A recombinant expression plasmid, recombinant Agrobacterium, and recombinant tobacco expression system, and a method for synthesizing paclitaxel using these components.

Abstract

This invention belongs to the fields of synthetic biology and pharmaceutical technology, and discloses a method for heterologous biosynthesis of paclitaxel using tobacco plants. Specifically, it discloses recombinant plant expression plasmids encoding glycosyl hydrolase P1-2 and acyltransferase ICM9-6, and recombinant Agrobacterium, thereby enabling the heterologous biosynthesis of paclitaxel using a tobacco plant expression system with 7-xylose-10-deacetylpaclitaxel (XDT) or 10-deacetylpaclitaxel (DT) as precursors. Compared with chemical synthesis methods, this method has the advantages of simple operation, mild transformation conditions, high efficiency and low toxicity, and has promising development prospects.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic biology and pharmaceutical technology, and relates to a method for heterologous biosynthesis of paclitaxel using tobacco as a chassis. Specifically, it relates to a plant recombinant expression plasmid that can encode glycosyl hydrolase P1-2 and acyltransferase ICM9-6, a recombinant Agrobacterium and recombinant tobacco expression system, and a method for heterologous biosynthesis of paclitaxel from tobacco plants. Background Technology

[0002] Paclitaxel Paclitaxel, a diterpenoid compound derived from the yew tree, has been the best-selling plant-based anti-tumor drug since its FDA approval in December 1992. It has been successfully used clinically to treat more than a dozen solid tumors, including ovarian cancer, breast cancer, lung cancer, malignant melanoma, colorectal cancer, head and neck cancer, lymphoma, and brain tumors. With the development and application of new formulations and indications, the demand for paclitaxel in both domestic and international pharmaceutical markets continues to increase. Like most plant-derived natural products, paclitaxel is present in extremely low concentrations in yew species (approximately 0.03%, in Taxus media). Currently, the main methods for producing paclitaxel in China are extraction from the branches and leaves of nursery-grown yew trees and the semi-synthetic chemical synthesis of paclitaxel using 10-DAB as a precursor. However, the extraction of paclitaxel often generates large amounts of 7-xylosyltaxanes as byproducts, with 7-xylosyl-10-deacetyltaxol (XDT) being a typical example. These byproducts are usually discarded as waste, leading to significant resource waste. Furthermore, the natural content of XDT in yew trees is approximately 0.5%, far exceeding the natural content of paclitaxel. Utilizing these byproducts to increase the supply of paclitaxel undoubtedly has significant economic, social, and environmental implications.

[0003] In our previous studies, we achieved the in vitro conversion of 7-xylose-10-deacetylpaclitaxel (XDT) to paclitaxel using an in vitro enzymatic catalytic experiment. The yield of paclitaxel in a 50 mL in vitro reaction system was 0.64 mg / mL [Li BJ, et al. Improving 10-deacetylbaccatin III-10-β-O-acetyltransferase catalytic fitness for Taxol production. Nat Commun., 2017, 8:15544-15556.]. However, the in vitro enzymatic catalytic experiment requires the addition of acetyl-CoA as the acyl donor for paclitaxel synthesis, resulting in higher costs. Furthermore, previous results showed that the glycosyl hydrolase P1-2 could be functionally expressed in yeast and Aspergillus nidulans expression systems, but the expression product in the E. coli expression system lost its activity. Acyltransferase ICM9-6 can only be functionally expressed in Escherichia coli, and the expression products in yeast expression systems and Aspergillus nidulans expression systems are inactive.

[0004] In recent years, the role of plant-based heterologous biosynthesis in the synthesis of complex natural products has gained increasing attention, leading to the successful de novo biosynthesis of several important compounds or their intermediates. The applicant of this invention constructed a functional co-expression system of two elements, P1-2 and ICM9-6, in tobacco chassis cells. While feeding the XDT substrate, acetyl-CoA synthesized by the tobacco cells themselves can be used as an acyl donor for the conversion reaction. This overcomes the aforementioned drawbacks and provides a feasible solution for the reuse of XDT and the biosynthesis of paclitaxel through non-natural pathways. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned problems by providing a method for heterologous biosynthesis of paclitaxel using plant expression plasmids transiently infecting tobacco.

[0006] To achieve the above objectives, the present invention includes the following technical solutions.

[0007] In a first aspect, the present invention provides a recombinant plant expression plasmid, wherein the plasmid encodes a glycosyl hydrolase P1-2 comprising the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80% identity with it and / or an acyltransferase ICM9-6 comprising the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% identity with it.

[0008] In a second aspect, the present invention provides a recombinant Agrobacterium, wherein the recombinant Agrobacterium is transformed with the recombinant plant expression plasmid or encoding the glycosyl hydrolase P1-2 and / or the acyltransferase ICM9-6.

[0009] Thirdly, the present invention provides a method for heterologous synthesis of paclitaxel from tobacco plants, comprising:

[0010] (1) Construct a recombinant plant expression plasmid, wherein the plasmid encodes a glycosyl hydrolase P1-2 containing the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80% identity with it and an acyltransferase ICM9-6 containing the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% identity with it.

[0011] (2) The recombinant expression plasmid was transformed into Agrobacterium to obtain recombinant Agrobacterium;

[0012] (3) Using the recombinant Agrobacterium to infect tobacco or a portion thereof, or tobacco cells;

[0013] (4) The infected tobacco or parts thereof, or tobacco cells, were fed with substrate and then paclitaxel was extracted.

[0014] Fourthly, this invention provides the use of a combination of glycosyl hydrolase P1-2 and acyltransferase ICM9-6, the recombinant plant expression plasmid, the recombinant Agrobacterium, and the recombinant tobacco expression system in the preparation of paclitaxel from XDT or DT. Beneficial technical effects:

[0015] The tobacco biosynthesis system provided by this invention, by expressing P1-2 and ICM9-6, can realize the heterologous biosynthesis of paclitaxel using XDT and DT as substrates, providing a new synthetic biology route for the production of paclitaxel, and has development and application potential. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the tobacco recombinant expression plasmids pEAQ-P1-2 and pEAQ-ICM9-6.

[0017] Figure 2 This is the LC-MS detection of DT and its precursor XDT in tobacco infected with Agrobacterium GV3101-pEAQ-P1-2.

[0018] Figure 3 This is the LC-MS detection result of paclitaxel and its precursor DT in tobacco infected with Agrobacterium GV3101-pEAQ-ICM9-6.

[0019] Figure 4This paper presents the LC-MS detection results of precursor XDT, intermediate product DT, and final product paclitaxel in tobacco co-infected with Agrobacterium GV3101-pEAQ-P1-2 and GV3101-pEAQ-ICM9-6.

[0020] Figure 5 This is the concentration-peak area standard curve of paclitaxel.

[0021] Figure 6 Chromatogram and mass spectrum of purified paclitaxel.

[0022] Figure 7 Paclitaxel 1 H NMR spectrum.

[0023] Figure 8 Paclitaxel 13 C NMR spectrum. Detailed Implementation

[0024] For those skilled in the art, various changes and improvements can be made to the specific embodiments of this application without departing from the spirit and scope of this application, which are also included within the protection scope of this application.

[0025] In some embodiments, the present invention provides a recombinant plant expression plasmid, wherein the plasmid encodes a glycosyl hydrolase P1-2 comprising the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80% identity with it, and an acyltransferase ICM9-6 comprising the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% identity with it.

[0026] In some embodiments, the glycosyl hydrolase P1-2 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:1.

[0027] In some embodiments, the acyltransferase ICM9-6 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:2.

[0028] In some embodiments, the plasmid contains separate or fused genes encoding the glycosyl hydrolase P1-2 and the acyltransferase ICM9-6.

[0029] In some embodiments, a plasmid contains one or more copies of the gene encoding the glycosyl hydrolase P1-2. In some embodiments, a plasmid contains one or more copies of the gene encoding the acyltransferase ICM9-6.

[0030] In some embodiments, the coding genes for the glycosyl hydrolase P1-2 and the coding genes for the acyltransferase ICM9-6 are contained in separate vectors. Alternatively, in some embodiments, the coding genes for the glycosyl hydrolase P1-2 and the coding genes for the acyltransferase ICM9-6 are contained in the same vector.

[0031] In some embodiments, the plasmid comprises a gene encoding glycosyl hydrolase P1-2 as shown in SEQ ID NO:3 and / or a gene encoding acyltransferase ICM9-6 as shown in SEQ ID NO:4. In this document, a single plasmid may comprise one or both of the gene encoding glycosyl hydrolase P1-2 as shown in SEQ ID NO:3 and the gene encoding acyltransferase ICM9-6 as shown in SEQ ID NO:4.

[0032] In some embodiments, the plasmid is an Agrobacterium expression plasmid, such as an Agrobacterium binary expression vector.

[0033] In some embodiments, the plasmid may be constructed from vectors selected from: pEAQ-HT, pCB302, pEff, PBI series expression vectors, pCAMBIA series expression vectors, and pGreen series expression vectors.

[0034] In some embodiments, the present invention provides a recombinant Agrobacterium, wherein the recombinant Agrobacterium is transformed with the recombinant plant expression plasmid or expresses the glycosyl hydrolase P1-2 and / or the acyltransferase ICM9-6.

[0035] In some embodiments, the Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

[0036] In some embodiments, a single Agrobacterium expresses one or both of the glycosyl hydrolase P1-2 and the acyltransferase ICM9-6.

[0037] In some embodiments, the Agrobacterium is integrated with a gene encoding glycosyl hydrolase P1-2 as shown in SEQ ID NO:3 and / or a gene encoding acyltransferase ICM9-6 as shown in SEQ ID NO:4. In this document, a single Agrobacterium is integrated with one or both of the gene encoding glycosyl hydrolase P1-2 as shown in SEQ ID NO:3 and the gene encoding acyltransferase ICM9-6 as shown in SEQ ID NO:4.

[0038] In some embodiments, the present invention provides a method for heterologous synthesis of paclitaxel from tobacco plants, comprising:

[0039] (1) Construct a recombinant plant expression plasmid, wherein the plasmid encodes a glycosyl hydrolase P1-2 containing the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80% identity with it and an acyltransferase ICM9-6 containing the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% identity with it.

[0040] (2) The recombinant expression plasmid was transformed into Agrobacterium to obtain recombinant Agrobacterium;

[0041] (3) Using the recombinant Agrobacterium to infect the tobacco plant or a part thereof, or tobacco cells;

[0042] (4) Feed the infected tobacco plants or parts thereof, or tobacco cells with substrate, and then extract paclitaxel.

[0043] In some embodiments, the Nicotiana plant is Nicotiana tabacum L. or Nicotiana benthamiana.

[0044] In some embodiments, in step (1), the recombinant plant expression plasmid is constructed using an Agrobacterium expression vector, such as an Agrobacterium binary expression vector.

[0045] In some embodiments, the plasmid may be constructed from vectors selected from: pEAQ-HT, pCB302, pEff, PBI series expression vectors, pCAMBIA series expression vectors, and pGreen series expression vectors.

[0046] In this paper, the coding genes for glycosyl hydrolase P1-2 and acyltransferase ICM9-6 can be ligated together with the vector using conventional methods such as enzyme digestion and ligation or homologous recombination.

[0047] In some embodiments, the glycosyl hydrolase P1-2 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:1.

[0048] In some embodiments, the acyltransferase ICM9-6 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:2.

[0049] In some embodiments, the plasmid contains separate or fused genes encoding the glycosyl hydrolase P1-2 and the acyltransferase ICM9-6.

[0050] In some embodiments, a plasmid contains one or more copies of the gene encoding the glycosyl hydrolase P1-2. In some embodiments, a plasmid contains one or more copies of the gene encoding the acyltransferase ICM9-6.

[0051] In some embodiments, the coding genes for the glycosyl hydrolase P1-2 and the coding genes for the acyltransferase ICM9-6 are contained in separate vectors. Alternatively, in some embodiments, the coding genes for the glycosyl hydrolase P1-2 and the coding genes for the acyltransferase ICM9-6 are contained in the same vector.

[0052] In some embodiments, the plasmid comprises a gene encoding glycosyl hydrolase P1-2 as shown in SEQ ID NO:3 and / or a gene encoding acyltransferase ICM9-6 as shown in SEQ ID NO:4. In this document, a single plasmid may comprise one or both of the gene encoding glycosyl hydrolase P1-2 as shown in SEQ ID NO:3 and the gene encoding acyltransferase ICM9-6 as shown in SEQ ID NO:4.

[0053] In this paper, any method known in the art (e.g., electroporation, CaCl2 method (heat shock method)) can be used to transform the recombinant expression plasmid into competent Agrobacterium cells.

[0054] In some embodiments, the Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

[0055] In some embodiments, a single Agrobacterium expresses one or both of the glycosyl hydrolase P1-2 and the acyltransferase ICM9-6.

[0056] In some embodiments, the Agrobacterium is integrated with a gene encoding glycosyl hydrolase P1-2 as shown in SEQ ID NO:3 and / or a gene encoding acyltransferase ICM9-6 as shown in SEQ ID NO:4. In this document, a single Agrobacterium is integrated with one or both of the gene encoding glycosyl hydrolase P1-2 as shown in SEQ ID NO:3 and the gene encoding acyltransferase ICM9-6 as shown in SEQ ID NO:4.

[0057] In some embodiments, in step (3), recombinant Agrobacterium tumefaciens expressing the glycosyl hydrolase P1-2 or the acyltransferase ICM9-6 are used to infect tobacco plants or parts thereof, or tobacco cells, respectively.

[0058] In some embodiments, in step (3), recombinant Agrobacterium species expressing either the glycosyl hydrolase P1-2 or the acyltransferase ICM9-6 are used to co-infect tobacco plants or parts thereof, or tobacco cells. In some embodiments, recombinant Agrobacterium species expressing either the glycosyl hydrolase P1-2 or the acyltransferase ICM9-6 are used to co-infect tobacco plants or parts thereof, or tobacco cells, at a cell ratio of 10:1 to 1:10.

[0059] In some embodiments, in step (3), a recombinant Agrobacterium that can express both the glycosyl hydrolase P1-2 and the acyltransferase ICM9-6 (i.e., the same strain that can express both the glycosyl hydrolase P1-2 and the acyltransferase ICM9-6) is used to infect a tobacco plant or a part thereof, or tobacco cells.

[0060] In some implementations, in step (4), the substrate is XDT or DT.

[0061] In some embodiments, in step (4), paclitaxel is extracted using an alcohol solvent (e.g., methanol, ethanol, butanol), chloroform, ethyl acetate, or acetone.

[0062] In some embodiments, the present invention provides the use of a combination of glycosyl hydrolase P1-2 and acyltransferase ICM9-6, the recombinant plant expression plasmid, the recombinant Agrobacterium, or the recombinant tobacco expression system in the preparation of paclitaxel from XDT or DT, wherein the glycosyl hydrolase P1-2 comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80% identity with it, and the acyltransferase ICM9-6 comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% identity with it.

[0063] In this paper, by constructing a recombinant Agrobacterium that can express glycosyl hydrolase P1-2 and acyltransferase ICM9-6 separately or simultaneously, and then infecting tobacco plants with this Agrobacterium, paclitaxel can be produced using tobacco plants as a biosynthetic system.

[0064] Example

[0065] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Those skilled in the art should understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention. The scope of protection of the present invention is limited only by the claims. Without departing from the scope of the claims, those skilled in the art can make various modifications and improvements to various aspects of the present invention, and these modifications and improvements also fall within the scope of protection of the present invention.

[0066] Additionally, it should be noted that, unless otherwise specified, all materials and reagents used in the following embodiments are commonly used in the art and can be obtained through conventional commercial means; all methods used are conventional methods known to those skilled in the art.

[0067] Example 1

[0068] Construction of recombinant expression plasmids pEAQ-P1-2 and pEAQ-ICM9-6:

[0069] Using Pichia pastoris recombinant expression plasmid pPIC3.5K-P1-2 (constructed according to Cheng, HL, et al. Cloning and characterization of the glycoside hydrolases that remove xylosyl groups from 7-β-xylosyl-10-deacetyltaxol and its analogues. Molecular & Cellular Proteomics. 2013, 12: 2236-2248.) and Escherichia coli recombinant expression plasmid pCWori-ICM9-6 (constructed according to the method described in Example 4 of CN116987681A regarding the construction of 2-283-P217K / F301V / E350M / S351D) as templates, specific primers were designed, and NEB Q5 High-Fidelity DNA was used. Polymerase amplified the target gene fragments P1-2 (GenBank accession number: JN167171.1) and ICM9-6 from the plasmids using the PCR amplification system and conditions shown below. The upstream and downstream primers used are listed in Table 1.

[0070] Table 1. Primers for PCR amplification

[0071]

[0072] PCR amplification system (50 μL):

[0073]

[0074] PCR amplification conditions: 98℃, 30s; 98℃, 10s; 71℃, 30s; 72℃, 45s; 35 cycles; 72℃, 2min; 4℃, ∞.

[0075] The pEAQ-HT vector, after double digestion with Nru I and Xho I, was detected by 1.0% agarose gel electrophoresis along with the PCR product. The linearized vector and PCR product were purified and recovered using the Transgen EasyPure Quick Gel Extraction Kit according to its manufacturer's instructions. Homologous recombination ligation of the recovered linearized vector and target gene fragment was performed using the ClonExpress II One Step Cloning Kit according to its manufacturer's instructions, and the resulting cells were transformed into DH5α competent E. coli cells. The cells were then plated onto LB agar containing 50 μg / mL kanamycin-resistant medium and incubated at 37°C for 12 h. Single colonies were picked for colony PCR screening, and positive transformants were sequenced for verification.

[0076] Example 2

[0077] Agrobacterium competent transformation:

[0078] Take 1 μg each of the correctly sequenced pEAQ-P1-2 and pEAQ-ICM9-6 recombinant expression plasmids obtained in Example 1, and add them to 100 μl of competent Agrobacterium tumefaciens strain GV3101 cells. Incubate on ice for 5 min, flash freeze in liquid nitrogen for 5 min, incubate in a 28°C water bath for 5 min, place on ice for 5 min, add 1 mL of antibiotic-free LB medium, and incubate at 28°C on a shaker for 4 h. Spread all bacterial cells on LB agar plates containing 50 μg / ml kanamycin (Kan) and 20 μg / ml rifamycin (Rif), and incubate at 28°C for 2-4 days. Select single-clone colonies for colony PCR screening to obtain positive clones of Agrobacterium tumefaciens strains GV3101-pEAQ-P1-2 and GV3101-pEAQ-ICM9-6.

[0079] The initial vector pEAQ-HT was also transformed into competent cells of Agrobacterium tumefaciens strain GV3101 using the same method, resulting in Agrobacterium tumefaciens strain GV3101-pEAQ-HT as a control.

[0080] Example 3

[0081] Agrobacterium infection of tobacco leaves:

[0082] (1) Single infection: Single colonies of Agrobacterium strains GV3101-pEAQ-HT, GV3101-pEAQ-P1-2 and GV3101-pEAQ-ICM9-6 obtained in Example 2 were picked and placed in LB liquid medium (containing 50 μg / ml Kan + 20 μg / ml Rif) and incubated overnight at 28°C until OD. 600 >2.0. Collect bacterial cells separately and resuspend them to OD500 using introduction buffer (10 mM MES, 10 mM MgCl2, and 100 μM acetylsylcholine). 600 =0.8 and incubate at room temperature for 2 hours, then inject tobacco leaves using a needleless syringe.

[0083] (2) Co-infection: Single colonies of GV3101-pEAQ-P1-2 and GV3101-pEAQ-ICM9-6 were picked and added to LB liquid medium (containing 50 μg / ml Kan + 20 μg / ml Rif) and incubated overnight at 28°C until OD. 600>2.0. Collect bacterial cells separately and resuspend them to OD500 using introduction buffer (10 mM MES, 10 mM MgCl2, and 100 μM acetylsylcholine). 600 =1.6, the two bacterial cells were mixed at a volume ratio of 1:1 after resuspension and incubated at room temperature for 2 hours, and then injected into tobacco leaves using a needleless syringe.

[0084] Example 4

[0085] Substrate feeding and extraction of products from tobacco leaves:

[0086] Three days after the transient infection of tobacco with Agrobacterium in Example 3, 100 μM of XDT substrate was fed to tobacco leaves infected with Agrobacterium strain GV3101-pEAQ-P1-2 alone; 100 μM of DT substrate was fed to tobacco leaves infected with Agrobacterium strain GV3101-pEAQ-ICM9-6 alone; 100 μM of XDT substrate was fed to tobacco leaves co-infected with Agrobacterium strains GV3101-pEAQ-P1-2 and GV3101-pEAQ-ICM9-6; similarly, 100 μM of XDT or DT was also fed to tobacco leaves infected with Agrobacterium strain GV3101-pEAQ-HT alone as a blank control. The tobacco leaves were harvested and freeze-dried 1.5 days after feeding.

[0087] Freeze-dried tobacco leaves were used, and the products and potential substrates were extracted from the tobacco at a ratio of 30 mL ethyl acetate per 1 g of dry leaf weight. After ultrasonic treatment for 30 min, the extract was filtered to obtain the filtrate. The filtrate was then treated in a rotary evaporator to evaporate the organic solvent. Then, 30 mL ethyl acetate was added to the leaves again for ultrasonic extraction, which was repeated 5 times. The extracts were combined and evaporated to dryness to obtain the rotary evaporation product. The rotary evaporation product was dissolved by adding 1.5 mL methanol per 1 g of dry leaf extract. The supernatant was filtered through a 0.22 μm filter into a liquid chromatography vial to obtain the sample to be tested.

[0088] Example 5

[0089] Detection and quantification of products in tobacco leaves:

[0090] The sample obtained in Example 4 was analyzed using LC-MS. The method was as follows: LC-MS analysis was performed using a Shimadzu LCMS-2020 mass spectrometer at 28℃ and 230nm. Data acquisition was performed in positive ion mode, with a scan range of m / z 700–1100. ESI source parameters were: capillary voltage -33V, dry gas temperature 250℃, dry gas flow rate 15L / min, sheath gas temperature 300℃, sheath gas flow rate 10L / min, nebulizer pressure 20psi, cone voltage 500V, and data acquisition frequency 1 spectrum / s. The chromatographic column was a SHIMADZU shim-pack GIST C18 HSS column (5 μm, 2.1 mm × 100 mm). The mobile phase was acetonitrile (solvent A) and water (solvent B). The flow rate was 0.3 mL / min. The solvent gradient was: 0–15 min, 28%–40% solvent A; 15–16 min, 40%–44% solvent A; 16–26 min, 44% solvent A; 26–36 min, 40%–44% solvent A. XDT, DT, and paclitaxel standards were used (XDT was purchased from Fujian Nanfang Pharmaceutical Co., Ltd., DT was prepared in the laboratory, and paclitaxel was purchased from Beijing Kaiguo Technology Co., Ltd.).

[0091] Based on LC-MS results, it can be found that compared to the blank control group (tobacco infected with Agrobacterium strain GV3101-pEAQ-HT):

[0092] In tobacco infected solely with Agrobacterium strain GV3101-pEAQ-P1-2 and fed with XDT, a more significant peak of the product DT was detected. In positive ion mode, the same major ion fragments (m / z 812.5, m / z 834.5, and m / z 850.5) as the DT standard were also present. In tobacco infected solely with Agrobacterium strain GV3101-pEAQ-ICM9-6 and fed with DT, a peak of the product paclitaxel, which was not present in the blank control group tobacco, was detected. In positive ion mode, the same major ion fragments (m / z 854.5, m / z 876.5, and m / z 850.5) as the paclitaxel standard were also present. 892.5; In tobacco co-infected with Agrobacterium strains GV3101-pEAQ-P1-2 and GV3101-pEAQ-ICM9-6, the peak of paclitaxel, a product not present in the blank control group, was also detected. In positive ion mode, the same major ion fragments (m / z 854.5, m / z 876.5, and m / z 892.5) as the paclitaxel standard were also present. This indicates that P1-2 and ICM9-6, whether expressed alone or co-expressed, can achieve specific catalysis of their respective non-natural substrates in tobacco leaves. Co-expression of P1-2 and ICM9-6 in tobacco leaves enables the conversion from XDT to paclitaxel, resulting in a high content of paclitaxel.

[0093] The product was quantified by HPLC. The method was as follows: HPLC analysis of the sample was performed using an Agilent Technologies 1200 Series and an OSAKA SODA CAPCELL PAK-C18 MG II (4.6 × 150 mm, 5 μm) column at 28℃ and 230 nm. A concentration-peak area standard curve of paclitaxel standards was determined under the same conditions. Based on the peak area of ​​the paclitaxel product in the sample, the paclitaxel content in tobacco leaves was calculated to be 53.84 μg / g (dry weight) when fed the above-mentioned amount of XDT substrate.

[0094] Example 6

[0095] Isolation and structural identification of paclitaxel:

[0096] Leaves weighing approximately 100g after freeze-drying were crushed and extracted twice with ultrasonic extraction using approximately 3L of ethyl acetate. The filtrates were combined and concentrated using a rotary evaporator to obtain a total extract, which was then freeze-dried to obtain a crude extract powder. The crude extract powder was soaked in petroleum ether to remove pigments, and the petroleum ether extract was discarded after filtration. The remaining sample was dissolved in methanol, and paclitaxel was prepared using a semi-preparative liquid chromatography system (mobile phase: 45% acetonitrile-water). The prepared sample was then dissolved in deuterated chloroform and subjected to further processing. 1 H-NMR and 13 C-NMR detection (results are shown in...) Figure 6 , Figure 7 and Figure 8 and table 2) below.

[0097]

[0098] Table 2. NMR spectral data of paclitaxel

[0099]

[0100] a Overlapping signals

[0101] Nuclear magnetic resonance (NMR) detection confirmed that tobacco co-expressing glycosyl hydrolase P1-2 and acyltransferase ICM9-6 of the present invention can convert XDT into paclitaxel, thereby achieving heterologous expression of paclitaxel.

[0102] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these examples without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A recombinant plant expression plasmid, wherein, The plasmid encodes a glycosyl hydrolase P1-2 containing the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80% identity with it, and an acyltransferase ICM9-6 containing the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% identity with it.

2. The recombinant plant expression plasmid as described in claim 1, wherein, The plasmid contains, separately or fused together, the encoding genes for the glycosyl hydrolase P1-2 and the encoding genes for the acyltransferase ICM9-6.

3. The recombinant plant expression plasmid as described in any one of claims 1 or 2, wherein, One of the plasmids contains one or more copies of the gene encoding the glycosyl hydrolase P1-2; and / or One of the plasmids contains one or more copies of the gene encoding the acyltransferase ICM9-6.

4. The recombinant plant expression plasmid according to any one of claims 1-3, wherein, The gene encoding the glycosyl hydrolase P1-2 and the gene encoding the acyltransferase ICM9-6 are respectively contained in separate vectors; or The encoding genes for the glycosyl hydrolase P1-2 and the acyltransferase ICM9-6 are contained in the same vector.

5. The recombinant plant expression plasmid according to any one of claims 1-4, wherein, The plasmid contains the coding gene for glycosyl hydrolase P1-2 shown in SEQ ID NO:3 and / or the coding gene for acyltransferase ICM9-6 shown in SEQ ID NO:

4.

6. The recombinant plant expression plasmid according to any one of claims 1-5, wherein, The plasmid is an Agrobacterium expression plasmid.

7. The recombinant plant expression plasmid according to any one of claims 1-5, wherein, The plasmid can be constructed from vectors selected from the following: pEAQ-HT, pCB302, pEff, PBI series expression vectors, pCAMBIA series expression vectors, and pGreen series expression vectors.

8. A recombinant Agrobacterium, wherein, The recombinant Agrobacterium is transformed with any one of the recombinant plant expression plasmids according to claims 1-7, or expresses glycosyl hydrolase P1-2 containing the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80% identity with it and / or acyltransferase ICM9-6 containing the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% identity with it.

9. The recombinant Agrobacterium as described in claim 8, wherein, The Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

10. The recombinant Agrobacterium as described in any one of claims 8 or 9, wherein, A single Agrobacterium expresses one or both of the glycosyl hydrolase P1-2 and the acyltransferase ICM9-6.

11. The recombinant Agrobacterium as described in any one of claims 8-11, wherein, The Agrobacterium is integrated with the gene encoding the glycosyl hydrolase P1-2 shown in SEQ ID NO:3 and / or the gene encoding the acyltransferase ICM9-6 shown in SEQ ID NO:

4.

12. A method for heterologous synthesis of paclitaxel from tobacco plants, comprising: (1) Construct a recombinant plant expression plasmid, wherein the plasmid encodes a glycosyl hydrolase P1-2 containing the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80% identity with it and an acyltransferase ICM9-6 containing the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% identity with it. (2) The recombinant expression plasmid was transformed into Agrobacterium to obtain recombinant Agrobacterium; (3) Using the recombinant Agrobacterium to infect tobacco or a portion thereof, or tobacco cells; (4) The infected tobacco or parts thereof, or tobacco cells, were fed with substrate and then paclitaxel was extracted.

13. The method of claim 12, wherein, The plants mentioned are tobacco (Nicotiana tabacum L.) or Nicotiana benthamiana.

14. The method of claim 12 or 13, wherein, The recombinant plant expression plasmid was constructed using the Agrobacterium expression vector.

15. The method according to any one of claims 12-14, wherein, The plasmid can be constructed from vectors selected from the following: pEAQ-HT, pCB302, pEff, PBI series expression vectors, pCAMBIA series expression vectors, and pGreen series expression vectors.

16. The method according to any one of claims 12-15, wherein, The plasmid contains, separately or fused together, the encoding genes for the glycosyl hydrolase P1-2 and the encoding genes for the acyltransferase ICM9-6.

17. The method according to any one of claims 12-16, wherein, One of the plasmids contains one or more copies of the gene encoding the glycosyl hydrolase P1-2; and / or One of the plasmids contains one or more copies of the gene encoding the acyltransferase ICM9-6.

18. The method according to any one of claims 12-17, wherein, The gene encoding the glycosyl hydrolase P1-2 and the gene encoding the acyltransferase ICM9-6 are respectively contained in separate vectors; or The encoding genes for the glycosyl hydrolase P1-2 and the acyltransferase ICM9-6 are contained in the same vector.

19. The method according to any one of claims 12-18, wherein, The plasmid contains the coding gene for glycosyl hydrolase P1-2 shown in SEQ ID NO:3 and / or the coding gene for acyltransferase ICM9-6 shown in SEQ ID NO:

4.

20. The method according to any one of claims 12-19, wherein, The Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

21. The method according to any one of claims 12-20, wherein, A single Agrobacterium expresses one or both of the glycosyl hydrolase P1-2 and the acyltransferase ICM9-6.

22. The method according to any one of claims 12-21, wherein, The Agrobacterium is integrated with the gene encoding the glycosyl hydrolase P1-2 shown in SEQ ID NO:3 and / or the gene encoding the acyltransferase ICM9-6 shown in SEQ ID NO:

4.

23. The method according to any one of claims 12-22, wherein, The recombinant Agrobacterium tumefaciens expressing the glycosyl hydrolase P1-2 or the acyl transferase ICM9-6 was used to infect the tobacco plant or a part thereof, or tobacco cells, respectively. The recombinant Agrobacterium species expressing either the glycosyl hydrolase P1-2 or the acyltransferase ICM9-6 were used to co-infect the tobacco plant or parts thereof, or tobacco cells; or Recombinant Agrobacterium expressing both the glycosyl hydrolase P1-2 and the acyltransferase ICM9-6 was used to infect the tobacco plant or a part thereof, or tobacco cells.

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