze a series of oxidation reactions related to the biosynthesis of natural products, including hydroxylation, epoxidation, dehydrogenation, and the formation of CC, CN and CS bonds. P450-catalyzed oxidative hydroxylation and cyclization are very important in the biosynthesis of many natural products. Among them, CYP71 is the largest subfamily of P450 oxidases. It has been reported in the literature that members of the CYP71 subfamily, such as CYP76AH and CYP76AK, are involved in the anabolism of diterpenoid compounds in Salvia miltiorrhiza, indicating that the CYP71 family may play a key role in the synthesis of diterpenes in medicinal plants.
[0003] As a diterpene compound, andrographolide has four isoprene units. Its synthetic precursors, isoprene pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), are mainly derived from the mevalonate pathway (MVA) and 2-C-methyl-D-erythritol-4-phosphate pathway (MEP). IPP and DMAPP synthesize geranyl geranyl pyrophosphate (GGPP) through condensation reaction, and then copalyl / labdadienyl diphosphate Synthases (CPS) are used to catalyze the formation of the precursor skeleton of andrographolide, ent-copalol. So far, no CYP450 has been found to be able to catalyze the intermediates in the synthesis pathway of andrographolide. It is worth mentioning that in the synthesis pathway of andrographolide, hydroxylation is the only modification method for the synthesis of andrographolide. In the early stage, our laboratory has isolated and prepared the key intermediate (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol (CAS: 37886-56-9) from Andrographolide, which is used to explore the key CYP450 in the synthesis pathway of andrographolide and provide an important reference for the study of hydroxylation modification of diterpenoids.
[0004] Andrographis paniculata is a traditional Chinese medicine. Its anti-inflammatory, anti-cancer and immunomodulatory properties have been widely recognized, which makes it effective in treating various inflammations and diseases such as arthritis, ulcerative colitis, inflammatory bowel disease, etc. Its diterpenoid component andrographolide is the main medicinal secondary metabolite. Although andrographolide has important medical value, its biosynthetic pathway has not yet been elucidated. Faced with a large market demand, the only source of andrographolide is still plant extraction. Therefore, there is an urgent need to elucidate the biosynthetic pathway of andrographolide. Summary of the invention
[0005] In view of the fact that the above-mentioned existing andrographolide is a traditional Chinese medicine, it plays an important role in combating upper respiratory tract diseases and is known as a natural antibiotic. Andrographolide is used in large quantities in clinical practice, but currently it can only be obtained by plant extraction, which leads to the problem of tight supply of andrographolide, and the present invention is proposed.
[0006] Therefore, the purpose of the present invention is to provide a process for the biosynthesis pathway of andrographolide, which aims to: use transcriptomic association analysis to dig out the CYP450 gene that can catalyze the continuous oxidation of (4R, 5S, 9R, 10S)-labda-8(17), 13-dien-15, 19-diol and finally form new andrographolide aglycones, and use tobacco transient expression system to verify the formation of lactone ring. Not only provide important gene elements for the biosynthesis of andrographolide, but also provide key gene sites for molecular design breeding of andrographolide.
[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: a process for the biosynthesis pathway of andrographolide, comprising:
[0008] Step 1: Through differential expression analysis of transcriptome data, it was found that ApCYP71BE90 was specifically expressed in stems and leaves, which was consistent with the accumulation site of Andrographis paniculata;
[0009] Step 2, collecting mature leaves of Andrographis paniculata, using RNA extraction kit (Adlai, RN38 EASYspinplus) to extract RNA from the mature leaves of Andrographis paniculata, and obtaining cDNA through reverse transcription after the test is qualified;
[0010] Step 3: Design primer sequences, select SalI and XhoI as the connection sites, and design primers, use the sequenced pEASY-Blunt-ApCYP71BE90 as the template, and use 2*keypo for gene amplification;
[0011] Step 4, ligation reaction: Use SalI and XhoI to cut the PEAQ backbone, cut the gel together with the PCR product of ApCYP71BE90 obtained by amplification, and then use T4 ligase to connect after recovery. After connection, transform into T1 competent state, apply to Kan resistance plate, screen single clones for bacterial liquid PCR, and extract the recombinant plasmid from the successfully sequenced positive clones for subsequent experiments.
[0012] As a preferred scheme of the process for the biosynthesis pathway of andrographolide described in the present invention, wherein: the designed primer sequence, using cDNA as a template, using 2*Keypo high-fidelity enzyme to clone the ApCYP71BE90 gene fragment (2*Keypo high-fidelity enzyme; the total volume of the PCR system is 50 μL: 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 2 μL template, 25 μL2*Keypo Mix and 19 μL water, program), with the help of pEASY-Blunt vector, the ApCYP71BE90 fragment is successfully connected to the vector (the total volume of the connection system is 2.5 μL: 0.5 μL pEASY-Blunt vector and 2.0 μL cDNA template, 25°C connection reaction for 1 hour), the connection system is directly transformed into TransT1 competent state, and the positive clones are selected for sequencing (the total volume of the colony PCR system is 25 μL: 13 μL 2×Taq PCR Mix, 1 μL template, 1 μL forward primer, 1 μL reverse primer and 9 μL water), compared with the reference sequence, the nucleotide sequence has a 100% similarity with the original data.
[0013] As a preferred scheme for the process for the biosynthesis pathway of andrographolide described in the present invention, the total volume of the system in step three is 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.
[0014] As a preferred scheme for the process for the biosynthesis pathway of andrographolide described in the present invention, wherein: in the step four, the backbone and the fragment are connected overnight at 16 degrees Celsius using T4 ligase, transferred into the TransT1 competent state, and pEAQ-HT-ApCYP71BE90 positive clones are screened on 50 mg / mL Kan-resistant LB solid culture medium and PCR detected.
[0015] As a preferred scheme for the process of andrographolide biosynthesis pathway described in the present invention, wherein: the ApCYP71BE90 contains 1500 nucleotides, encodes a protein of 499 amino acids, and is officially named ApCYP71BE90.
[0016] The beneficial effects of the present invention are as follows: by utilizing transcriptomic association analysis, a CYP450 gene capable of catalyzing the continuous oxidation of (4R, 5S, 9R, 10S)-labda-8(17), 13-dien-15, 19-diol and ultimately forming a new andrographolide aglycone was discovered, and the formation of the lactone ring was verified using a tobacco transient expression system, which not only provides important gene elements for the biosynthesis of andrographolide, but also provides key gene sites for the molecular design and breeding of andrographolide. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. 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 creative labor. Among them:
[0018] Figure 1 The present invention provides a primer sequence table for cloning genes used in the process of andrographolide biosynthesis pathway.
[0019] Figure 2 The present invention provides a primer sequence table for constructing a vector for andrographolide biosynthesis process.
[0020] Figure 3 The 2*Keypo high-fidelity enzyme PCR reaction program table for the andrographolide biosynthesis pathway process of the present invention is shown.
[0021] Figure 4 The following is a colony PCR reaction program chart for the andrographolide biosynthesis pathway process of the present invention.
[0022] Figure 5 This is a diagram of the tissue location and MeJA response of ApCYP71BE90 used in the andrographolide biosynthesis pathway process of the present invention.
[0023] Figure 6 This is a GC-MS chart for the identification of (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol used in the andrographolide biosynthesis pathway process of the present invention.
[0024] Figure 7 This is a GC-MS chart for identifying the new product of ApCYP71BE90 used in the andrographolide biosynthesis pathway process of the present invention.
[0025] Figure 8 It is a comparison diagram of product fragments used in the process of andrographolide biosynthesis pathway of the present invention.
[0026] Fig. 9 The present invention provides a molecular structural formula diagram of (4R, 5S, 9R, 10S)-labda-8(17), 13-dien-15, 19-diol, neoandrographolide aglycone and andrographolide used in the andrographolide biosynthesis process.
[0027] Fig.10 This is a diagram of the ApCYP71BE90 gene cloning and vector construction for the andrographolide biosynthesis pathway process of the present invention. DETAILED DESCRIPTION
[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.
[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0030] Reference Figure 1-10 , as an embodiment of the present invention, provides a process for the biosynthesis pathway of andrographolide, and the process for the biosynthesis pathway of andrographolide comprises:
[0031] Step 1: Andrographolide has the characteristic of accumulating in large quantities in stems and leaves, so the transcriptome sequencing of different tissues of Andrographis paniculata plants was performed using the Illumina platform. Through differential expression analysis of transcriptome data, it was found that ApCYP71BE90 was highly expressed in stems and leaves, which is consistent with the accumulation site of Andrographis paniculata. (e.g. Figure 5 shown);
[0032] Step 2, collecting mature leaves of Andrographis paniculata, using RNA extraction kit (Adlai, RN38 EASYspinplus) to extract RNA from the mature leaves of Andrographis paniculata, and obtaining cDNA through reverse transcription after the test is qualified;
[0033] Step 3: Design primer sequences, select SalI and XhoI as the ligation sites, and design primers (such as Figure 2 As shown, wherein ApCYP71BE90-T4-F is SEQ ID NO.5, and ApCYP71BE90-T4-R is SEQ ID NO.6), the sequenced pEASY-Blunt-ApCYP71BE90 is used as a template, and 2*keypo is used for gene amplification;
[0034] Step 4, ligation reaction: Use SalI and XhoI to cut the PEAQ backbone, and cut and recover it together with the PCR product of ApCYP71BE90 obtained by amplification. After gel recovery, use T4 ligase to connect. After connection, transform into T1 competent state, apply to Kan resistance plate, screen single clones for bacterial liquid PCR, and extract recombinant plasmids from positive clones with successful sequencing for subsequent experiments.
[0035] The designed primer sequences (such as Figure 1 As shown, wherein the sequence of ApCYP71BE90-F is SEQ ID NO.3; the sequence of ApCYP71BE90-R is SEQ ID NO.4), cDNA was used as a template, and the ApCYP71BE90 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 program is as follows Figure 3 As shown in the figure, with the help of pEASY-Blunt vector, the ApCYP71BE90 fragment was successfully connected to the 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 TransT1 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, as shown in the figure). Figure 4 Compared with the reference sequence, the nucleotide sequence has a 100% similarity with the original data.
[0036] The total volume of the system in step 3 is 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 (such as Figure 3 shown).
[0037] In the step 4, the backbone and fragments were ligated overnight at 16°C using T4 ligase, and then transferred into TransT1 competent medium. Positive clones of pEAQ-HT-ApCYP71BE90 were screened on 50 mg / mL Kan-resistant LB solid medium and detected by PCR.
[0038] The ApCYP71BE90 contains 1500 nucleotides, encodes a protein of 499 amino acids, and is officially named ApCYP71BE90. The specific information is as follows:
[0039] Gene nucleotide sequence information-SEQ ID NO.1:
[0040]
[0041] Amino acid sequence - SEQ ID NO.2:
[0042] METTTIQLVILASFLFSILMLFKQAKKSRPNLPPGPPKLPLIGNLHNLIGGGMPHHAMHQLALKYGPLMHLKLGEVSTVVVSSPDAARQVMKTHDLNFASRPPLLAAEILNYGKSGVVFSPYGES WRHLRKICTLELLSNKRVQSFRPVRQKVHLELARMIAAGEGTAVDLSSKLYSSTYDVVSRAAVGESNKSRKARLMEIINEASELSAGFNIAELYPSIKVLGKITGLEKKLRKLHRETDELLEGII GDHKEAPEEEDGKKVDDLVDVLLKLQGEEHQSSLTSNSIKSVIFDILGAGSETSATTLDWAMTELLKNPPVMDRAQREVRAVFDRKGHVDESDINELKYLNSVIKETLRLHNPGPLLIPRMCIEA CRINGCDIPENTRVIVNAWAINRDPNYWQDPLSFTPERFLDSAMDYNGNSFEFLPFGAGRRICPGISFGVANIQNPLAVLLYHFDWKLPDGMKPEDIDMSEKFGVTTRRLINLRVVPVITRPLP*.
[0043] Verification of gene function
[0044] Refer to the experimental instructions of GV3101 Agrobacterium chemical transformation of Weidi Biotechnology for Agrobacterium transformation:
[0045] (1) Take out GV3101 Agrobacterium competent cells from the -80℃ refrigerator, put them in ice for 5 minutes, wait for them to melt, add 1μL of pEAQ-HT-ApCYP71BE90 positive plasmid DNA, put them in ice bath for 5 minutes, put them in liquid nitrogen for 5 minutes, put them in a 37℃ water bath for 5 minutes, put them in ice bath for 5 minutes, add 500μL of non-resistant LB liquid culture medium, shake them at 200rpm for 3 hours at 28℃, take them out, centrifuge them at 6000rpm for one minute to collect the bacteria, keep about 100μL of supernatant, gently blow to resuspend the bacteria and spread them on LB plates containing 50mg / L Kan and 50mg / L Rif antibiotics, and place them upside down in a 28℃ incubator for 2-3 days.
[0046] (2) Single colonies were picked from the LB plate with a sterilized toothpick and cultured 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 h. The upstream universal primers of pEAQ-HT-DEST and the downstream primers of ApCYP71BE90 were selected for PCR identification, and the positive strains were used for the next tobacco transient expression test.
[0047] Tobacco transient expression and product identification are as follows:
[0048] (1) Injection of Agrobacterium tumefaciens GV3101 into Nicotiana benthamiana leaves
[0049] The pEAQ-HT-ApCYP71BE90 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 in a shaking incubator at 28°C until OD600 was about 1.0; centrifuged at 4000 g for 5 min, the supernatant was discarded, the bacterial precipitate was collected, and resuspended with an equal volume of MMA solution (a mixed solution of 10 mM MES, 10 Mm MgCl2, and 200 μM acetosyringone) to OD600 of 0.2, and placed at room temperature for 3 h; select Nicotiana benthamiana with good growth, and gently pierce a small hole on the surface of the leaf with a syringe needle;
[0050] The control group was injected with only the substrate (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol (CAS: 37886-56-9), and the experimental group was injected with pEAQ-HT-ApCYP71BE90 and (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol. Three tobacco leaves were injected into each combination as a replicate. The injected tobacco leaves were cultured in the dark for 1 day, then taken out and cultured in the light for 4 days. The leaves injected with Agrobacterium were collected, freeze-dried for 48 hours, and then tested on the machine.
[0051] (2) Analysis and identification of catalytic products
[0052] The injected tobacco leaves were ground into powder in liquid nitrogen, and about 0.1 g of the powder was taken into 500 uL of ethyl acetate, and the product was extracted by ultrasonication for 10 min. After centrifugation at 6000 rpm for 5 min, 200 μL of the supernatant was transferred to an Agilent sample bottle for GC-MS detection.
[0053] Agilent 8250 was used for GC-MS detection. The gas chromatograph column was hb-5 (30m×0.25mm×0.25um, Aglient). The heating program was as follows: 50°C for 2 min, then heated to 280°C at a rate of 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 detector was Agilent 8250-Q-Tof, and the mass spectrometer scanning range was 50-500u.
[0054] GC-MS analysis results are as follows Figure 6-8 As shown in Figure 2, (4R,5S,9R,10S)-labda-8(17)13-dien-15,19-diol (CAS: 37886-56-9) is a standard obtained by our laboratory after separation and purification from Andrographis paniculata. When (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol was injected into tobacco alone, its peak was 24.935 (as shown in Figure 2). Figure 6 shown);
[0055] When ApCYP71BE90 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 consumed and a product peak with 205 as a characteristic fragment appeared at 26.677 (e.g. Figure 7 The peak time and characteristic fragments are consistent with those of the new andrographolide aglycone standard (CAS: 82209-74-3) (as shown in Figure 8 shown);
[0056] It can be speculated that ApCYP71BE90 can oxidize (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol and form new andrographolide aglycones.
[0057] The present invention utilizes transcriptomic association analysis to discover the CYP450 gene that can catalyze the continuous oxidation of (4R,5S,9R,10S)-labda-8(17),13-dien-15,19-diol and ultimately form new andrographolide aglycones, and uses a tobacco transient expression system to verify the formation of the lactone ring, which not only provides important gene elements for the biosynthesis of andrographolide, but also provides key gene sites for the molecular design and breeding of andrographolide.
[0058] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.
Claims
1. A process for the biosynthesis of andrographolide, characterized in that: The steps include: Step 1: Through differential expression analysis of transcriptome data, it was found that ApCYP71BE90 was specifically expressed in stems and leaves, which was consistent with the accumulation site of Andrographis paniculata; Step 2, collecting mature leaves of Andrographis paniculata, using RNA extraction kit (Adlai, RN38 EASYspin plus) to extract RNA from the mature leaves of Andrographis paniculata, and obtaining cDNA through reverse transcription after the test is qualified; Step 3: Design primer sequences, select SalI and XhoI as the connection sites, and design primers, use the sequenced pEASY-Blunt-ApCYP71BE90 as the template, and use 2*keypo for gene amplification; Step 4, ligation reaction: Use SalI and XhoI to cut the PEAQ backbone, and cut and recover it together with the PCR product of ApCYP71BE90 obtained by amplification. After gel recovery, use T4 ligase to connect. After connection, transform into T1 competent state, apply to Kan resistance plate, screen single clones for bacterial liquid PCR, and extract recombinant plasmids from positive clones with successful sequencing for subsequent experiments.
2. The process for the biosynthesis of andrographolide according to claim 1, characterized in that: The primer sequences were designed, cDNA was used as a template, and 2*Keypo high-fidelity enzyme was used to clone the ApCYP71BE90 gene fragment (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, program), with the help of pEASY-Blunt vector, the ApCYP71BE90 fragment was successfully connected to the vector (the total volume of the connection system was 2.5 μL: 0.5 μL pEASY-Blunt vector and 2.0 μL cDNA template, 25°C connection reaction for 1 hour), the connection system was directly transformed into TransT1 competent state, 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), compared with the reference sequence, the nucleotide sequence has a 100% similarity with the original data.
3. The process for the biosynthesis of andrographolide according to claim 1, characterized in that: The total volume of the system in step 3 is 50 μL: 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 2 μL template, 25 μL 2*KeypoMix and 19 μL water.
4. The process for the biosynthesis of andrographolide according to claim 1, characterized in that: In the step 4, the backbone and fragments were ligated overnight at 16°C using T4 ligase, and then transferred into TransT1 competent medium. Positive clones of pEAQ-HT-ApCYP71BE90 were screened on 50 mg / mL Kan-resistant LB solid medium and detected by PCR.
5. The process for the biosynthesis of andrographolide according to claim 1, characterized in that: The ApCYP71BE90 contains 1500 nucleotides, encodes a protein of 499 amino acids, and is officially named ApCYP71BE90.