Method for promoting ginsenoside biosynthesis based on cooperative expression of MVA pathway and saponin synthesis module

Abstract

The invention relates to a method for promoting biosynthesis of ginsenoside (Rg3 / Rh2) based on cooperative expression of an MVA pathway and a saponin synthesis module. According to the method, efficient synthesis is realized through a double-vector synergistic expression strategy: an MVA pathway module vector is constructed, and terpene precursor IPP supply is enhanced; an Rg3 synthesis module carrier is constructed, and a ginsenoside synthesis route is directionally catalyzed. A gene module is assembled by adopting a TransGene Stacking II system, and the nicotiana benthamiana is transiently co-transformed through agrobacterium tumefaciens mediation. Through MVA module single expression, the IPP content of tobacco leaves reaches 533 ng / g and is improved by 43% compared with that of a wild type (372 ng / g); the metabolic flux is synergistically activated through double-module co-expression, the yield of Rg3 reaches 23.70 mu g / g and is increased by 53.90% compared with that of a single Rg3 module (15.04 mu g / g), and Rh2 (0.37 mu g / g) is synthesized in tobacco for the first time. The bottleneck of insufficient supply of plant chassis precursors is broken through, efficient synthesis of ginsenoside Rg3 / Rh2 is achieved through modular design, and a new normal form is provided for green biological manufacturing of rare triterpenoid saponin.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for promoting the biosynthesis of ginsenosides (Rg3 / Rh2) based on the synergistic expression of the MVA pathway and the saponin synthesis module. This method is based on the synergistic expression of the MVA pathway and the saponin synthesis module to increase the content of the plant terpene precursor IPP, thereby further promoting the efficient biosynthesis of ginsenosides Rg3 / Rh2. Background Technology

[0002] Terpenoids, as the most abundant secondary metabolites in nature, have significant value in the pharmaceutical field. Among them, dammarane-type triterpenoid saponins such as ginsenosides Rg3 and Rh2 have been proven to possess antitumor and neuroprotective activities. However, traditional plant extraction methods are limited by extremely low content (often less than one ten-thousandth of dry weight), and chemical synthesis is cumbersome and costly. While microbial heterologous synthesis has made progress, it faces challenges such as exogenous enzyme folding errors, insufficient post-translational modifications, and poor product tolerance, leading to difficulties in industrialization. For example, microbial chassis exhibit low efficiency in expressing the activity of cytochrome P450 enzymes (CYPs) and glycosyltransferases (UGTs), failing to meet demand.

[0003] Plant chassis, with their naturally compartmentalized metabolic environment, are theoretically better suited for the synthesis of complex terpenoids. However, their practical application is still limited by a core bottleneck—insufficient precursor supply. The synthesis of ginsenoside Rg3 / Rh2 depends on the IPP / DMAPP precursor generated via the mevalonate (MVA) pathway, which is then further catalyzed by eight enzymes, including squalene synthase (SS) and squalene epoxidase (SE). However, the expression level of endogenous MVA pathway genes in plants is low, and competing pathways (such as sterol synthesis) consume large amounts of precursors, resulting in limited accumulation of the target product. While existing patented technologies (such as CN116694672A) achieve multi-gene co-expression (such as IDI, FPS, PPDS, etc.), they do not specifically enhance the precursor supply module, resulting in extremely low Rg3 content in transgenic tobacco, failing to overcome the yield bottleneck.

[0004] To address the aforementioned issues, this invention proposes an innovative synergistic expression strategy: for the first time, the MVA pathway module (containing AACT, HMGS, HMGR, MK, PMK, and MVD6 genes) and the Rg3 synthesis module (SS, SE, UGT, etc. genes) are integrated through a dual-vector system, achieving simultaneous optimization of precursor supply and transformation pathways. This design, on the one hand, utilizes modular assembly technology to overexpress key enzymes of the MVA pathway to increase the IPP / DMAPP library capacity, thereby improving the synthesis efficiency of ginsenosides such as Rg3; on the other hand, it uses a tobacco chassis transient transformation system for validation, leveraging its advantages of rapid growth, mature operation, and low cost, providing economic feasibility for industrialization.

[0005] This strategy overcomes the dual limitations of "precursor insufficiency and pathway competition" in traditional technologies, providing a new paradigm for the efficient biosynthesis of rare ginsenosides. By synergistically enhancing precursor supply and targeted catalysis modules, it not only significantly increases Rg3 / Rh2 yield but also verifies the potential of plant chassis in the large-scale production of complex terpenes, laying the foundation for the subsequent development of green biomanufacturing technologies that combine high yield and economic efficiency. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art by providing a method for promoting ginsenoside biosynthesis based on the synergistic expression of the MVA pathway and a saponin synthesis module. This method achieves efficient synthesis through a dual-vector synergistic expression strategy: an MVA pathway module vector (containing AtAACT, AtHMGS, AtHMGR, AtMK, AtPMK, and AtMVD genes) is constructed to enhance the supply of the terpene precursor IPP; an Rg3 synthesis module vector (containing PgSS1, PgSE, PgDDS, PgPPDS, Pn1-31, Pn3-31, tAtHMGR, AtIDI, and AtMVD genes) is constructed to directionally catalyze the ginsenoside synthesis pathway.

[0007] Existing methods for heterologous synthesis of ginsenoside Rg3 in plants do not provide a strategy for supplying the Rg3 precursor, resulting in low levels. This invention significantly improves the supply of isopentenyl pyrophosphate (IPP), a core precursor in terpene synthesis, by co-expressing a mevalonate pathway (MVA) module and a rare ginsenoside Rg3 synthesis gene module in tobacco. Increased IPP levels further promote the synthesis of ginsenosides Rg3 and Rh2. This invention solves the key problem of low synthesis efficiency of rare ginsenosides due to insufficient terpene substrates in the plant substrate.

[0008] The objective of this invention can be achieved through the following methods: A method for promoting ginsenoside biosynthesis based on the synergistic expression of the MVA pathway and saponin synthesis module includes the following steps: S1. Construct a recombinant vector containing the following gene modules: MVA pathway module recombinant vector: containing AtAACT2, AtHMGS, AtHMGR, AtMK, AtPMK, AtMVD, Cre, HPT genes, with the vector backbone based on the TransGene Stacking II system (such as the recipient vector pYLTAC380GW). The Rg3 synthesis module recombinant vector contains AtIDI, AtMVD, Pn1-31 (UGT glycosyltransferase gene) and Pn3-31 (UGT glycosyltransferase gene), PgDDS (dammarene diol synthase gene), PgPPDS (protopanaxadiol synthase gene), PgSE (squalene epoxidase gene), PgSS1 (squalene synthase gene), tAtHMGR, and HPT. The vector backbone is based on the TransGene Stacking II system (such as the recipient vector pYLTAC380GW). S2. The MVA pathway module recombinant vector and the Rg3 synthesis module recombinant vector were introduced into the plant chassis via Agrobacterium-mediated transformation, transiently co-expressing the MVA and Rg3 modules, activating terpene precursor metabolism and synthesizing ginsenosides Rg3 and Rh2.

[0009] In one embodiment of the present invention, in the MVA pathway module recombinant vector, the AtAACT2, AtHMGS, AtHMGR, AtMK, AtPMK, AtMVD, Cre, and HPT genes are sequentially linked. Specifically, each of the AtAACT2, AtHMGS, AtHMGR, AtMK, AtPMK, and AtMVD genes has an upstream linker connected to a P gene. 35S The promoter and downstream are both connected to T NOS Terminator. The HPT gene upstream of the P gene is linked. Bnm1 Promoter, downstream connection T NOS Terminator. The Cre gene upstream of the P gene is linked. 35S Promoter, downstream connection T 35S Termination of contract.

[0010] In one embodiment of the present invention, in the MVA pathway module recombinant vector, the genes AtIDI, AtMVD, Pn1-31, Pn3-31, PgDDS, PgPPDS, PgSE, PgSS1, tAtHMGR, and HPT are sequentially linked. Specifically, each of the AtIDI, AtMVD, Pn1-31, Pn3-31, PgDDS, PgPPDS, PgSE, PgSS1, and tAtHMGR genes has an upstream linker connected to a P… 35S The promoter and downstream are both connected to T NOS Terminator. The HPT gene upstream of the P gene is linked. Bnm1 Promoter, downstream connection T NOS Termination of contract.

[0011] The donor vector (pYL322d1-35S) contains a 35S promoter and a terminator.

[0012] As one embodiment of the present invention, in step S1, the AtAACT2, AtHMGS, AtHMGR, AtMK, AtPMK, and AtMVD genes are derived from Arabidopsis thaliana.

[0013] As one embodiment of the present invention, in step S1, the AtIDI, tAtHMGR, and AtMVD genes are derived from Arabidopsis thaliana; PgDDS (dammarene diol synthase gene), PgPPDS (protopanaxadiol synthase gene), PgSE (squalene epoxidase gene), and PgSS1 (squalene synthase gene) are derived from ginseng; and Pn1-31 (UGT glycosyltransferase gene) and Pn3-31 (UGT glycosyltransferase gene) are derived from Panax notoginseng.

[0014] As one embodiment of the present invention, the recombinant vector is constructed by using the TransGeneStacking II multi-gene aggregation vector system to sequentially assemble the above-mentioned genes into a recipient vector (such as pYLTAC380GW).

[0015] In one embodiment of the present invention, in step S1, donor vectors containing the AtAACT2, AtHMGS, AtHMGR, AtMK, and AtMVD+AtPMK genes are co-transformed sequentially with recipient vectors. Then, the hygromycin selection marker gene HPT and the Cre gene from the pYLMFH vector are recombined using a BP reaction to obtain an MVA pathway module recombinant vector. The recipient vector is pYLTAC380GW.

[0016] After each co-transformation of the donor and recipient vectors, a mixed plasmid is obtained. This plasmid is then screened, and erroneous plasmids are removed by enzyme digestion. The enzyme digestion uses homing endonuclease I-Sce I.

[0017] The BP reaction involves reacting the co-transformed plasmid vector with PYLMFH (the HPT-Cre gene is on PYLMFH), and recombining the hygromycin selection marker gene HPT and the Cre gene from the pYLMFH vector into the co-transformed plasmid vector. The reaction yields 380MF-AtAACT2-AtHMGS-AtHMGR-AtMK-AtMVD-AtPMK-Cre-HPT (MVA).

[0018] This invention utilizes the Gibson Assembly method to construct the corresponding genes into donor vectors pYL322d1-35S and pYL322d2-35S, respectively. Subsequently, the genes are sequentially assembled into a 380GW vector using the TransGene Stacking II multi-gene aggregation vector system, resulting in pYLTAC380GW-AtAACT2-AtHMGS-AtHMGR-AtMK-AtMVD-AtPMK. Then, the hygromycin selection marker gene and the Cre gene from the pYLMFH vector are recombined using a bp recombination reaction, ultimately yielding the corresponding expression vector 380MF-AtAACT2-AtHMGS-AtHMGR-AtMK-AtMVD-AtPMK-Cre-HPT (MVA).

[0019] In one embodiment of the present invention, in step S1, donor vectors containing the genes tAtHMGR, PgSS1, PgSE, PgPPDS+PgDDS, and Pn1-31+Pn3-31 are co-transformed with recipient vectors sequentially. Then, through a BP recombination reaction, the AtIDI-AtMVD dual-gene module and the hygromycin selection marker gene HPT are integrated into the vector to obtain the Rg3 pathway module recombinant vector. The recipient vector is pYLTAC380GW.

[0020] The AtIDI-AtMVD dual-gene module and the hygromycin selection marker gene HPT were integrated into the co-transformed plasmid vector by reacting the co-transformed plasmid vector with PYLMFH-35S-AtIDI-35S-AtMVD via a BP recombination reaction.

[0021] Preparation of PYLMFH-35S-AtIDI-35S-AtMVD: The AtIDI+AtMVD gene fragment was ligated into the XbaI and HindIII restriction sites on the PYLMFH vector to replace the Cre gene in the original vector, thus obtaining PYLMFH-35S-AtIDI-35S-AtMVD.

[0022] The gene fragments AtAACT2, AtHMGR, AtPMK+AtMVD, tAtHMGR, PgSS1, and Pn1-31+Pn3-31 were inserted into the multiple cloning sites BamHI and EcoRI of the donor vector pYL322d1-35S (which contains a 35S promoter and terminator), respectively, to obtain six d1 donor vectors: pYL322d1-35S-AtAACT2, pYL322d1-35S-AtHMGR, pYL322d1-35S-AtMVD+35S-AtPMK, pYL322d1-35S-tAtHMGR, pYL322d1-35S-PgSS1, and pYL322d1-35S-Pn1-31-35S-Pn3-31.

[0023] The gene fragments AtHMGS, AtMK, AtPgSE, and PgDDS+PgPPDS were inserted into the multiple cloning sites BamHI and EcoRI of the donor vector pYL322d2-35S (which contains a 35S promoter and terminator), respectively, to obtain four d2 donor vectors: pYL322d2-35S-AtHMGS, pYL322d2-35S-AtMK, pYL322d2-35S-PgSE, and pYL322d2-35S-PgDDS+35S-PgPPDS.

[0024] In one embodiment of the present invention, in step S1, all genes are regulated by the 35S constitutive promoter and assembled into a 380GW vector using the TransGene Stacking II system; respectively, the following MVA modules are obtained: 380MF-AtAACT2-AtHMGS-AtHMGR-AtMK-AtPMK-AtMVD-Cre-HPT ( Figure 1 ) and Rg3 module: 380MF-tAtHMGR-PgSS1-PgSE-PgPPDS-PgDDS-Pn1-31-Pn3-31-AtMVD-AtIDI-HPT ( Figure 3 ).

[0025] In one embodiment of the present invention, in step S2, the co-expression ratio of the MVA module and the Rg3 module is 1:3 (OD600=1.0).

[0026] In one embodiment of the present invention, in step S2, Agrobacterium containing the target vector is inoculated into LB medium containing antibiotics (such as kanamycin Km, chloramphenicol Chl, or ampicillin Amp) and cultured in a shaker at 28°C for 24 hours. The culture is then diluted 100-fold and inoculated into fresh LB medium, with the addition of MES (0.5 M, pH 5.6) and AS (100 mM). The culture is continued in a shaker at 28°C until the OD600 reaches 0.6-1.0. The cells are collected by centrifugation at 4,000 rpm for 10 minutes, resuspended in 10 mM MgCl2 until the OD600 reaches 1.0, and 2 μl of AS is added per milliliter of bacterial suspension. The mixture is then allowed to stand for 3 hours. The Agrobacterium suspension is injected into the abaxial surface of leaves of approximately 5-week-old Nicotiana benthamiana plants using a syringe without the needle, ensuring full penetration of the bacterial suspension into the mesophyll cells. Finally, the tobacco plants are cultured in a light incubator for 3-5 days to detect the expression of the target gene or the accumulation of metabolites.

[0027] In one embodiment of the present invention, in step S2, the plant substrate is tobacco. The tobacco receptor is *Nicotiana benthamiana*, and the *Agrobacterium* strain is GV3101.

[0028] Transgenic plants: Plants transformed through the recombinant vector, with the recipient plant being tobacco, have significantly higher IPP content than the wild type.

[0029] Compared with existing technologies, this invention solves the core problem of insufficient IPP precursor supply in the synthesis of traditional terpenoid compounds in plant chassis through the above technical solution, and achieves the following significant effects: (1) The IPP content in tobacco leaves was significantly increased by transient conversion of MVA vector: the IPP content in wild-type control tobacco leaves was 372 ng / g, while the IPP content in tobacco leaves transiently converted by MVA vector reached 533 ng / g, an increase of about 43%. Figure 5 ).

[0030] (2) The instantaneous conversion of tobacco leaves by the Rg3 carrier can produce ginsenoside Rg3. The MVA carrier and the Rg3 carrier can instantaneously convert tobacco leaves together. Due to the increase in the substrate IPP content, the Rg3 content in tobacco leaves increases by about 53.90%, and ginsenoside Rh2 can also be produced. Attached Figure Description

[0031] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram of an MVA carrier. Figure 2Gel image for Not I restriction enzyme digestion verification of MVA vector; Figure 3 A schematic diagram of the Rg3 carrier; Figure 4 The gel image was verified by Not I restriction enzyme digestion of the Rg3 vector. Figure 5 For the detection of IPP content in tobacco leaves by transient conversion using MVA carrier; Figure 6 The results of LC-MS / MS detection of transient expression of Rg3 and MVA vector in tobacco are shown; A represents Rg3; B represents Rh2. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0033] This invention significantly improves the supply of isopentenyl pyrophosphate (IPP), a terpene precursor, by co-expressing a mevalonate pathway (MVA) module and a rare ginsenoside Rg3 synthesis gene module in tobacco. IPP is a core precursor for the synthesis of terpene compounds, and its increased content can further promote the synthesis of ginsenosides Rg3 and Rh2.

[0034] This invention selects six genes from *Arabidopsis thaliana* (nives) for heterologous expression in *Nicotiana benthamiana*: AtAACT2, AtHMGS, AtHMGR, AtMK, AtPMK, and AtMVD. Based on the results of a query for related gene transcripts at https: / / www.ncbi.nlm.nih.gov / , appropriate primers were designed to amplify the target genes from *Arabidopsis thaliana* leaf cDNA.

[0035] The sequence information for AtAACT2 can be found in the gene accession number: NM_180823.3. It is the full-length CDS sequence of the Arabidopsis AACT gene, with a sequence length of 1197 bp. The sequence information for AtHMGS can be found in the gene accession number: NM_117251.3. It is the full-length CDS sequence of the Arabidopsis thaliana HMGS gene, with a sequence length of 1386 bp. The sequence information for AtHMGR can be found in the gene accession number: NM_106299.4. It is the full-length CDS sequence of the Arabidopsis thaliana HMGR gene, with a sequence length of 1929 bp. The sequence information for AtMK can be found in the gene accession number: NM_122627.6. It is the full-length CDS sequence of the Arabidopsis thaliana MK gene, with a sequence length of 1137 bp. The sequence information for AtMVD can be found in the gene accession number: NM_129427.5. It is the full-length CDS sequence of the Arabidopsis thaliana MVD gene, with a sequence length of 1239 bp. The sequence information for AtPMK can be found in the gene accession number: NM_102927.3. It is the full-length CDS sequence of the Arabidopsis PMK gene, with a sequence length of 1518 bp.

[0036] This invention also selects key gene modules from Arabidopsis thaliana, Panax ginseng, and Panax notoginseng for heterologous expression in Nicotiana benthamiana to reconstruct the biosynthetic pathway of ginsenoside Rg3. These genes include core enzyme genes in the ginsenoside pathway: tAtHMGR (modified 3-hydroxy-3-methylglutaryl-CoA reductase, with transmembrane domain removed), PgSE (squalene epoxidase), PgSS1 (squalene synthase), PgDDS (dammarene diol synthase), PgPPDS (protopanaxadiol synthase), Pn1-31 (uridine diphosphate glycosyltransferase), Pn3-31 (uridine diphosphate glycosyltransferase), AtIDI (isopentene diphosphate isomerase), and AtMVD (mevalerate pyrophosphate decarboxylase). Based on the query results of relevant gene transcripts in the NCBI gene database (https: / / www.ncbi.nlm.nih.gov / ), specific primers (with added Gibson Assembly homologous arms) were designed to amplify the target gene fragment from ginseng root cDNA, Panax notoginseng root cDNA, and Arabidopsis thaliana leaf cDNA.

[0037] The sequence information for each gene can be found in the gene accession numbers below, and the specific CDS sequences (based on NCBI queries) are as follows: The tAtHMGR gene, accession number NM_106299.4, is the CDS sequence (with transmembrane domain removed) of the Arabidopsis thaliana AtHMGR gene, as shown in SEQ ID NO:1, with a sequence length of 1293 bp. SEQ ID NO: 1 PgSE gene: accession number AB265170.1, is the CDS sequence of the ginseng PgSE gene, with a sequence length of 1611 bp; PgSS1 gene: accession number AB265171.1, is the CDS sequence of the ginseng PgSS gene, with a sequence length of 1248 bp; PgDDS gene: accession number AB022914.1, is the CDS sequence of the ginseng PgDDS gene, with a sequence length of 2310 bp; The PgPPDS gene, accession number AB034803.2, is the CDS sequence of the ginseng PgPPDS gene, with a sequence length of 1449 bp. Pn1-31 gene: accession number MT551198.1, is the CDS sequence of the Panax notoginseng Pn1-31 gene, with a sequence length of 1368 bp; Pn3-31 gene: accession number MT551200.1, is the CDS sequence of the Panax notoginseng Pn3-31 gene, with a sequence length of 1329 bp; AtIDI gene: Accession number NP_197148.3 is the CDS sequence of the Arabidopsis thaliana AtIDI gene, with a sequence length of 876 bp.

[0038] Example 1 1. Construction of donor vectors d1 / d2: Using a one-step homologous recombination kit (NovaZyn Biotech Inc.), the gene fragments AtAACT2, AtHMGR, AtPMK+AtMVD, tAtHMGR, PgSS1, and Pn1-31+Pn3-31 were inserted into the multiple cloning sites BamHI and EcoRI of the donor vector pYL322d1-35S (which contains a 35S promoter and terminator), respectively, resulting in six d1 donor vectors: pYL322d1-35S-AtAACT2, pYL322d1-35S-AtHMGR, pYL322d1-35S-AtMVD+35S-AtPMK, pYL322d1-35S-tAtHMGR, pYL322d1-35S-PgSS1, and pYL322d1-35S-Pn1-31-35S-Pn3-31.

[0039] Similarly, by homologous recombination, the gene fragments AtHMGS, AtMK, AtPgSE, and PgDDS+PgPPDS were inserted into the multiple cloning sites BamHI and EcoRI of the donor vector pYL322d2-35S (which contains a 35S promoter and terminator), respectively, to obtain four d2 donor vectors: pYL322d2-35S-AtHMGS, pYL322d2-35S-AtMK, pYL322d2-35S-AtPgSE, and pYL322d2-35S-PgDDS-35S-PgPPDS.

[0040] By homologous recombination, the AtIDI+AtMVD gene fragment was ligated to the XbaI and HindIII restriction sites on the PYLMFH vector to replace the Cre gene in the original vector, resulting in PYLMFH-35S-AtIDI-35S-AtMVD.

[0041] 2. Schematic diagram of MVA pathway module recombinant carrier as shown below Figure 1 As shown, the specific construction process is as follows: (1) Select the donor vectors d1 constructed in step 1: pYL322d1-35S-AtAACT2, pYL322d2-35S-AtHMGS, pYL322d1-35S-AtHMGR, pYL322d2-35S-AtMK, pYL322d1-35S-AtMVD+35S-AtPMK.

[0042] (2) The donor vector pYL322d1-35S-AtAACT2 and the recipient vector pYLTAC380GW (at a ratio of 2:1) were mixed in NS3529 competent cells for cotransformation. The heat shock method was used: ice bath for 30 min, heat shock for 90 s, ice bath for 2 min, and recovery in antibiotic-free LB at 37°C and 200 rpm for 2 h. The mixture was then plated on LA plates containing kanamycin (Km, 25 mg / L) and chloramphenicol (Chl, 15 mg / L). After about 18 h, single clones grew. All single clones were washed into tubes with ddH2O and the mixed plasmid was extracted.

[0043] (3) Take about 50 ng of the mixed plasmid and digest it with 0.5 μL of homing endonuclease I-Sce I (NEB) in a 10 μL system (50 ng mixed plasmid; 0.5 μL I-Sce I: 1 μL 10×CutSmart Buffer; add ddH2O to 10 μL) for 4 h. Transform it into Escherichia coli strain NEB10-β, plate it on LA plate containing kanamycin (Km, 25 mg / L), incubate at 37℃ for 15 h, then pick single clones and culture them in LB (containing 25 mg / L Km and 0.5 mM IPTG). Perform bacterial PCR identification. Using Green Taq Mix, extract plasmids that can amplify bright bands. Take 300 ng of each plasmid and digest it with 0.3 μL Not l (NEB) in a 20 μL reaction system (300 ng plasmid; 0.3 μL Not l: 2 μL 10×CutSmart Buffer). Enzyme digestion in Buffer (add ddH2O to 20 μL) showed three bands, and the positive clone pYLTAC380GW-AtAACT2 containing the target gene of 3.84 kbp was the desired positive clone.

[0044] (4) The donor vector pYL322d2-AtHMGS and the recipient vector pYLTAC380GW-AtAACT2 from (3) (at a ratio of 2:1) were mixed in NS3529 competent cells for cotransformation. The transformation was carried out according to the method in (2), and the mixture was plated on LA plates containing kanamycin (Km, 25 mg / L) and ampicillin (Amp, 70 mg / L). After about 18 hours, single clones grew. All single clones were washed into tubes with ddH2O and the mixed plasmid was extracted.

[0045] (5) Take about 50 ng of mixed plasmid and digest it with 0.5 μL PI-Sce I (NEB) and 0.5 μL BSA in a 10 μL system (50 ng mixed plasmid; 0.5 μL PI-Sce I: 1 μL 10×CutSmart Buffer; add ddH2O to 10 μL) for 4 h. Then transform and verify according to the method in (3). Four bands appear. The positive clone pYLTAC380GW-AtAACT2-AtHMGS contains the target gene 3.84 kbp AtAACT2 and 2.22 kbp AtHMGS.

[0046] (6) Multiple rounds of recombination were carried out, and the donor vectors pYL322d1-35S-AtHMGR (d1), pYL322d2-35S-AtMK (d2), and pYL322d1-35S-AtMVD+35S-AtPMK (d1) (using d1 and d2 donor vectors containing different genes in a crossover) were co-transformed with the recipient vectors constructed in the previous round to construct 380GW-AtAACT2-AtHMGS-AtHMGR-AtMK-AtMVD-AtPMK; (7) Finally, the reaction was carried out by BP reaction at 25°C. 380GW-AtAACT2-AtHMGS-AtHMGR-AtMK-AtMVD-AtPMK plasmid and PYLMFH plasmid were reacted in a 10 μL reaction system (200 ng 380GW-AtAACT2-AtHMGS-AtHMGR-AtMK-AtMVD-AtPMK plasmid; 100 ng PYLMFH plasmid; 2 μL 5×BP enzyme mixture; ddH2O added to 10 μL) for 5 hours. Then, 1 μL proteinase K solution was added to terminate the reaction at 37°C for 10 minutes. The cells were then transferred to NEB10-β (Bomaide Biotechnology Co., Ltd.) competent cells, and single clones were selected for identification.

[0047] Detection using Not1 enzyme digestion revealed a correct positive final vector: 380MF-AtAACT2-AtHMGS-AtHMGR-AtMK--AtPMK-AMVD-Cre-HPT (MVA). Figure 1 There are 9 DNA bands ( Figure 2 Positive clones were selected for whole-plasmid sequencing analysis. The correct MVA plasmid was selected and transformed into Agrobacterium GV3101 (denoted as MVA Agrobacterium). The transformed Agrobacterium strain was stored at -80 ℃ for later use.

[0048] 3. Rg3 pathway module recombination vectors, such as Figure 3 As shown, the specific construction process is as follows: (1) Select the donor vectors d2 constructed in step 1: pYL322d1-35S-tAtHMGR, pYL322d2-35S-PgSS1, pYL322d1-35S-PgSE, pYL322d2-35S-PgPPDS-35S-PgDDS (double gene module), pYL322d1-35S-Pn1-31-35S-Pn3-31 (double gene module) and PYLMFH-35S-AtIDI-35S-AtMVD (double gene module).

[0049] (2) Using the TransGene Stacking II multi-gene aggregation vector system, referring to steps (2)-(6) in step 1, the above gene modules were sequentially assembled into the recipient vector pYLTAC380GW. The assembly order was: tAtHMGR module → PgSS1 module → PgSE module → PgPPDS-PgDDS dual module → Pn1-31-Pn3-31 dual module. The correctness of each round of assembly was ensured by co-transformation, antibiotic screening (kanamycin Km), and enzyme digestion verification (Not I), to obtain the intermediate vector pYLTAC380GW-tAtHMGR-PgSS1-PgSE-PgPPDS-PgDDS-Pn1-31-Pn3-31.

[0050] (3) Then, through BP recombination reaction, the AtIDI-AtMVD double gene module and the hygromycin selection marker gene HPT on the donor vector PYLMFH-35S-AtIDI-35S-AtMVD were integrated into the intermediate vector pYLTAC380GW-tAtHMGR-PgSS1-PgSE-PgPPDS-PgDDS-Pn1-31-Pn3-31. The reaction system is as follows: 25℃, react for 5 hours in a 10μL reaction system (200ng pYLTAC380GW-tAtHMGR-PgSS1-PgSE-PgPPDS-PgDDS-Pn1-31-Pn3-31 plasmid; 100ng PYLMFH-35S-AtIDI-35S-AtMVD plasmid; 2μL 5×BP enzyme mixture; ddH2O added to 10μL). Then, 1 μL of proteinase K solution was added to terminate the reaction at 37°C for 10 minutes. The cells were then transformed into NEB10-β (Bomaide Biotechnology Co., Ltd.) competent cells, and single clones were selected for identification. After transformation, the expression vector pYLTAC380GW-tAtHMGR-PgSS1-PgSE-PgPPDS-PgDDS-Pn1-31-Pn3-31-AtMVD-AtIDI-HPT (abbreviated as Rg3 vector) was obtained through Not I digestion and whole-plasmid sequencing verification (the expected fragment includes all gene modules and HPT). Figure 3 The vector successfully constructed a large fragment expression module with a size of 40.458 kb, and it was verified by enzyme digestion and electrophoresis. Figure 4 The correctness was confirmed by sequencing. The correct Rg3 plasmid was selected and transformed into Agrobacterium GV3101 (denoted as Agrobacterium Rg3). The transformed Agrobacterium strain was stored at -80 ℃ for later use.

[0051] 4. Recombinant carrier introduced into plant substrate By introducing recombinant vectors of the MVA pathway module and the Rg3 synthesis module into the plant chassis via Agrobacterium-mediated transformation, the MVA and Rg3 modules are transiently co-expressed, thereby activating terpene precursor metabolism and synthesizing Rg3 and Rh2.

[0052] Effect verification: (1) Detection of IPP content in transiently converted tobacco Agrobacterium containing the target vector (MVA) obtained in step 2 of Example 1 was inoculated into LB medium containing kanamycin and cultured in a shaker at 28°C for 24 hours. The culture was then diluted 100-fold and inoculated into fresh LB medium, with the addition of MES (0.5 M, pH 5.6) and AS (100 mM). The culture was continued in a shaker at 28°C until the OD600 reached 0.6-1.0. The bacterial cells were collected by centrifugation at 4,000 rpm for 10 minutes and resuspended in 10 mM MgCl2 until the OD600 reached 1.0. 2 μl of AS was added per milliliter of the bacterial suspension, and the mixture was allowed to stand for 3 hours. The Agrobacterium suspension was injected into the abaxial surface of approximately 5-week-old leaves of Nicotiana benthamiana using a syringe without the needle, ensuring full penetration of the bacterial suspension into the mesophyll cells. Finally, the tobacco plants were cultured in a light incubator for 4 days to detect the expression of the target gene or the accumulation of metabolites.

[0053] MVA vectors were transferred into Agrobacterium GV3101, and then transiently transferred into tobacco leaves. Using wild-type tobacco as a control, the IPP content was determined three days after injection using a Plant Isoprene Pyrophosphate (IPP) ELISA kit from Shanghai Fantai Biotechnology Co., Ltd. The kit employed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) with double antibodies. Samples, standards, and HRP-labeled detection antibodies were added sequentially to the microwells pre-coated with isopentenyl pyrophosphate (IPP) antibodies, followed by incubation and thorough washing. The substrate TMB was used for color development; TMB was converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity was positively correlated with the IPP content in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader. A linear regression curve was plotted with the standard concentration on the x-axis and the corresponding OD value on the y-axis, and the concentration of each sample was calculated according to the curve equation.

[0054] The results showed that the IPP content in tobacco leaves after transient transformation (CK, transient transformation with empty vector Agrobacterium tobacco control) and MVA was 372±11 ng / g and 533±10 ng / g, respectively. Figure 5 Compared with the control (CK), the IPP content in tobacco leaves increased by 43% after transient conversion to MVA, which preliminarily verified the effectiveness of the MVA expression module in increasing the IPP content in plants.

[0055] (2) Detection of the content of ginsenosides Rh2 and Rg3 in tobacco during transient conversion Agrobacterium tumefaciens Rg3 and MVA suspensions were inoculated into 50 mL centrifuge tubes and cultured until the OD600 reached 0.6-1.0. The culture was then resuspended in 10 mM MgCl2 until the OD600 reached 1.0. 2 μl of AS was added per mL of culture, and the mixture was allowed to stand for 3 hours to obtain Agrobacterium tumefaciens Rg3 and MVA, respectively. The Agrobacterium tumefaciens suspension (Rg3 and MVA) was injected into approximately 5-week-old *Nicotiana benthamiana* (Rg3+MVA) plants using a syringe without the needle. Wild-type plants served as the control, while Rg3, MVA, and Rg3+MVA were used as experimental groups. Leaf metabolites were extracted after 4 days for LC-MS / MS analysis.

[0056] The detection method is as follows: ① Liquid chromatography conditions: The mobile phase mixtures are shown in Table 1, where mobile phase A is acetonitrile and mobile phase B is formic acid water. The chromatographic column was a Waters ACQUITY UPLC BEH C18, 1.7 μm, 2.1 mm × 100 mm, with a flow rate of 0.2 mL / min, and the injection volume was 2 μL for standard and 10 μL for sample.

[0057] Table 1. Elution of mobile phase (V / V)

[0058] ②Mass spectrometry conditions: Electrospray ionization (ESI) was used as the ion source; negative ion scanning multiple reaction monitoring was employed. Mode (MRM) detection; Ion spray voltage: -4500 V. Auxiliary heater temperature (TEM) 450 °C, curtain gas (CUR) flow rate 35 psi; atomizing gas (ion source gas1, GS1) 50 psi, auxiliary heating gas (ion source gas2, GS2) 50 psi.

[0059] ③ Preparation of Ginsenoside Standard Curve: Accurately weigh Rh2 and Rg3 standard reference powders using an analytical balance and dissolve them in mass spectrometry-grade methanol to prepare a stock solution with a mass concentration of 5 mg / mL. Dilute the stock solution with methanol to prepare standard solutions with mass concentrations of 0.16, 0.8, 1.6, 4.0, 8.0, 20.0, and 100.0 μg / mL. After filtration through a 0.22 μm organic membrane, the solutions are injected for analysis at different concentrations. For each concentration of standard, the peak area of ​​the target peak is collected as the response value. A scatter plot is plotted with the mass concentration of the standard solution as the independent variable (x-axis) and the response value as the dependent variable (y-axis). Based on the trend of the scatter plot, a suitable fitting model is selected for linear regression analysis to obtain the equation and parameters of the standard curve.

[0060] The product peaks of standard solutions at different concentrations were integrated in LC-MS / MS, and scatter plots were plotted to obtain the peak areas at the corresponding concentrations. The horizontal axis represents the standard concentration (μg / mL), and the vertical axis represents the peak area in‰. The standard curves for Rh2 and Rg3 were fitted and are shown in Table 2. Table 2 shows the linear regression equations for Rh2 and Rg3.

[0061] The test results found that, Figure 6 As shown, wild-type tobacco cannot produce ginsenosides Rg3 and Rh2; the content of ginsenoside Rg3 in the leaves of Rg3-transformed tobacco was 15.04 μg / g, but ginsenoside Rh2 was undetectable; while the content of Rg3 in the leaves of MVA+Rg3-transformed tobacco was 23.70 μg / g and the content of Rh2 was 0.37 μg / g. These results demonstrate that the Rg3 expression module can effectively synthesize ginsenoside Rg3 in plants, and further verify that the MVA expression module can promote ginsenoside synthesis.

[0062] Application value: By stably creating plant chassis with high IPP content (such as rice and tobacco) through genetics, it can support the biosynthesis of downstream terpenoid compounds (such as ginsenosides and precursors of the anticancer drug paclitaxel), overcoming the limitations of high cost of chemical synthesis and large carbon source consumption of microbial fermentation.

[0063] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for promoting ginsenoside biosynthesis based on the synergistic expression of the MVA pathway and saponin synthesis module, characterized in that, Includes the following steps: S1. Construct a recombinant vector containing the following gene modules: MVA pathway module recombinant vector: containing AtAACT2, AtHMGS, AtHMGR, AtMK, AtPMK, AtMVD, Cre, and HPT genes, with the vector backbone based on the TransGene Stacking II system; Rg3 synthesis module recombinant vector: containing AtIDI, AtMVD, Pn1-31, Pn3-31, PgDDS, PgPPDS, PgSE, PgSS1, tAtHMGR, HPT, vector backbone based on TransGene Stacking II system; S2. The MVA pathway module recombinant vector and the Rg3 synthesis module recombinant vector were introduced into the plant chassis via Agrobacterium-mediated transformation, transiently co-expressing the MVA and Rg3 modules, activating terpene precursor metabolism and synthesizing ginsenosides Rg3 and Rh2.

2. The method according to claim 1, characterized in that, Construction of MVA pathway module recombinant vector in step S1: Donor vectors containing AtAACT2, AtHMGS, AtHMGR, AtMK, and AtMVD+AtPMK genes respectively were co-transformed with recipient vectors in sequence. Then, the hygromycin selection marker gene HPT and Cre gene on the pYLMFH vector were recombined by BP reaction to obtain MVA pathway module recombinant vector.

3. The method according to claim 2, characterized in that, Construction of donor vectors containing the AtAACT2, AtHMGS, AtHMGR, AtMK, AtMVD+AtPMK genes: The AtAACT2, AtHMGR, and AtPMK+AtMVD gene fragments were inserted into the multiple cloning sites BamHI and EcoRI of the donor vector pYL322d1-35S, respectively, to obtain three d1 donor vectors: pYL322d1-35S-AtAACT2, pYL322d1-35S-AtHMGR, and pYL322d1-35S-AtMVD-35S-AtPMK; The AtHMGS and AtMK gene fragments were inserted into the multiple cloning sites BamHI and EcoRI of the donor vector pYL322d2-35S, respectively, to obtain two d2 donor vectors: pYL322d2-35S-AtHMGS and pYL322d2-35S-AtMK.

4. The method according to claim 2, characterized in that, The BP reaction involves reacting the co-transformed plasmid vector with PYLMFH to recombine the hygromycin selection marker gene HPT and the Cre gene from the pYLMFH vector.

5. The method according to claim 1, characterized in that, Construction of the Rg3 synthesis module recombinant vector in step S1: Donor vectors containing the genes tAtHMGR, PgSE, PgSS1, PgDDS+PgPPDS, and Pn1-31+Pn3-31 were co-transformed with recipient vectors in sequence. Then, through BP recombination reaction, the AtIDI-AtMVD dual gene module and the hygromycin selection marker gene HPT were integrated into the co-transformed plasmid vector to obtain the Rg3 pathway module recombinant vector.

6. The method according to claim 5, characterized in that, Construction of donor vectors containing the genes tAtHMGR, PgSE, PgSS1, PgDDS+PgPPDS, and Pn1-31+Pn3-31: The tAtHMGR, PgSS1, and Pn1-31+Pn3-31 gene fragments were inserted into the multiple cloning sites BamHI and EcoRI of the donor vector pYL322d1-35S, respectively, to obtain three d1 donor vectors: pYL322d1-35S-tAtHMGR, pYL322d1-35S-PgSS1, and pYL322d1-35S-Pn1-31-35S-Pn3-31. The AtPgSE and PgDDS+PgPPDS gene fragments were inserted into the multiple cloning sites BamHI and EcoRI of the donor vector pYL322d2-35S, respectively, to obtain two d2 donor vectors: pYL322d2-35S-PgSE and pYL322d2-35S-PgDDS+35S-PgPPDS.

7. The method according to claim 5, characterized in that, The AtIDI-AtMVD dual-gene module and the hygromycin selection marker gene HPT were integrated into the co-transformed plasmid vector by reacting the co-transformed plasmid vector with PYLMFH-35S-AtIDI-35S-AtMVD via a BP recombination reaction. Preparation of PYLMFH-35S-AtIDI-35S-AtMVD: The AtIDI+AtMVD gene fragment was ligated into the XbaI and HindIII restriction sites on the PYLMFH vector to replace the Cre gene in the original vector, thus obtaining PYLMFH-35S-AtIDI-35S-AtMVD.

8. The method according to claim 1, characterized in that, In step S2, the co-expression ratio of the MVA pathway module recombinant vector and the Rg3 synthesis module recombinant vector is 1:

3.

9. The method according to claim 1, characterized in that, In step S2, the Agrobacterium-mediated method is as follows: Agrobacterium containing the target vector is inoculated into LB medium containing antibiotics and cultured on a shaker; after dilution, it is inoculated into a new LB medium, and MES and AS are added, and cultured on a shaker until OD600 reaches 0.6-1.0; the bacterial cells are collected by centrifugation, resuspended until OD600=1.0, AS is added to the bacterial solution, and the solution is allowed to stand.

10. The method according to claim 1, characterized in that, In step S2, the plant substrate is tobacco.

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