Methods for synthesis of megastigmatrienone using a chassis plant, compositions, kits and uses thereof

Abstract

This invention relates to the field of soybean trienone synthesis technology, specifically to a method, composition, kit, and application for synthesizing soybean trienone using a chassis plant. The method involves introducing multiple polynucleotide constructs into at least one plant cell of the chassis plant. These multiple polynucleotide constructs contain nucleotide sequences encoding enzymes NtDXR, NtDXS1, NtGGPPS1, NtPSY1, and NtCCD1-1, the nucleotide sequences of which are shown in the text. This invention establishes a highly efficient soybean trienone plant synthesis system. By co-expressing five enzyme genes, it enhances the supply of isoprene precursors, GGPP synthesis, carotenoid precursor generation, and lutein-directed cleavage, forming a metabolic flux-directed regulatory network. The yield is nearly 2.3 times higher than that of the wild group, achieving efficient and green production of soybean trienone.

Description

Technical Field

[0001] This invention belongs to the field of stigmatatrienone synthesis technology, specifically relating to methods, compositions, kits and applications for synthesizing stigmatatrienone from basal plants. Background Technology

[0002] Megastigmatrienone is an important synthetic flavoring agent, appearing as a pale yellow to yellow oily liquid. It possesses a long-lasting, sweet tobacco aroma, dried fruit notes, and a spicy undertone. It has been listed as a Generally Recognized As Safe (GRAS) substance by the Food Flavor and Extract Manufacturers Association (FEMA) and is included in the Food Flavor List (#4663). It is widely used in the development of specialty high-quality tobacco leaves, cosmetic perfume formulation, wine quality monitoring, and flavorings. Megastigmatrienone is the most abundant carotenoid degradation product among the neutral aroma components of tobacco, formed from the degradation of lutein, and makes a significant contribution to the aroma of tobacco leaves. It plays a particularly significant role in enhancing flavor and removing off-flavors; even small amounts can greatly improve tobacco quality, making it an indispensable flavoring agent in the tobacco industry. Megastigmatrienones were isolated and their structures identified from Burley tobacco and aromatic tobacco in 1972, and they are distributed in various flowers, fruits, and woods. Its structure is quite unique, with eight isomers. To date, only the first five isomers have been detected in natural products, while the remaining three are only found in synthetic samples or intermediates. Since megastigmatrienones are present in very small amounts in plants, relying solely on natural product extraction is clearly insufficient to meet market demand. Therefore, chemical synthesis is its primary source.

[0003] Chinese invention patent application CN119822939A discloses a method for preparing megastigmatrienone. The method includes the following steps: using 5-bromo-1,3-pentadiene as a starting material, reacting it with trivalent phosphine to prepare a phosphine salt; obtaining a Wittig reagent through alkali treatment, which then reacts with acetylacetin acetate to obtain an acetate-substituted conjugated tetraene; under the co-catalysis of copper and a sterically hindered ligand, it undergoes a Diels-Alder reaction with isobutylene to construct a six-membered ring; subsequently, it is hydrolyzed to obtain an alcohol, which is then oxidized to a ketone; under acidic conditions, the terminal conjugated diene undergoes rearrangement isomerization to obtain megastigmatrienone. This application uses simple, inexpensive, and readily available raw materials and reagents, has a short reaction procedure, high yield, and easy separation, making it suitable for large-scale production. However, the above patent application pertains to the chemical synthesis technology of megastigmatrienone and does not involve genetic engineering, enzyme regulation, or the construction of plant synthesis systems.

[0004] Chinese invention patent application CN120843588A discloses the application of the LOX2 gene in increasing the aroma content of tobacco. It utilizes homologous cloning technology to obtain a tobacco lipoxygenase 2 (LOX2) encoding gene from tobacco, constructs an overexpression vector for this gene, transforms it into tobacco plants, and obtains tobacco plants overexpressing the LOX2 gene. Detection of these LOX2-overexpressing tobacco plants reveals that the LOX2 gene has significant application potential in cultivating tobacco with high contents of 2-hexenal, solanone, damascene, and megastigmatrienone, laying the foundation for creating tobacco varieties with increased aroma content through genetic engineering. However, the above application only overexpresses the LOX2 gene (lipoxygenase gene) and does not involve a combination of multiple key metabolic enzyme genes. The regulatory pathway is the lipoxygenase pathway, not the isoprene-carotenoid metabolic pathway, and it does not enhance the entire metabolic flow of precursor synthesis and cleavage.

[0005] Therefore, there is an urgent need to develop a synthetic method for stigmasterones via a plant-based synthetic system that is regulated by multiple enzymes and utilizes the isoprene-carotenoid metabolic pathway. Summary of the Invention

[0006] To address the technical problems existing in the prior art, this invention provides a method for synthesizing megalotrienone using chassis plants. This method co-expresses five enzyme genes, including NtDXR, NtDXS1, NtGGPPS1, NtPSY1, and NtCCD1-1, in chassis plants, respectively enhancing the supply of isoprene precursors (NtDXR / NtDXS1), GGPP synthesis (NtGGPPS1), carotenoid precursor production (NtPSY1), and lutein-directed cleavage (NtCCD1-1), forming a metabolic flux-directed regulatory network to generate megalotrienone, with a yield nearly 2.3 times higher than that of the wild group.

[0007] This invention is achieved through the following technical solutions:

[0008] A first aspect of the invention relates to a method for synthesizing stigmatatrienones using a chassis plant, comprising: co-expressing a set of enzymes in the chassis plant to generate stigmatatrienones; said enzymes including NtDXR, NtDXS1, NtGGPPS1, NtPSY1, and NtCCD1-1, said method comprising introducing a plurality of polynucleotide constructs into at least one plant cell of the chassis plant, said plurality of polynucleotide constructs comprising nucleotide sequences encoding said set of enzymes, wherein, The nucleotide sequence encoding the NtDXR enzyme is as shown in SEQ 1, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ 1; The nucleotide sequence encoding the NtDXS1 enzyme is as shown in SEQ 2, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ 2; The nucleotide sequence encoding the NtGGPPS1 enzyme is as shown in SEQ 3, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ 3; The nucleotide sequence encoding the NtPSY1 enzyme is as shown in SEQ 4, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ 4; The nucleotide sequence encoding the NtCCD1-1 enzyme is as shown in SEQ 5, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ 5.

[0009] In some embodiments of the invention, each polynucleotide construct comprises a nucleotide sequence encoding an enzyme and a promoter operatively linked to express the nucleotide sequence in a plant.

[0010] In some embodiments of the present invention, the chassis plant is tobacco.

[0011] A second aspect of the invention relates to a composition comprising a plurality of polynucleotide constructs, the plurality of polynucleotide constructs comprising nucleotide sequences encoding a set of enzymes NtDXR, NtDXS1, NtGGPPS1, NtPSY1 and NtCCD1-1 as described above.

[0012] A third aspect of the invention relates to a kit comprising the above-described composition.

[0013] A fourth aspect of the invention relates to the use of the method, composition, or kit described herein in the synthesis of megastigmatrienones.

[0014] The beneficial effects of this invention are: 1) It establishes an efficient plant synthesis system for stigmatatrienones, laying the foundation for efficient production of stigmatatrienones; 2) By co-expressing five enzyme genes, it strengthens the supply of isoprene precursors (NtDXR / NtDXS1), GGPP synthesis (NtGGPPS1), carotenoid precursor generation (NtPSY1), and lutein directional cleavage (NtCCD1-1), forming a metabolic flux directional regulation network to achieve efficient and green production. Attached Figure Description

[0015] Figure 1 Gas chromatographic analysis of the synthesis of stigmasterone from tobacco; Figure 2 For the yield analysis of megastigmatrienone; Figure 3 This is the mass spectrum of the stigmatatrienone standard; Figure 4 This is the mass spectrum of the stigmatatrienone synthesized from tobacco in this invention; wherein... Figure 1 , Figure 2 In the figure, NtMEG represents the co-expression of NtDXR, NtDXS1, NtGGPPS1, NtPSY1 and NtCCD1-1; the control is wild-type tobacco TN90 leaves. Detailed Implementation

[0016] The present invention will be further described below with reference to embodiments. It should be understood that the following embodiments are only for explanation and illustration of the present invention and do not limit the scope of the present invention in any way.

[0017] Experimental materials: The plant material used in the following examples is tobacco TN90, which was cultivated in our laboratory; The Escherichia coli used in the following examples ( Escherichia coli DH5α and Agrobacterium ( Agrobacterium tumefaciens GV3101 was purchased from Shanghai Weidi Biotechnology Co., Ltd. The plant expression vector pEAQ-HT used in the following examples was preserved in our laboratory and is commercially available.

[0018] Main reagents: MES was purchased from Sigma-Aldrich, CAS No.: 4432-31-9, item number: M3671; Acetyleugenol was purchased from Sigma-Aldrich, CAS No.: 2478-38-8, Product No.: D134406; Ethyl acetate was purchased from Sigma-Aldrich, CAS No.: 141-78-6, catalog number: 270989; The megalotrienone was purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS No.: 13215-88-8, molecular formula: C13H18O, product number: S31653.

[0019] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and are of laboratory purity. Unless otherwise specified, the experimental methods and conditions used in the following examples are conventional experimental methods and conditions in the art, and can be found in relevant experimental manuals, public literature, or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] Example 1: Establishment of a method for synthesizing megastigmatrienone using a tobacco chassis Candidate genes NtDXR, NtDXS1, NtGGPPS1, NtPSY1, and NtCCD1-1 related to stigmatatrienone synthesis were identified and cloned from tobacco TN90. Functional validation of these genes was performed using transient expression technology in tobacco, and a method for high-yield stigmatatrienone production was constructed. Specific experiments are as follows.

[0021] 1. RNA extraction and reverse transcription Total RNA was extracted from tobacco TN90 leaves using the RNAprep Pure Plant Kit (catalog number: DP441) from Tiangen Biotech, following the product instructions. Using the extracted total RNA as a template, cDNA was synthesized using the SuperScript III Reverse Transcriptase Kit (Invitrogen, catalog number: 18080085) according to the product instructions. The steps are as follows: (1) The components and amounts used for RNA template denaturation are shown in Table 1.

[0022] Table 1

[0023] Heat at 65°C for 5 minutes, then quickly place on ice to cool, and let stand on ice for 2 minutes.

[0024] (2) The components and amounts used in the synthesis of the first-strand cDNA are shown in Table 2.

[0025] Table 2

[0026] Briefly centrifuge to mix. React at 55℃ for 60 min, then heat at 70℃ for 15 min to terminate the reaction. Obtain tobacco leaf cDNA and store at -20℃.

[0027] 2. Cloning of the target gene Using 5-fold diluted tobacco TN90 leaf cDNA as a template, the target genes NtDXR, NtDXS1, NtGGPPS1, NtPSY1 and NtCCD1-1 were amplified using the corresponding primers in Table 3.

[0028] Table 3

[0029] The genes amplified by each primer in Table 3 are as follows: NtDXR-F and NtDXR-R were used to amplify the NtDXR gene in tobacco (SEQ1).

[0030] NtDXS1-F and NtDXS1-R were used to amplify the NtDXS1 gene in tobacco (SEQ2).

[0031] NtGGPPS1-F and NtGGPPS1-R were used to amplify the NtGGPPS1 gene in tobacco (SEQ3).

[0032] NtPSY1-F and NtPSY1-R were used to amplify the NtPSY1 gene in tobacco (SEQ4).

[0033] NtCCD1-1-F and NtCCD1-1-R were used to amplify the NtCCD1-1 gene in tobacco (SEQ5).

[0034] The PCR reaction was performed using 2×Phanta Max Master Mix high-fidelity enzyme (vazyme, catalog number: P515-02). The reaction system is shown in Table 4.

[0035] Table 4

[0036] Reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 33 cycles; 72℃ final extension for 7 min. After the reaction, the PCR products were detected by 1% agarose gel electrophoresis. Using the Gel Extraction Kit (Omega, catalog number: D2500-02), each target gene fragment was recovered from the gel according to the product instructions.

[0037] 3. Construction of expression vector Using restriction endonucleases Xho I and Sal The pEAQ-HT vector was digested using Thermo Fisher enzyme. The digestion reaction system is shown in Table 5.

[0038] Table 5

[0039] The reaction was carried out at 37℃ for 30 min to obtain the pEAQ-HT linearized vector.

[0040] Then, using the ClonExpress II One Step Cloning Kit (Vazyme), the recovered target gene fragments were cloned into the pEAQ-HT vector according to the product instructions. The reaction system is shown in Table 6.

[0041] Table 6

[0042] The reaction can be carried out at 37°C for 30 minutes to obtain the ligation product.

[0043] The ligation products of each target gene fragment and the pEAQ-HT linearized vector were transformed into Escherichia coli DH5α using the heat shock method. The steps are as follows: Take 100 μL of competent DH5α cells thawed in an ice bath, add the ligation product, mix gently, and place in an ice bath for 30 min; heat shock in a 42℃ water bath for 60 s, and quickly transfer the centrifuge tube to an ice bath for 2 min; add 200 μL of antibiotic-free LB medium to the centrifuge tube, mix well, and incubate at 37℃ and 180 rpm for 1 h to revive the cells; aspirate the reviving cells and add them to LB agar medium containing kanamycin (50 mg / L), spread the cells evenly, blow off the liquid on the surface of the medium, and incubate upside down at 37℃ overnight; pick several single colonies and add them to 500 μL of LB liquid medium containing kanamycin (50 mg / L), and incubate at 37℃ and 180 rpm for 4 h; perform bacterial PCR identification using primers pEAQ-F and pEAQ-R (Table 1); send the PCR-identified positive clones to Ruiboxingke Biotechnology Co., Ltd. for sequencing.

[0044] Plasmids from correctly sequenced positive clones were extracted using the EZNA Plasmid Mini Kit I (omega, catalog number: D6942-02) according to the product instructions. Expression vectors for each target gene were obtained, denoted as pEAQ-NtDXR, pEAQ-NtDXS1, pEAQ-NtGGPPS1, pEAQ-NtPSY1, and pEAQ-NtCCD1-1, respectively.

[0045] 4. Agrobacterium-mediated transformation The expression vectors of the above-mentioned target genes were transformed into Agrobacterium GV3101 cells. The steps are as follows: Take Agrobacterium GV3101 competent cells stored at -80 ℃ and allow them to partially thaw at room temperature for a short time. When they are in an ice-water mixture, place them in ice. Take a 1.5 ml tube, add 20 μL of competent cells and 500 ng of pEAQ-HT recombinant plasmid containing the target gene; mix well with a pipette, place in ice for 5 min, immerse in liquid nitrogen for 5 min, 37 ℃ for 5 min, and place back in ice for 5 min. Add 700 ml of antibiotic-free LB liquid medium and incubate at 28 ℃ with shaking for 2-3 hours. Centrifuge at 5000 rpm for 1 min; discard the supernatant, mix the precipitated cells, and add them to LB agar medium containing kanamycin (Kana), gentamicin (Gent), and rifampicin (Rif) (Kana: selection concentration 50 mg / L; Gent: selection concentration 50 mg / L; Rif: selection concentration 20 mg / L). Spread the cells evenly, blow off the liquid on the surface of the medium, and incubate upside down at 28 ℃ for 2-3 days. Pick several single colonies and add them to 500 μL of LB liquid medium containing kanamycin (Kana), gentamicin (Gent), and rifampicin (Rif) (Kana: selection concentration 50 mg / L; Gent: selection concentration 50 mg / L; Rif: selection concentration 20 mg / L), and incubate at 28 ℃ and 180 rpm for 12 h. Perform PCR identification of the bacterial culture using primers pEAQ-F and pEAQ-R (Table 1). The strains that expressed the target genes were identified as GV3101-pEAQ-NtDXR, GV3101-pEAQ-NtDXS1, GV3101-pEAQ-NtGGPPS1, GV3101-pEAQ-NtPSY1 and GV3101-pEAQ-NtCCD1-1, respectively.

[0046] 5. Instantaneous transformation of tobacco The expression strains of each target gene were inoculated into LB liquid medium (Kana: 50 mg / L selection concentration; Gent: 50 mg / L selection concentration; Rif: 20 mg / L selection concentration) and cultured at 28 °C with shaking for 24 h (25 ml of bacterial culture in a 100 ml Erlenmeyer flask). The Agrobacterium culture was centrifuged at 5500 rpm for 10 min at room temperature (25 ml of bacterial culture in a 50 ml centrifuge tube), the supernatant was discarded, and the bacterial pellet was resuspended in 5 ml Agromix [10 mM MgCl2, 10 mM MES (pH 5.6 KOH), 150 μM acetylsylcholine]. The Agrobacterium suspension in the Agromix was incubated in the dark at room temperature for at least 2 h. The absorbance of the culture at 600 nm was measured using a spectrophotometer, and the culture was diluted to 0.2 OD / mL (600 nm) to obtain the expression bacterial dilutions for each target gene.

[0047] Using transient expression technology in tobacco, the function of the target gene was validated through a stepwise screening method. Equal volumes of the expression bacterial solutions for the target gene were mixed to obtain a mixed bacterial solution, which was then co-infiltrated into tobacco TN90 leaves. The injection method was as follows: a hole was punched in the tobacco leaf using a sterile pipette tip, and then the mixed bacterial solution was infiltrated into the tobacco leaf using a 5 mL syringe without the needle. The infiltrated tissue leaves were harvested approximately 6 days later (depending on the expressed gene) and then freeze-dried directly.

[0048] 6. Extraction and GC-MS analysis of megastigmatrienones (1) Extraction of megastigmatrienone Weigh 100 mg of lyophilized tobacco leaves into a 2 mL tube. Add 1.5 mL of 10% NaOH, vortex until the powder sample is completely wetted, add 15 mL of n-hexane, vortex for 1 min, sonicate at 30-40 ℃ for 30 min (pay attention to water temperature during the process), centrifuge at 3000 rpm for 10 min, collect 3 mL of the supernatant, and dry under nitrogen. Dissolve in 1 mL of ethyl acetate and perform GC-MS analysis.

[0049] (2) GC-MS analysis Instrument: Agilent Technologies Gas Chromatography-Mass Spectrometry (GC-MS) Chromatographic column: Agilent 19091S-433UI HP-5ms Ultra Inert column (30 m x 250 μm x 0.25 μm) Set the mass spectrometer to scan mode, scanning all masses from 60 to 800, with a solvent delay of 14 minutes. Inlet temperature: 280 °C. Temperature cycling: Initial oven temperature: 40 °C, 2 minutes. Ramp 1: 40 °C to 150 °C, 10 °C / min, finally held at 150 °C for 2 minutes. Ramp 2: 150 °C to 250 °C, 10 °C / min, finally held at 250 °C for 5 minutes.

[0050] 7. Experimental Results We identified and cloned candidate genes NtDXR, NtDXS1, NtGGPPS1, NtPSY1, and NtCCD1-1 related to steroid compound synthesis from tobacco, and validated their functions using transient expression technology in tobacco. NtDXSR The gene encodes 1-deoxy-D-xylitol-5-phosphate reductase. NtDXS1 The gene encodes 1-deoxy-D-xylitol-5-phosphate synthase. NtDXR , NtDXS1 , NtGGPPS1 , NtPSY1 and NtCCD1-1 The genes encode 1-deoxy-D-xyulose-5-phosphate reductase, 1-deoxy-D-xyulose-5-phosphate synthase, geraniolyl pyrophosphate synthase, phytoene synthase, and carotenoid lysin, respectively.

[0051] like Figures 1-4 As shown, gas chromatography-mass spectrometry (GC-MS) analysis revealed that NtDXR, NtDXS1, NtGGPPS1, NtPSY1, and NtCCD1-1 were co-expressed in tobacco leaves, enabling the synthesis of megastigmatrienone with a yield of 850.11 ug / g (dry weight). For ease of description, the combined expression of NtDXR, NtDXS1, NtGGPPS1, NtPSY1, and NtCCD1-1 is abbreviated as NtMEG. Figure 2 The results showed that the yield of NtMEG megalotrienone was 2.3 times that of the wild TN90 group.

[0052] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for synthesizing megalotrienone using basal plants, wherein the method involves co-expressing a group of enzymes in basal plants to generate megalotrienone, characterized in that, The set of enzymes includes NtDXR, NtDXS1, NtGGPPS1, NtPSY1, and NtCCD1-1. The method includes introducing a plurality of polynucleotide constructs into at least one plant cell of the chassis plant, wherein the plurality of polynucleotide constructs contain nucleotide sequences encoding the set of enzymes. The nucleotide sequence encoding the NtDXR enzyme is as shown in SEQ 1, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ 1; The nucleotide sequence encoding the NtDXS1 enzyme is as shown in SEQ 2, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ 2; The nucleotide sequence encoding the NtGGPPS1 enzyme is as shown in SEQ 3, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ 3; The nucleotide sequence encoding the NtPSY1 enzyme is as shown in SEQ 4, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ 4; The nucleotide sequence encoding the NtCCD1-1 enzyme is as shown in SEQ 5, or has at least 90% sequence identity with the nucleotide sequence shown in SEQ 5.

2. The method according to claim 1, characterized in that, Each of the polynucleotide constructs contains a nucleotide sequence encoding an enzyme and an operable promoter for expressing the nucleotide sequence in a plant.

3. The method according to any one of claims 1-2, characterized in that, The plant on the chassis is tobacco.

4. A composition, characterized in that, The composition comprises a plurality of polynucleotide constructs, the plurality of polynucleotide constructs comprising nucleotide sequences encoding a set of enzymes as described in claim 1.

5. A reagent kit, characterized in that, The kit comprises the composition of claim 4.

6. The use of the method according to any one of claims 1-3, the composition according to claim 4, or the kit according to claim 5 in the synthesis of megastigmatrienone.

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