Method for synthesizing various diterpenoid compounds from beginning by utilizing nicotiana benthamiana and protein combination used by method

Abstract

The invention discloses a method for synthesizing various diterpenoid compounds from beginning by utilizing nicotiana benthamiana and a protein combination used by the method. According to the present invention, through precursor optimization, diterpene synthase gene combination screening and CYP450 region chamber transformation, the protein combination used for synthesizing a variety of diterpenoid compounds is obtained, wherein the protein combination comprises TwGGPPS1, TwDXS, AstHMGR, RoCPS1, SmKSL1, CfTPS3, tpS3 / tpTS-CYP82D274, tpS3 / tpTS-CYP71BE86, tpS3 / tpTS-tCYP71BE85 and tpS3 / tpTS-tCYP82D213; the protein combination is utilized to successfully realize de novo synthesis of a plurality of tripterygium wilfordii terpenoid active ingredients (triptolide ketone, triptophenolide, triptonin B, hypotanshinone diene, arosin triene and 14-hydroxy arosin triene) in the Bensi tobacco.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for de novo synthesis of various diterpenoid compounds using Nicotiana benthamiana and the protein combination used therein. Background Technology

[0002] Tripterygium wilfordii (a medicinal plant) Tripterygium wilfordii The dried root of *Tripterygium wilfordii* (also known as *Hook. f.*) is a traditional Chinese medicine used for its anti-inflammatory and antirheumatic properties, with a medicinal history dating back to the Ming Dynasty (1476 AD). This plant is rich in apinane-type diterpenoids, which are the main components contributing to its clinical efficacy. These include triptolide, which has potential as a non-hormonal male contraceptive; triptolide and triptolide B, used to treat rheumatoid arthritis; and 14-hydroxyascorbic acid triene and ascorbic acid triene, which possess cytotoxic, immunomodulatory, antifungal, and antiviral activities. However, these compounds are present in extremely low concentrations within the plant (e.g., triptolide is only 0.0001%). Traditional extraction and separation methods not only damage valuable plant resources but also suffer from low extraction efficiency and high costs. Furthermore, the complex chiral structures of these compounds pose significant challenges to chemical synthesis, thus limiting further research and drug development.

[0003] Currently, existing research has elucidated that the aforementioned diterpenoid compounds originate from the same biosynthetic pathway—including a cyclization reaction catalyzed by diterpene synthases (diTPSs) to generate arosinane-type parent nuclei, tanshinone diene and arosintriene, followed by four CYP450-mediated reactions: hydroxylation, methyl transfer, lactone formation, and triepoxidation, sequentially generating 14-hydroxyaspintriene, triptolide B, triptolide lactone, and triptolide ketone. Based on a thorough understanding of the biosynthetic pathway, current synthetic biology primarily utilizes heterologous plant chassis or microbial chassis to rapidly and greenly produce target active ingredients. However, the arosinane-type diterpenoid compounds in this application exhibit weak CYP450 gene catalytic activity in their biosynthetic pathway and show characteristics of expression in different compartments of the cell compared to upstream diTPSs, posing a significant challenge to the de novo synthesis of these compounds using a microbial chassis. (Note: The last sentence about *Nicotiana benthamiana* appears to be unrelated and likely refers to a different application.) Nicotiana benthamiana Plant chassis systems, such as those for diterpenoids, have less difference from the plants from which these compounds originate. They can provide the protein microenvironment and cellular compartments required for compound synthesis to the greatest extent possible, making them a better choice for de novo synthesis of these complex and high-value diterpenoid active ingredients. Summary of the Invention

[0004] The purpose of this invention is to provide a method for de novo synthesis of various diterpenoid compounds using *Nicotiana benthamiana* and the protein combination used therein.

[0005] This invention first provides a protein combination.

[0006] The protein combination provided by this invention includes TwGGPPS1 protein, TwDXS protein, and AstHMGR protein.

[0007] Furthermore, the protein combination also includes any one of the following protein combinations A1)-A3): A1) RoCPS1 protein and SmKSL1 protein; A2) RoCPS1 and CfTPS3 proteins; A3) RoCPS1 protein, SmKSL1 protein, and CfTPS3 protein.

[0008] Furthermore, the protein combination also includes any one or a combination of proteins from B1) to B3): B1) CYP82D274 protein; B2) CYP82D274 protein, CYP71BE86 protein, CYP71BE85 protein and CYP82D213 protein; B3) CYP82D274 protein with N-terminal fusion of chloroplast signal peptide, CYP71BE86 protein with N-terminal fusion of chloroplast signal peptide, tCYP71BE85 protein with N-terminal fusion of chloroplast signal peptide, and tCYP82D213 protein with N-terminal fusion of chloroplast signal peptide.

[0009] Furthermore, the chloroplast signal peptide is chloroplast signal peptide tpS3 or chloroplast signal peptide tpTS.

[0010] The amino acid sequence of the tpS3 signal peptide is shown in Sequence 2.

[0011] The amino acid sequence of the tpTS signal peptide is shown in Sequence 4.

[0012] The amino acid sequence of any of the TwGGPPS1 proteins described above is shown in the NCBI reference sequence number AKQ99275.1.

[0013] The amino acid sequence of any of the TwDXS proteins described above is shown in the NCBI reference sequence number AKP20998.1.

[0014] The amino acid sequence of any of the AstHMGR proteins described above is shown in Sequence 6.

[0015] The amino acid sequence of any of the RoCPS1 proteins described above is shown in NCBI reference sequence number AHL67261.1.

[0016] The amino acid sequence of any of the SmKSL1 proteins described above is shown in the NCBI reference sequence number ABV08817.1.

[0017] The amino acid sequence of any of the CfTPS3 proteins described above is shown in the NCBI reference sequence number AHW04048.1.

[0018] The amino acid sequence of any of the CYP82D274 proteins described above is shown in the NCBI reference sequence number UTK45969.1.

[0019] The amino acid sequence of any of the CYP71BE86 proteins described above is shown in the NCBI reference sequence number UTK45967.1.

[0020] The amino acid sequence of any of the CYP71BE85 proteins described above is shown in the NCBI reference sequence number UTK45966.1.

[0021] The amino acid sequence of any of the tCYP71BE85 proteins described above is the sequence obtained by removing amino acids 1-26 from the amino acid sequence of the CYP71BE85 protein.

[0022] The amino acid sequence of any of the CYP82D213 proteins described above is shown in the NCBI reference sequence number UTK45968.1.

[0023] The amino acid sequence of any of the tCYP82D213 proteins mentioned above is the sequence obtained by removing amino acids 1-70 from the amino acid sequence of the CYP82D213 protein.

[0024] In some specific embodiments, the protein combination consists of TwGGPPS1 protein, TwDXS protein, AstHMGR protein, RoCPS1 protein, SmKSL1 protein, and CYP82D274 protein.

[0025] In some specific embodiments, the protein combination consists of TwGGPPS1 protein, TwDXS protein, AstHMGR protein, RoCPS1 protein, CfTPS3 protein, and CYP82D274 protein.

[0026] In some specific embodiments, the protein combination consists of TwGGPPS1 protein, TwDXS protein, AstHMGR protein, RoCPS1 protein, SmKSL1 protein, CfTPS3 protein, and CYP82D274 protein.

[0027] In some specific embodiments, the protein assembly consists of TwGGPPS1 protein, TwDXS protein, AstHMGR protein, RoCPS1 protein, SmKSL1 protein, CfTPS3 protein, CYP82D274 protein, CYP71BE86 protein, CYP71BE85 protein, and CYP82D213 protein.

[0028] In some preferred embodiments, the protein combination comprises TwGGPPS1 protein, TwDXS protein, AstHMGR protein, RoCPS1 protein, SmKSL1 protein, CfTPS3 protein, tpS3-CYP82D274 protein, tpS3-CYP71BE86 protein, tpS3-tCYP71BE85 protein, and tpS3-tCYP82D213 protein.

[0029] In some preferred embodiments, the protein combination consists of TwGGPPS1 protein, TwDXS protein, AstHMGR protein, RoCPS1 protein, SmKSL1 protein, CfTPS3 protein, tpTS-CYP82D274 protein, tpTS-CYP71BE86 protein, tpTS-tCYP71BE85 protein, and tpTS-tCYP82D213 protein.

[0030] The present invention also provides a nucleic acid molecule encoding the above-mentioned protein combination.

[0031] The nucleic acid molecule can be a DNA molecule, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be an RNA molecule, such as mRNA or hnRNA.

[0032] The nucleotide sequence of the DNA molecule encoding the TwGGPPS1 protein is shown in NCBI reference sequence number KM978332.1.

[0033] The nucleotide sequence of the DNA molecule encoding the above-mentioned TwDXS protein is shown in the NCBI reference sequence number KM879186.1.

[0034] The nucleotide sequence of the DNA molecule encoding the AstHMGR protein is shown in Sequence 5.

[0035] The nucleotide sequence of the DNA molecule encoding the RoCPS1 protein is shown in Sequence 7.

[0036] The nucleotide sequence of the DNA molecule encoding the SmKSL1 protein is shown in Sequence 8.

[0037] The nucleotide sequence of the DNA molecule encoding the CfTPS3 protein is shown in Sequence 9.

[0038] The nucleotide sequence of the DNA molecule encoding the CYP82D274 protein is shown in Sequence 10.

[0039] The nucleotide sequence of the DNA molecule encoding the CYP71BE86 protein is shown in Sequence 11.

[0040] The nucleotide sequence of the DNA molecule encoding the CYP71BE85 protein is shown in Sequence 12.

[0041] The nucleotide sequence of the DNA molecule encoding the tCYP71BE85 protein is obtained by removing bases 1-78 from the nucleotide sequence of the DNA molecule encoding the CYP71BE85 protein.

[0042] The nucleotide sequence of the DNA molecule encoding the CYP82D213 protein is shown in Sequence 13.

[0043] The nucleotide sequence of the DNA molecule encoding the tCYP82D213 protein is obtained by removing bases 1-210 from the nucleotide sequence of the DNA molecule encoding the CYP82D213 protein.

[0044] The nucleotide sequence encoding the above-mentioned tpS3 signal peptide is shown in Sequence 1.

[0045] The nucleotide sequence encoding the above-mentioned tpTS signal peptide is shown in Sequence 3.

[0046] The present invention also provides biological materials containing the above-mentioned nucleic acid molecules; the biological materials are expression cassettes, recombinant vectors, recombinant microorganisms, transgenic plant cell lines, transgenic plant tissues, or transgenic plant organs.

[0047] The expression cassette described above may include a promoter, the aforementioned nucleic acid molecule, and a terminator. Promoters that can be used in this invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Furthermore, the expression cassette may also include an enhancer sequence.

[0048] The vector described above refers to a vector capable of delivering the aforementioned nucleic acid molecules into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). In some embodiments, the vector is pEAQ-HT.

[0049] The recombinant vector described above refers to a recombinant DNA molecule constructed by linking the aforementioned nucleic acid molecule to the vector in vitro. Existing plant expression vectors can be used to construct recombinant vectors containing the aforementioned nucleic acid molecules. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as by adding genes (GFP gene, GUS gene, luciferase gene, etc.) that can be expressed in plants and encode enzymes or luminescent compounds that produce color changes.

[0050] In some embodiments, the recombinant vector is the recombinant expression vector pEAQ-HT:: as described in the examples below. TwGGPPS1 pEAQ-HT:: TwDXS pEAQ-HT:: AstHMGR pEAQ-HT:: RoCPS1 pEAQ-HT:: SmKSL1 pEAQ-HT:: CfTPS3 pEAQ-HT:: CYP82D274 pEAQ-HT:: CYP71BE86 pEAQ-HT:: CYP71BE85 and pEAQ-HT:: CYP82D213 .

[0051] In some embodiments, the recombinant vector is the recombinant expression vector pEAQ-HT::( in the examples below). tpS3-CYP82D274 ), pEAQ-HT::( tpS3-CYP71BE86 ), pEAQ-HT::( tpS3-tCYP71BE85 ), pEAQ-HT::( tpS3-tCYP82D213 ), pEAQ-HT::( tpTS-CYP82D274 ), pEAQ-HT::( tpTS-CYP71BE86 ), pEAQ-HT::( tpTS-tCYP71BE85 ) and pEAQ-HT::( tpTS-tCYP82D213 ).

[0052] The microorganisms mentioned above can be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. In some embodiments, the bacteria are Agrobacterium (such as Agrobacterium GV3101).

[0053] The recombinant microorganisms mentioned above refer to those obtained by manipulating and modifying the genes of a target microorganism, thereby altering its function. For example, recombinant microorganisms obtained after introducing the aforementioned recombinant vector into a target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring may not necessarily be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0054] In some embodiments, the recombinant microorganism is a microorganism containing the recombinant expression vector pEAQ-HT:: described in the examples below. TwGGPPS1 pEAQ-HT:: TwDXS pEAQ-HT:: AstHMGR pEAQ-HT:: RoCPS1 pEAQ-HT:: SmKSL1 pEAQ-HT:: CfTPS3 pEAQ-HT:: CYP82D274 pEAQ-HT:: CYP71BE86 pEAQ-HT:: CYP71BE85 pEAQ-HT:: CYP82D213 pEAQ-HT::( tpS3-CYP82D274 ), pEAQ-HT::( tpS3-CYP71BE86 ), pEAQ-HT::( tpS3-tCYP71BE85 ), pEAQ-HT::( tpS3-tCYP82D213 ), pEAQ-HT::( tpTS-CYP82D274 ), pEAQ-HT::( tpTS-CYP71BE86 ), pEAQ-HT::( tpTS-tCYP71BE85 ) and pEAQ-HT::( tpTS- tCYP82D213 Agrobacterium GV3101.

[0055] The transgenic plant tissues described above may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0056] The organs of any of the transgenic plants mentioned above may be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0057] The transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs described above may or may not include propagation material.

[0058] This invention also provides new uses for the above-mentioned protein combinations, nucleic acid molecules, or biological materials.

[0059] This invention provides the application of the above-mentioned protein combination, nucleic acid molecule, or biological material in the synthesis of diterpenoid compounds or the preparation of products containing synthetic diterpenoid compounds.

[0060] In some embodiments, a protein combination consisting of TwGGPPS1 protein, TwDXS protein, AstHMGR protein, RoCPS1 protein, SmKSL1 protein and CYP82D274 protein can be used to synthesize 14-hydroxyascorbic acid triene.

[0061] In some implementations, a protein combination consisting of TwGGPPS1, TwDXS, AstHMGR, RoCPS1, CfTPS3, and CYP82D274 proteins can be used to synthesize 14-hydroxyascorbic acid triene.

[0062] In some embodiments, a protein combination consisting of TwGGPPS1, TwDXS, AstHMGR, RoCPS1, SmKSL1, CfTPS3, and CYP82D274 proteins can be used to synthesize 14-hydroxyascorbic acid triene.

[0063] In some embodiments, a protein combination consisting of TwGGPPS1, TwDXS, AstHMGR, RoCPS1, SmKSL1, CfTPS3, CYP82D274, CYP71BE86, CYP71BE85, and CYP82D213 proteins can be used to synthesize triptolide, triptolide, triptolide B, tanshinone diene, asparagine, and 14-hydroxyasparagine.

[0064] In some embodiments, a protein combination consisting of TwGGPPS1 protein, TwDXS protein, AstHMGR protein, RoCPS1 protein, SmKSL1 protein, CfTPS3 protein, tpS3-CYP82D274 protein, tpS3-CYP71BE86 protein, tpS3-tCYP71BE85 protein, and tpS3-tCYP82D213 protein can be used to synthesize triptolide, triptolide, triptolide B, tanshinone diene, asparagine, and 14-hydroxyasparagine.

[0065] In some embodiments, a protein combination consisting of TwGGPPS1, TwDXS, AstHMGR, RoCPS1, SmKSL1, CfTPS3, tpTS-CYP82D274, tpTS-CYP71BE86, tpTS-tCYP71BE85, and tpTS-tCYP82D213 proteins can be used to synthesize triptolide, triptolide, triptolide B, tanshinone diene, asparagine, and 14-hydroxyasparagine.

[0066] Finally, this invention also provides a method for synthesizing diterpenoid compounds.

[0067] The method for synthesizing diterpenoids provided by the present invention includes the following steps: increasing the content and / or activity of proteins in the above-mentioned protein combination in plants to obtain transgenic plants; and isolating diterpenoids from the transgenic plants.

[0068] In the above method, the method for increasing the content and / or activity of the proteins in the above protein combination in the plant is to transiently express the proteins in the above protein combination in the plant.

[0069] In the above method, the transient expression is Agrobacterium-mediated transient expression.

[0070] In the above method, the plant is Nicotiana benthamiana.

[0071] The diterpenoids mentioned above include Tripterygium wilfordii terpenoid active ingredients.

[0072] In some embodiments, the tripterygium terpenoid active ingredient includes at least one of tripterygium lactone, tripterygium lactone, tripterygium phenanthrene, tanshinone diene, asparagine triene, and 14-hydroxyasparagine triene.

[0073] This invention, through precursor optimization, diterpene synthase gene combination screening, and CYP450 compartmentalization modification, obtained the optimal protein combination for the de novo synthesis of various triptolide active components from tobacco: TwGGPPS1, TwDXS, AstHMGR, RoCPS1, SmKSL1, CfTPS3, tpS3 / tpTS-CYP82D274, tpS3 / tpTS-CYP71BE86, tpS3 / tpTS-tCYP71BE85, and tpS3 / tpTS-tCYP82D213. Experiments demonstrated that this optimal protein combination can successfully achieve the de novo synthesis of various triptolide active components (triptolide lactone, triptolide, triptolide B, tanshinone diene, asparagine triene, and 14-hydroxyasparagine triene) from tobacco. This invention provides a basis for further research and synthesis of... T. wilfordii These compounds, as well as other bio-similar diterpenoid active compounds, provide suitable methods. Attached Figure Description

[0074] Figure 1 This method utilizes the Benzoic tobacco chassis to synthesize various diterpenoid compounds de novo. Note: In the diagram, "1" represents triptolide; "2" represents tanshinone diene; "3" represents asarin triene; "4" represents 14-hydroxyasarin triene; "5" represents triptolide B; and "6" represents triptolide. Detailed Implementation

[0075] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0076] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0077] The experimental reagents used in the following examples were obtained from the following sources: gene cloning and plasmid construction were performed using *E. coli*. Trans 1-T1 cells were used (Beijing TransGen Biotechnology Co., Ltd.); transient transformation of tobacco was performed using Agrobacterium GV3101 (pSoup-p19) competent cells (Shanghai Weidi Biotechnology Co., Ltd.); functional verification and de novo biosynthesis were performed using the pEAQ-HT vector (Biovector NTCC), and subcellular localization experiments were performed using the pCAMBIA1300-GFP vector (Biovector NTCC); gene PCR amplification was performed using KOD DNA polymerase (Toyobo Co., Ltd., Japan); PCR product purification was performed using an agarose gel DNA extraction kit (Thermo Fisher Scientific, USA); the chemical standards triptolide, triptolide, and triptolide B were all products of Shanghai Yuanye Biotechnology Co., Ltd. The chemical standards tanshinone diene, acrylonitrile, and 14-hydroxyacrylonitrile were prepared and preserved in our laboratory.

[0078] The plant (Nicotiana benthamiana) material culture method in the following examples is as follows: Nicotiana benthamiana plants were sown and cultivated using sterile nutrient soil. Before harvest, the plants were continuously placed in a constant temperature incubator at 25°C, using red and blue light as the light source, with a light cycle of 16 hours of light and 8 hours of darkness alternating.

[0079] The method for transient expression of Agrobacterium-mediated transgenic expression in *Nicotiana benthamiana* in the following examples is as follows: The constructed recombinant plasmid was transformed into GV3101 (pSoup-p19) competent cells. Transformed cells were seeded on LB agar plates containing 50 mg / L kanamycin and 50 mg / L rifampin and cultured at 30°C for 2-3 days. Single colonies were then inoculated into liquid LB medium containing kanamycin and rifampin. Agrobacterium strains carrying the target recombinant plasmid were cultured with shaking at 30°C for approximately 12 hours as seed culture, followed by amplification at a 1:10 ratio. The OD of each culture was determined based on the total number of infected strains. 600 Value, in equal OD 600 Mix all strains and increase total OD 600The concentrations were added together to 1, and then centrifuged at 4000×g for 5 min. The precipitate was collected and resuspended in tobacco conversion buffer (200 μM AS, 10 mM MES, 10 mM MgCl2), and incubated at 30℃ for 1–3 h before infection. To ensure sample sufficiency and result reliability, each suspension was used to infect multiple *Nicotiana benthamiana* plants in parallel. During infection, a 1 mL needleless syringe was used to inject the suspension into the underside of the leaves of 4–5 week old tobacco plants until the leaf surface was evenly saturated. After infection, the plants were grown in darkness for 12 h, followed by red-blue cyclical light to promote further growth, and continued to be cultured under light for 6 days before sample collection and subsequent processing.

[0080] The bioinformatics analysis methods in the following embodiments are as follows: transmembrane domains are predicted using TMHMM 2.0 software (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ); subcellular localization is predicted using the Plant-mPLoc module of Cell-PLoc2.0 (http: / / www.csbio.sjtu.edu.cn / bioinf / plant-multi / ).

[0081] The subcellular localization analysis method in the following examples is as follows: After infiltration with Agrobacterium for 36 to 48 hours, a small piece of leaf tissue was taken from the infiltrated area and placed on a glass slide with the back side facing up. A small amount of purified water was added, a coverslip was placed on top, and the sample was then inverted and placed on the microscope stage. Fluorescence imaging was performed using a Zeiss LSM880 ZEN laser confocal microscope with the following specific parameter settings: green GFP fluorescence was collected in the wavelength range of 493-574 nm using a 488 nm laser; red m-Cherry fluorescence was collected in the wavelength range of 587-610 nm using a 543 nm laser; and chloroplast autofluorescence was collected in the wavelength range of 658-683 nm using a 543 nm laser.

[0082] The plant sample collection and metabolite extraction methods in the following examples are as follows: Tobacco samples were collected on day 6 post-infection. The sample processing procedure is as follows: After processing, tobacco leaves were immediately flash-frozen in liquid nitrogen and stored at -80°C for at least 4 hours. After lyophilization for 48-72 hours, the frozen tissues were ground into a uniform fine powder using a grinder. All powder samples were extracted twice with 8 times their volume of ethyl acetate, concentrated, and divided into two equal portions. One portion was reconstituted with 100 μL of chromatographic grade ethyl acetate, and the other portion was reconstituted with 100 μL of chromatographic grade methanol. The above test solutions were then analyzed.

[0083] The qualitative and quantitative detection methods for the products in the following examples are as follows: Volatile products tanshinone diene, acrylonitrile, and 14-hydroxyacrylonitrile in the samples were analyzed using GC-TQ-MS / MS. Tripterygium lactone, triptolide B, and triptolide were detected using UPLC-qTOF-MS and UPLC-TQ-MS / MS, respectively.

[0084] GC-TQ-MS / MS analysis was performed using an Agilent 7890B GC system equipped with a 7000C GC / MS triple quadrupole and a DB-5MS (30m × 0.25mm × 0.1μm) column. The temperature program was set as follows: initial temperature 50℃ held for 1 min, then increased to 240℃ at 50℃ / min, then increased to 255℃ at 1.5℃ / min, and finally increased to 300℃ at 50℃ / min and held for 1 min. Helium was used as the carrier gas at a flow rate of 1 mL / min. The injection port temperature was set to 300℃, the ion trap temperature to 250℃, the electron energy to 70 eV, and the mass spectrometry data acquisition range was [missing information]. m / z 10-400. Data analysis was performed using qualitative analysis software (version B.07.00).

[0085] UPLC-qTOF-MS (Waters) was performed using an Acquity UPLC HSS T3 column (2.1 mm × 100 mm × 1.8 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. The gradient program was set as follows: 0-3 min, 5% B; 3-4 min, 5-45% B; 4-8 min, 45-50% B; 8-15 min, 50-55% B; 15-21 min, 55-65% B; 21-23 min, 65-100% B; 23-28 min, isocratic hold-up, 100% B. The flow rate was set to 0.4 mL / min. Each run analyzed 2 μL of sample, and the column temperature was 30℃. Mass spectrometry was performed in positive and negative ion modes, and the scan range was [not specified]. m / z The mass spectrometry range was 50-1200 V, with a single scan time of 0.2 s. The mass spectrometry parameters were set as follows: analyzer mode; normal dynamic range; collision energy gradient 10-50 V; cone voltage 40 V. Data analysis was performed using MassLynx v4.2 software.

[0086] UPLC-TQ-MS / MS analysis was performed using a UPLC-TQ-TRAP 6500+QQQ-MS (Epoch Semiconductor, USA). Chromatographic separation was performed on a T3 column (2.1×100 mm, 1.8 μm, Waters) at 40 °C. The mobile phase consisted of acetonitrile (A) and an aqueous solution (B) containing 0.1% formic acid, with a flow rate of 0.4 mL / min and the following gradient program: 0–3.5 min, A phase 30%–80%; 3.5–4 min, A phase 80%–95%; 4–4.5 min, A phase 95%–30%; 4.5–6.5 min, A phase maintained at 30%. Specific test parameters: Tripterygium wilfordii B, Q1 Mass (DA) 315.100, Q3 Mass (DA) 227.100, declustering voltage DP (volts) 177.000, collision energy CE (volts) 27.00, collision chamber outlet voltage CXP (volts) 7.000; Tripterygium wilfordii lactone, Q1 313.100, Q3 225.200, DP (volts) 157.000, CE 28.00, CXP 7.000; Tripterygium wilfordii lactone, Q1 359.000, Q3 143.400, DP 182.000, CE 40.00, CXP 15.000.

[0087] Example 1: A method for de novo synthesis of various tripterpenoid active ingredients using Nicotiana benthamiana. I. Preliminary Functional Study To verify that tobacco can serve as a heterologous expression system for the complex diterpenoid synthases (diTPSs) and CYP450s from Tripterygium wilfordii, transient expression of these genes was performed via de novo substrate synthesis. The results showed that all genes catalyzed the formation of their respective products. Therefore, the diterpenoid synthases TwCPS1 (the TwCPS1 gene sequence is shown in Sequence 14, and the amino acid sequence of the encoded TwCPS1 protein is shown in Sequence 15) and TwMS (the TwMS gene sequence is shown in Sequence 16, and the amino acid sequence of the encoded TwMS protein is shown in Sequence 17), along with downstream CYP82D274, CYP71BE86, CYP71BE85, and CYP82D213, were co-transformed into tobacco to integrate the entire metabolic pathway. However, except for triptolide (…), Figure 1 (A) The fact that most intermediates in the biosynthetic pathways were detectable indicates that the low content of precursor substances limits the biosynthesis of subsequent products. To achieve de novo synthesis of the target diterpenoid compound, a multi-level modification strategy is required.

[0088] II. Optimization of Isoprene Precursors Geraniylgeraniyl pyrophosphate (GGPP), as a universal precursor of diterpenoids, plays a crucial role in the overall flux of metabolic pathways. To improve GGPP synthesis efficiency, this invention optimizes the methyl erythritol phosphate (MEP) and mevalonic acid (MVA) pathways, designing a synthesis from Tripterygium wilfordii (GGPP). T. wilfordii TwGGPPS1, TwGGPPS8, TwDXS and rough oats (from) Avena strigosa Multiple combinations of AstHMGR from various sources (see details for specific combinations) Figure 1 B), and co-infected tobacco plants with TwCPS1 and TwMS. Meanwhile, tobacco plants infected only with TwCPS1 and TwMS served as a control group.

[0089] Experimental results showed that overexpression of the rate-limiting enzyme in the MVA / MEP pathway significantly increased the yield of diterpenoid skeletons. The optimal combination, TwGGPPS1+TwDXS+AstHMGR, increased the total diterpenoid skeleton flux of asarintriene and tanshinone diene by 9.25 times compared to the control group without optimized GGPP. Specifically, the yields of asarintriene and tanshinone diene increased by 14.60 times and 3.89 times, respectively. Figure 1 B).

[0090] III. Screening of Diterpene Synthase Gene Combinations After obtaining sufficient precursor GGPP, this invention further focused the optimization steps on synthesizing the diterpenoid skeletons acrylonitrile and tanshinone diene, and screened for the optimal combination of diterpenoid synthases (diTPSs). This invention integrates enzymes derived from Tripterygium wilfordii (Tripterygium wilfordii) T. wilfordii )of TwCPS1 , TwMS Danshen ( Salvia miltiorrhiza )of SmCPS1 , SmKSL1 Greek sage ( S. fruticose )of SfKSL Hairy throat coleopterus ( Coleus forskohlii )of CfTPS1 , CfTPS3 , CfTPS4 , European summer solstice grass ( Marrubium vulgare )of MvCPS3 , MvELS and rosemary ( Rosmarinus officinalis )of RoCPS1 , RoKSL1 , RoKSL2 Thirteen candidate diterpenoid synthase genes were selected to evaluate the effect of combinations of class II and class I diterpenoid synthases on the synthesis of diterpenoid skeletons in *Nicotiana benthamiana*. Preliminary screening revealed that five diterpenoid synthase combinations significantly increased the yields of the products tanshinone diene and asarin triene. Figure 1C), and then these five combinations were co-expressed with CYP82D274 to evaluate their ability to modify diterpenes. Among them, the RoCPS1+SmKSL1 and RoCPS1+CfTPS3 combinations produced the highest content of 14-hydroxyarsinotriene, which was 4.59 times and 2.87 times higher than the control group TwCPS1+TwMS, respectively. Figure 1 D).

[0091] This invention also discovered that co-expressing the three diterpene synthases RoCPS1+SmKSL1+CfTPS3 significantly increased the yield of 14-hydroxyascorbic acid triene, reaching 17.16 times that of the control group TwCPS1+TwMS. This further enhances OD... 600 Subsequently, it was found that the yield of 14-hydroxyascorbic acid triene increased by 29.41 times. Figure 1 D). Thus, it was finally confirmed that the combination of the six genes TwGGPPS1, TwDXS, AstHMGR, RoCPS1, SmKSL1, and CfTPS3 optimized the supply of CYP450 precursors.

[0092] IV. Subcellular localization analysis of DiTPSs and CYP450s The subcellular localization of CYP450s and diTPSs was predicted using an online website. The predictions showed that all diTPSs were located in chloroplasts, while all CYP450s were located in the endoplasmic reticulum (ER). Subcellular localization was further confirmed by fusing this gene with green fluorescent protein (GFP) and transforming it into *Nicotiana benthamiana* leaves. This confirmed that four CYP450s were located in the ER, while all diTPSs were located in chloroplasts. Figure 1 F and 1G original positioning).

[0093] V. Research on the Zonal Remodeling of CYP450s This invention employs a chloroplast compartmentalization metabolic engineering strategy to relocate CYP450 enzymes from the endoplasmic reticulum to the chloroplasts containing diterpenoid synthases, reducing the physical distance between intermediate metabolites and catalytic enzymes to improve transformation efficiency. The specific method is as follows: Chloroplast signal peptide tpS3 (positions 1-43 of the N-terminus of CfTPS3 protein, denoted as tpS3, amino acid sequence as shown in Sequence 2, encoding nucleotide sequence as shown in Sequence 1) and chloroplast signal peptide tpTS (positions 1-59 of the N-terminus of taxadiene synthase TS protein, denoted as tpTS, amino acid sequence as shown in Sequence 4, encoding nucleotide sequence as shown in Sequence 3) are fused with CYP82D274, CYP71BE86, truncated CYP71BE85 (tCYP71BE85), and truncated CYP82D213 (tCYP82D213), and co-expressed with GFP to observe their subcellular localization. Experimental results showed that these compartmentalized CYP450 enzymes all overlapped with the autofluorescent region of chloroplasts, but separated from their original endoplasmic reticulum locations, confirming that tpS3 / tpTS-CYP82D274, tpS3 / tpTS-CYP71BE86, tpS3 / tpTS-tCYP71BE85, and tpS3 / tpTS-tCYP82D213 were successfully transferred to chloroplasts. Figure 1 (After G was modified).

[0094] VI. De novo synthesis of various Tripterygium wilfordii terpenoid active ingredients using Nicotiana benthamiana. After the above optimization, the optimal enzyme combination used in this invention for the de novo synthesis of various Tripterygium wilfordii terpenoid active ingredients from Nicotiana benthamiana is as follows: TwGGPPS1, TwDXS, AstHMGR, RoCPS1, SmKSL1, CfTPS3, tpS3 / tpTS-CYP82D274, tpS3 / tpTS-CYP71BE86, tpS3 / tpTS-tCYP71BE85, and tpS3 / tpTS-tCYP82D213.

[0095] The nucleotide sequence of the TwGGPPS1 gene is shown in NCBI reference sequence number KM978332.1 (submission date: 11-AUG-2016), and the amino acid sequence of the TwGGPPS1 protein it encodes is shown in NCBI reference sequence number AKQ99275.1 (submission date: 11-AUG-2016).

[0096] The nucleotide sequence of the TwDXS gene is shown in NCBI reference sequence number KM879186.1 (submission date: 24-NOV-2015), and the amino acid sequence of the TwDXS protein it encodes is shown in NCBI reference sequence number AKP20998.1 (submission date: 24-NOV-2015).

[0097] The nucleotide sequence of the AstHMGR gene is shown in Sequence 5, and the amino acid sequence of the AstHMGR protein it encodes is shown in Sequence 6.

[0098] The nucleotide sequence of the RoCPS1 gene is shown in Sequence 7, and the amino acid sequence of the RoCPS1 protein it encodes is shown in NCBI reference sequence number AHL67261.1 (submission date: 16-MAR-2014).

[0099] The nucleotide sequence of the SmKSL1 gene is shown in Sequence 8, and the amino acid sequence of the SmKSL1 protein it encodes is shown in NCBI reference sequence number ABV08817.1 (submission date: 20-DEC-2016).

[0100] The nucleotide sequence of the CfTPS3 gene is shown in Sequence 9, and the amino acid sequence of the CfTPS3 protein it encodes is shown in NCBI reference sequence number AHW04048.1 (submission date: 09-APR-2014).

[0101] The nucleotide sequence of the CYP82D274 gene is shown in Sequence 10, and the amino acid sequence of the CYP82D274 protein it encodes is shown in NCBI reference sequence number UTK45969.1 (submission date: 18-JUL-2022).

[0102] The nucleotide sequence of the CYP71BE86 gene is shown in Sequence 11, and the amino acid sequence of the CYP71BE86 protein it encodes is shown in NCBI reference sequence number UTK45967.1 (submission date: 18-JUL-2022).

[0103] The nucleotide sequence of the CYP71BE85 gene is shown in Sequence 12, and the amino acid sequence of the CYP71BE85 protein it encodes is shown in NCBI reference accession number UTK45966.1 (submission date: 18-JUL-2022). The nucleotide sequence of the tCYP71BE85 gene is obtained by removing bases 1-78 from the CYP71BE85 gene nucleotide sequence, and the amino acid sequence of the tCYP71BE85 protein it encodes is obtained by removing amino acids 1-26 from the CYP71BE85 protein amino acid sequence.

[0104] The nucleotide sequence of the CYP82D213 gene is shown in Sequence 13, and the amino acid sequence of the CYP82D213 protein it encodes is shown in NCBI reference accession number UTK45968.1 (submission date: 18-JUL-2022). The nucleotide sequence of the tCYP82D213 gene is obtained by removing bases 1-210 from the CYP82D213 gene nucleotide sequence, and the amino acid sequence of the tCYP82D213 protein it encodes is obtained by removing amino acids 1-70 from the CYP82D213 protein amino acid sequence.

[0105] 1. Construction of recombinant expression vectors The Golden Gate assembly method was used to respectively... TwGGPPS1 , TwDXS , AstHMGR , RoCPS1 , SmKSL1 , CfTPS3 , CYP82D274 , CYP71BE86 , CYP71BE85 and CYP82D213 The gene fragment was integrated into the BsaI restriction site of the pEAQ-HT vector to obtain the recombinant expression vector pEAQ-HT:: TwGGPPS1 pEAQ-HT:: TwDXS pEAQ-HT:: AstHMGR pEAQ-HT:: RoCPS1 pEAQ-HT:: SmKSL1 pEAQ-HT:: CfTPS3 pEAQ-HT:: CYP82D274 pEAQ-HT:: CYP71BE86 pEAQ-HT:: CYP71BE85 and pEAQ-HT:: CYP82D213 .

[0106] The Golden Gate assembly method was used to respectively... tpS3-CYP82D274 , tpS3-CYP71BE86 , tpS3- tCYP71BE85 , tpS3-tCYP82D213 , tpTS-CYP82D274 , t pTS-CYP71BE86 , tpTS-tCYP71BE85 and tpTS-tCYP82D213 The gene fragment was integrated into the BsaI restriction site of the pEAQ-HT vector to obtain the recombinant expression vector pEAQ-HT::( tpS3-CYP82D274 ), pEAQ-HT::( tpS3-CYP71BE86 ), pEAQ-HT::( tpS3- tCYP71BE85 ), pEAQ-HT::( tpS3-tCYP82D213), pEAQ-HT::( tpTS-CYP82D274 ), pEAQ-HT::( tpTS-CYP71BE86 ), pEAQ-HT::( tpTS-tCYP71BE85 ) and pEAQ-HT::( tpTS-tCYP82D213 ).in, tpS3-CYP82D274 Gene fragments are sequentially composed of tpS3 Signal peptide genes and CYP82D274 Gene sequence composition, tpS3- CYP71BE86 Gene fragments are sequentially composed of tpS3 Signal peptide genes and CYP71BE86 Gene sequence composition, tpS3-tCYP71BE85 Gene fragments are sequentially composed of tpS3 Signal peptide genes and tCYP71BE85 Gene sequence composition, tpS3-tCYP82D213 Gene fragments are sequentially composed of tpS3 Signal peptide genes and tCYP82D213 Gene sequence composition, tpTS-CYP82D274 Gene fragments are sequentially composed of tpTS Signal peptide genes and CYP82D274 Gene sequence composition, tpTS-CYP71BE86 Gene fragments are sequentially composed of tpTS Signal peptide genes and CYP71BE86 Gene sequence composition, tpTS-tCYP71BE85 Gene fragments are sequentially composed of tpTS Signal peptide genes and tCYP71BE85 Gene sequence composition, tpTS-tCYP82D213 Gene fragments are sequentially composed of tpTS Signal peptide genes and tCYP82D213 Gene sequence composition.

[0107] The above recombinant expression vectors were transformed into Escherichia coli. Trans Plasmids were extracted from 1-T1 competent cells after resistance screening and sequencing verification.

[0108] 2. Transient expression in tobacco The recombinant plasmids from the following groups were transiently expressed in *Nicotiana benthamiana* using Agrobacterium-mediated transient expression: Control: The recombinant plasmid pEAQ-HT:: TwGGPPS1 pEAQ-HT:: TwDXS pEAQ-HT:: AstHMGR pEAQ-HT:: RoCPS1 pEAQ-HT:: SmKSL1 pEAQ-HT:: CfTPS3 pEAQ-HT:: CYP82D274 pEAQ-HT:: CYP71BE86 pEAQ-HT:: CYP71BE85and pEAQ-HT:: CYP82D213 Transient expression occurs in tobacco.

[0109] tpS3 modification group: recombinant plasmid pEAQ-HT:: TwGGPPS1 pEAQ-HT:: TwDXS pEAQ-HT:: AstHMGR pEAQ-HT:: RoCPS1 pEAQ-HT:: SmKSL1 pEAQ-HT:: CfTPS3 pEAQ-HT::( tpS3- CYP82D274 ), pEAQ-HT::( tpS3-CYP71BE86 ), pEAQ-HT::( tpS3-tCYP71BE85 ) and pEAQ-HT::( tpS3-tCYP82D213 Transient expression occurs in tobacco.

[0110] tpTS modification group: recombinant plasmid pEAQ-HT:: TwGGPPS1 pEAQ-HT:: TwDXS pEAQ-HT:: AstHMGR pEAQ-HT:: RoCPS1 pEAQ-HT:: SmKSL1 pEAQ-HT:: CfTPS3 pEAQ-HT::( tpTS- CYP82D274 ), pEAQ-HT::( tpTS-CYP71BE86 ), pEAQ-HT::( tpTS-tCYP71BE85 ) and pEAQ-HT::( tpTS-tCYP82D213 Transient expression occurs in tobacco.

[0111] tpS3+tpTS modified group: recombinant plasmid pEAQ-HT:: TwGGPPS1 pEAQ-HT:: TwDXS pEAQ-HT:: AstHMGR pEAQ-HT:: RoCPS1 pEAQ-HT:: SmKSL1 pEAQ-HT:: CfTPS3 pEAQ-HT::( tpS3- CYP82D274 ), pEAQ-HT::( tpS3-CYP71BE86 ), pEAQ-HT::( tpS3-tCYP71BE85 ), pEAQ-HT::( tpS3-tCYP82D213 ), pEAQ-HT::( tpTS-CYP82D274 ), pEAQ-HT::( tpTS-CYP71BE86 ), pEAQ-HT::(tpTS-tCYP71BE85 ) and pEAQ-HT::( tpTS-tCYP82D213 Transient expression occurs in tobacco.

[0112] Samples were collected on the 6th day after inoculation, and metabolites were extracted and tested.

[0113] The results showed that, compared with the unmodified CYP450s enzyme, modification with either tpS3 or tpTS alone directed metabolic flux towards the final product, significantly increasing the yield of triptolide and other intermediates. However, co-infection with the tpS3 and tpTS combined modification inhibited the yield increase, possibly due to metabolic pathway confusion caused by an excessive number of genes. The results also showed significant differences between the two chloroplast signal peptides. The tpS3 signal peptide exhibited better regulation of upstream intermediates tanshinone diene, asarin triene, 14-hydroxyasarin triene, and triptolide B, increasing their yields by 4.65, 7.85, 8.94, and 3.23 times, respectively, compared to the unmodified control group. In contrast, the tpTS signal peptide was better at promoting metabolic flux, increasing the yield of the final product triptolide by 4.5 times. Figure 1 H). The highest yields of diterpenoid compounds triptolide B, 14-hydroxyascorbic acid triene, and ascorbic acid triene produced from *Tobacco Bunsenii* were 2238.77 μg / g, 545.12 μg / g, and 396.66 μg / g, respectively. Figure 1 The levels of triptolide and triptolide were significantly higher than those of endogenous triptolide in *Tripterygium wilfordii*, limited by the weak catalytic activity of CYP450s in the final step. The highest yields of triptolide and triptolide were 0.0172 μg / g and 0.3152 μg / g, respectively. Figure 1 I and Table 1).

[0114] Table 1. Yield of Tripterygium wilfordii diterpenes produced from tobacco substrate (μg / g)

[0115] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A protein assembly comprising TwGGPPS1 protein, TwDXS protein, and AstHMGR protein.

2. The protein assembly according to claim 1, characterized in that: The protein combination also includes any one of the following protein combinations A1)-A3): A1) RoCPS1 protein and SmKSL1 protein; A2) RoCPS1 and CfTPS3 proteins; A3) RoCPS1 protein, SmKSL1 protein, and CfTPS3 protein.

3. The protein combination according to claim 1 or 2, characterized in that: The protein combination also includes any one or a combination of proteins from B1) to B3): B1) CYP82D274 protein; B2) CYP82D274 protein, CYP71BE86 protein, CYP71BE85 protein and CYP82D213 protein; B3) CYP82D274 protein with N-terminal fusion of chloroplast signal peptide, CYP71BE86 protein with N-terminal fusion of chloroplast signal peptide, tCYP71BE85 protein with N-terminal fusion of chloroplast signal peptide, and tCYP82D213 protein with N-terminal fusion of chloroplast signal peptide.

4. The protein assembly according to claim 3, characterized in that: The chloroplast signal peptide is either chloroplast signal peptide tpS3 or chloroplast signal peptide tpTS.

5. A nucleic acid molecule encoding a combination of proteins according to any one of claims 1-4.

6. A biological material containing the nucleic acid molecule of claim 5; wherein the biological material is an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue, or a transgenic plant organ.

7. The use of any protein combination according to claims 1-4, the nucleic acid molecule according to claim 5, or the biomaterial according to claim 6 in the synthesis of diterpenoids or the preparation of products containing synthetic diterpenoids.

8. The application according to claim 7, characterized in that: The diterpenoid compound is a tripterpenoid active ingredient from Tripterygium wilfordii. Alternatively, the active terpenoid components of Tripterygium wilfordii include at least one of Tripterygium wilfordii lactone, Tripterygium wilfordii lactone, Tripterygium wilfordii B, tanshinone diene, asparagine triene, and 14-hydroxyasparagine triene.

9. A method for synthesizing diterpenoid compounds, comprising the following steps: increasing the content and / or activity of proteins in any one of the protein combinations of claims 1-4 in a plant to obtain a transgenic plant; and isolating diterpenoid compounds from the transgenic plant.

10. The method according to claim 9, characterized in that: The diterpenoid compound is a tripterpenoid active ingredient from Tripterygium wilfordii. Alternatively, the active terpenoid components of Tripterygium wilfordii include at least one of Tripterygium wilfordii lactone, Tripterygium wilfordii lactone, Tripterygium wilfordii B, tanshinone diene, asparagine triene, and 14-hydroxyasparagine triene.

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