Composite elicitor for promoting taxus chinensis embryonic stem cells to produce paclitaxel and application

Abstract

The invention discloses a composite elicitor for promoting taxus chinensis embryogenic stem cells to produce paclitaxel and application, and belongs to the technical field of biology. The compound elicitor consists of methyl jasmonate, indolebutyric acid and salicylic acid, and the working concentrations of the methyl jasmonate, the indolebutyric acid and the salicylic acid in a culture medium are respectively as follows: 50-800 mu mol / L of methyl jasmonate, 12.5-50 mu mol / L of indolebutyric acid and 0.1-0.5 mg / L of salicylic acid. Preferably, the jasmonic acid methyl ester is 200 [mu] mol / L, the indolebutyric acid is 25 [mu] mol / L, the salicylic acid is 0.5 mg / L, and 0.1-0.5 mg / L arachidonic acid can be selectively added. The composite elicitor is added in the early stage of the exponential growth phase of the taxus chinensis embryogenic stem cells, and the taxus chinensis embryogenic stem cells are continuously cultured for 5 days, so that the yield of paclitaxel reaches 27.52 mg / L and is 7.86 times that of blank control, and meanwhile, the maximum biomass is obtained. According to the invention, the synergistic interaction of the three elicitors is realized for the first time, the production period is remarkably shortened, a set of efficient and stable paclitaxel production system is established, and an innovative solution is provided for large-scale production of paclitaxel.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an inducer composition for plant stem cell culture, and more specifically, to the application of a composite inducer for promoting the production of paclitaxel from yew embryonic stem cells. Background Technology

[0002] Taxol (also known as Pacific taxol) is a diterpenoid alkaloid compound isolated from the bark of the yew tree, a gymnosperm. As a highly effective, low-toxicity, and broad-spectrum natural anticancer drug, it has been widely used in the clinical treatment of breast cancer, ovarian cancer, and some head and neck cancers and lung cancers. Due to its unique chemical structure, significant biological activity, unique mechanism of action, and scarce natural resources, this compound has received continuous attention from the fields of botany, chemistry, pharmacology, and molecular biology since the late 20th century, becoming an important research subject in anticancer drug development.

[0003] With global population growth and rising cancer rates, the clinical demand for paclitaxel is increasing dramatically. Currently, clinical and research paclitaxel primarily relies on direct extraction from yew trees. However, the content of this component in plants is extremely low; even in the bark of the short-leaved yew, which is recognized as having the highest content, it contains only 0.069%, requiring approximately 13.6 kilograms of bark to extract just 1 gram of paclitaxel. It is estimated that treating one ovarian cancer patient would require consuming 3 to 12 century-old yew trees, and this unsustainable method of acquisition is leading to the near depletion of yew resources. Although the biosynthetic pathway of paclitaxel has been largely elucidated, its industrialization has not yet been achieved due to stringent reaction conditions, low yield, and high cost. While the relatively mature semi-synthetic method can improve resource utilization efficiency, it still consumes large amounts of yew raw materials and cannot fundamentally solve the raw material shortage problem. Therefore, developing new paclitaxel acquisition pathways is urgently needed.

[0004] Numerous studies have shown that yew cell culture is one of the most effective methods for producing paclitaxel. Using elicitors to increase paclitaxel levels in yew cells can multiply their content. Elicitors, as plant growth regulatory signals, can regulate plant metabolic pathways, promote the normal growth and development of callus tissue, and thus increase the accumulation of secondary metabolites, making them widely applicable in agricultural production. Utilizing elicitors to promote the synthesis of plant natural products has become a research hotspot in recent years for the in vitro cell culture production of useful natural products, and has demonstrated high inducibility in suspension culture cell lines of various plants.

[0005] The effects of paclitaxel induction on paclitaxel levels in yew cells are particularly significant. For example, studies have shown that 10 µmol / L methyl jasmonate and 5 mg / L ethylene increased the paclitaxel content in suspension cells of *Taxus chinensis* from 0.19 mg / L to 3.4 mg / L (19-fold). Adding 200 µmol / L methyl jasmonate on day 7 of the cell cycle resulted in a peak paclitaxel content of 36.0 mg / L seven days after induction. Adding 2 mL of crude extract of *Penicillium citrinum* mycelium to 100 mL of culture medium on day 20 (end of the exponential growth phase) of *Taxus chinensis* suspension culture resulted in the best paclitaxel induction effect, reaching 199.1 µg / gDW (dry weight). Adding 0.1 mg / L salicylic acid as a inducer to yew cell culture increased the paclitaxel content from 3.1 mg / L to 13.5 mg / L after 10 days of culture. The concentration of paclitaxel was 3 times higher than that of the control. When 0.1 mg / L of arachidonic acid was added to the yew cell culture medium for induction, the paclitaxel content was highest on day 10, reaching 4 times that of the control.

[0006] However, existing technologies have the following limitations: 1. Induction efficiency needs to be improved: Most schemes are simple combinations of one or two inducers, which have limited yield increases and cannot meet the ultimate pursuit of efficiency in industrial production.

[0007] 2. Long cultivation cycle: Many induction methods require the addition of inducers in the middle and late stages of cultivation, and it takes a long time (e.g., more than 10 days) to reach peak yield after induction, resulting in high production costs.

[0008] 3. Neglecting the balance between growth and metabolism: Powerful stress inducers often promote secondary metabolism while inhibiting cell growth, leading to a decrease in biomass, thus offsetting the benefits of increased yield per unit area.

[0009] Currently, there are no reports on combining methyl jasmonate, indolebutyric acid, and salicylic acid and utilizing their synergistic effect to achieve efficient and rapid accumulation of paclitaxel in Taxus chinensis embryonic stem cell culture. Summary of the Invention

[0010] The purpose of this invention is to provide a composite inducer that can significantly and efficiently increase the yield of paclitaxel in yew embryonic stem cells and its application method, so as to solve the problems of insufficient efficiency and excessively long cycle of existing induction methods.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a composite inducer for promoting the production of paclitaxel by yew embryonic stem cells, wherein the final concentration of the composite inducer in the culture medium during use is: methyl jasmonate 50-800 μmol / L, indolebutyric acid 12.5-50 μmol / L, and salicylic acid 0.1-0.5 mg / L.

[0012] Preferably, the final concentration of the composite inducer in the culture medium during use is: methyl jasmonate 150-400 μmol / L, indolebutyric acid 20-30 μmol / L, and salicylic acid 0.3-0.5 mg / L.

[0013] More preferably, the final concentration of the composite inducer in the culture medium during use is: methyl jasmonate 200 μmol / L, indolebutyric acid 25 μmol / L, and salicylic acid 0.5 mg / L.

[0014] As a preferred extension, the composite inducer is further supplemented with arachidonic acid at a final concentration of 0.1-0.5 mg / L in the culture medium during use.

[0015] Secondly, the present invention provides a method for increasing the yield of paclitaxel in yew embryonic stem cells, the method comprising: adding an effective amount of the composite inducer as described above to the culture system during the exponential growth phase of yew embryonic stem cells.

[0016] Preferably, the composite inducer is added between 40 and 56 hours after the start of culture.

[0017] Preferably, after adding the composite inducer, the mixture is cultured for another 3 to 7 days to harvest paclitaxel.

[0018] Preferably, a method for increasing paclitaxel production in yew embryonic stem cells includes the following steps: Step 1: Determine the growth curve of Taxus chinensis embryonic stem cells: Divide the growth curve into five phases: lag phase, exponential growth phase, deceleration phase, quiescent phase, and death phase. Step 2: Resuscitate Taxus embryonic stem cells: Take Taxus embryonic stem cells, inoculate them into MS liquid culture medium, seal them with sealing film, and place them in a shaking incubator for two days at 25℃ and 100-110 rpm. Step 3: Adding compound inducers for culture: At the 48th hour of yew embryonic stem cell culture, add methyl jasmonate (200 μmol / L), indolebutyric acid (25 μmol / L), and salicylic acid (0.5 mg / L) to the culture medium. Seal the culture medium with sealing film and continue to culture in a shaking incubator for 5 days. Step 4: Collection and processing of Taxus embryonic stem cells: Transfer the culture medium to a sterile collection bottle, balance and centrifuge; discard the supernatant, resuspend the cells in sterile deionized water and wash 2 to 3 times; collect the cells and weigh the wet weight, then dry them in an electric heating drying oven at 45-50℃, grind the dried cells into powder and weigh the dry weight. Step 5: Determine the paclitaxel content using high performance liquid chromatography.

[0019] Thirdly, the present invention provides a yew embryonic stem cell culture, which is a paclitaxel-rich culture obtained by the method described above.

[0020] The beneficial effects of this invention are: 1. Unprecedented Synergistic Effect: This invention is the first to combine methyl jasmonate (stress signal), indolebutyric acid (growth signal), and salicylic acid (defense signal) in a ternary combination, producing an unexpected synergistic effect. Experiments have shown that the optimal combination (Group E) resulted in a paclitaxel yield of 27.52 mg / L, which is 7.86 times that of the control group. This increase in yield far exceeds the expectations of those skilled in the art regarding the effects of conventional inducer combinations.

[0021] 2. Dual optimization of growth and metabolism: This invention breaks the technical prejudice that "high induction is always accompanied by low growth." While achieving the highest paclitaxel yield, it also obtained the largest cell biomass (dry weight), realizing efficient synergy between cell proliferation and secondary metabolite synthesis, laying a solid foundation for industrial-scale production.

[0022] 3. Revolutionary Improvement in Production Efficiency: This invention, by adding inducers in the early stages of exponential growth, requires only 5 more days of cultivation to reach peak yield, compared to the traditional method of 10 days or more, shortening the production cycle by more than 50%. This significantly reduces energy consumption, time costs, and equipment occupancy, demonstrating significant industrial application value.

[0023] 4. Clear formulation and controllable cost: The inducers used in this invention are all clearly defined chemical components, not complex fungal extracts. Indolebutyric acid is a synthetic auxin analogue with high stability, low phytotoxicity, and low synthesis cost. This ensures batch-to-batch stability and reproducibility of results. Furthermore, the raw materials are readily available and inexpensive, making it highly suitable for large-scale applications. Attached Figure Description

[0024] Figure 1 It is the fluid of Taxus chinensis embryonic stem cells in a centrifuge tube; Figure 2 This describes the growth of embryonic stem cells in the control group and group E in the conical flask during the induction process; Figure 3It is the yew stem cell powder that has been induced, collected and dried in centrifuge tubes and is used for the next step of paclitaxel determination; Figure 4 This is an analysis diagram of the wet weight of Taxus stem cells in the comparative example of this invention; Figure 5 This is an analysis diagram of the dry weight of Taxus stem cells in the comparative example of this invention; Figure 6 This is an analytical graph showing the paclitaxel content in the comparative example of this invention. Detailed Implementation

[0025] Example 1 A method for promoting paclitaxel production from Taxus chinensis embryonic stem cells using a complex inducer includes the following steps: Step 1: Determining the growth curve of yew embryonic stem cells: Take a tube of yew embryonic stem cell cryopreservation solution from the plant embryonic stem cell bank, thaw it in a 36°C water bath, pour it into a sterile 1L Erlenmeyer flask in a biosafety cabinet, add 400mL of MS liquid culture medium, seal with sealing film, and culture on a shaker. During culture, aspirate cell fluid every 8 hours and count cells on a cell counting plate. Based on the data, plot the growth curve of yew cells, dividing the growth curve into five phases: lag phase, exponential growth phase (initial cell number 310,000 cells / ml, culture time 2-4 days is the exponential growth phase), deceleration phase, quiescent phase, and death phase. According to the growth habits of plant stem cells, adding an inducer during the exponential growth phase can better increase the content of secondary metabolites. The exponential growth phase is divided into three stages: early stage (48h of culture), middle stage (72h), and late stage (96h).

[0026] Step 2: Resuscitate Taxus embryonic stem cells: Take one tube of Taxus stem cells free from contamination and evenly distribute them into three sterile 1L Erlenmeyer flasks in a biosafety cabinet. Add an equal amount of MS liquid culture medium (to the 400ml mark), seal with sealing film, and place in a vertical double-door shaking incubator for two days. The incubator speed is 100-110 rpm and the temperature is 25℃.

[0027] Step 3: Adding inducers to culture yew stem cells: At the early stage of the exponential growth phase of yew stem cells (48 hours of culture), add methyl jasmonate, 25 μmol / L indolebutyric acid and 0.5 mg / L salicylic acid to a final concentration of 200 μmol / L, seal with sealing film, and continue to culture in a shaking incubator for 5 days.

[0028] Step 4: Collect and dry yew stem cells: Pour the yew embryonic stem cell solution into a sterile collection bottle, balance the liquid, and centrifuge at 2400 rpm for 4 minutes in a benchtop large-capacity centrifuge. Discard the supernatant, add 200-300 ml of sterile deionized water to resuspend and wash the cells, balance the liquid again, and centrifuge at 2400 rpm for 4 minutes. Repeat this washing process 2-3 times. Place the collected stem cells in an electric heating drying oven and dry them at 45-50℃. After drying, grind the cells into powder using a mortar and pestle, and weigh the dry weight of the cells.

[0029] Step 5: Determination of paclitaxel content using high-performance liquid chromatography (HPLC): Weigh 1g of stem cell powder and add 10ml of methanol solvent for ultrasonic extraction for 30min. Filter, repeat twice, and combine the filtrates. Rotary evaporate to dryness, dilute to 10ml with methanol, and filter through a 0.22μm filter membrane. Inject 20μL of HPLC and calculate the content of the experimental sample by comparing with the paclitaxel standard curve.

[0030] Results: The stem cell dry weight of the treatment group in this embodiment reached 2.33 ± 0.05 g, and the paclitaxel yield was as high as 27.52 ± 0.16 mg / L, which were 1.85 times and 7.86 times that of the blank control group, respectively.

[0031] Example 2: Application of a composite inducer containing arachidonic acid The basic steps are the same as in Example 1, except that in step two, when adding the inducer, in addition to adding methyl jasmonate, indolebutyric acid and salicylic acid to the concentration of Example 1, arachidonic acid stock solution is also added to make its final concentration in the culture medium 0.3 mg / L.

[0032] Results: This treatment also effectively increased paclitaxel production, verifying the effectiveness of adding arachidonic acid as an alternative to the core ternary combination.

[0033] Comparative Example: Validation of the Effects of Different Concentration Ratios of Compound Elicitors on Paclitaxel Production from Taxus Embryonic Stem Cells I. Resuscitation of Taxus stem cells: Take 12 tubes of Taxus stem cells free from contamination and mix them in a sterile Erlenmeyer flask in a biosafety cabinet. Then, evenly distribute them into 36 sterile 1L Erlenmeyer flasks, add an equal amount of MS liquid culture medium (to the 400ml mark), seal with sealing film, and place in a vertical double-door shaking incubator for two days. The incubator speed is 110 rpm and the temperature is 25℃.

[0034] II. Adding appropriate elicitors: At the early stage of the exponential growth phase of Taxus chinensis stem cells (48 hours of culture), add the elicitor stock solution, namely 100 mM / L methyl jasmonate, 1 mg / mL indolebutyric acid, 10 mg / L salicylic acid, and 10 mg / L arachidonic acid. An orthogonal experimental design was used to determine the working concentrations of each factor: methyl jasmonate 0 μmol / L, 200 μmol / L, 400 μmol / L, 800 μmol / L; indolebutyric acid 0 μmol / L, 12.5 μmol / L, 25 μmol / L; salicylic acid... 0 mg / L, 0.1 mg / L, 0.3 mg / L, 0.5 mg / L; arachidonic acid 0 mg / L, 0.1 mg / L, 0.3 mg / L, 0.5 mg / L. An orthogonal array 16.4.4 was initially proposed, and orthogonal experimental analysis was performed using the combination method, resulting in 12 experimental groups. These were named blank group, group A, group B, group C, group D, group E, group F, group G, group H, group I, group J, and group K, with 3 bottles in each group, for a total of 36 bottles. The samples were incubated in a shaking incubator for 5 days, and subsequent experimental tests were conducted.

[0035] Table 1. Orthogonal Experimental Design Table 16.4.4 III. Collection and Drying of Taxus chinensis Stem Cells: Taxus chinensis stem cell solutions from different treatments were poured into sterile collection bottles and balanced. The bottles were centrifuged at 2400 rpm for 4 minutes in a benchtop centrifuge. After centrifugation, the supernatant was discarded, and the cells were resuspended in 300 ml of sterile deionized water to wash them. The mixture was balanced again and centrifuged at 2400 rpm for 4 minutes, for a total of three washes. The resulting cell solids were scooped out with a spatula and placed in labeled glass dishes. The wet weight of each treatment cell was weighed using an electronic balance. The extracted stem cells were placed in an electric drying oven and dried at 50°C. After the Taxus chinensis stem cells were thoroughly dried, they were ground into powder using a mortar and pestle, and the dry weight of the cells was weighed.

[0036] IV. Determination of paclitaxel content by high-performance liquid chromatography (HPLC): Weigh 1g of stem cells and add 10ml of methanol solvent for ultrasonic extraction for 30min. Filter, repeat twice, and combine the filtrates. Rotary evaporate to dryness, dilute to 10ml with methanol, and filter through a 0.22μm filter membrane. Inject 20μL of HPLC and calculate the content of the experimental sample by comparing with the paclitaxel standard curve.

[0037] V. Organize the experimental results: Table 2. Experimental treatments and results Table 3. Data Analysis Results Note: Data format: mean ± standard deviation (relative standard deviation RSD) The MS liquid culture medium components and hormone formulations described in this invention are as follows: Table 4: Composition and Hormone Ratio of MS Liquid Culture Medium The specific preparation method of the inducing mother liquor described in this application is as follows: 1. 100 mmol / L Methyl jasmonate: In a biosafety cabinet, use a pipette to draw 2292 μL of 95% methyl jasmonate solution and add it to a sterile conical flask. Add 5 ml of anhydrous ethanol to dissolve it, and finally add sterile deionized water to make up to 100 ml. Filter the solution through a 0.22 μm filter membrane into a 100 ml sterile brown reagent bottle. 2. 1 mg / ml Indolebutyric Acid (IBA): Accurately weigh 0.1 g of IBA powder using an electronic balance in a sterile room, add it to a sterile conical flask in a biosafety cabinet, add 5 ml of anhydrous ethanol to dissolve it, and finally add sterile deionized water to make up to 100 ml. Filter it through a 0.22 μm filter membrane into a 100 ml sterile brown reagent bottle.

[0038] 3. 10 mg / L salicylic acid: Accurately weigh 0.072 g of salicylic acid using a 0.01% balance, add a small amount of anhydrous ethanol and stir until completely dissolved, transfer the dissolved solution to a 1L volumetric flask and dilute to volume with anhydrous ethanol.

[0039] 4. 10 mg / L arachidonic acid: Accurately weigh 10 mg of arachidonic acid (purity ≥ 99%), add an appropriate amount of anhydrous ethanol and stir until completely dissolved, transfer to a 100 ml volumetric flask, and dilute to volume with anhydrous ethanol.

[0040] VI. Results Analysis: 1. Determination and Effect of Optimal Combination Based on the experimental results in Tables 2 and 3, Figure 4-6 The results showed that, among all test combinations, experimental group E (i.e., inducer composition E) exhibited unprecedented paclitaxel induction efficiency. The paclitaxel yield treated with this composition reached 27.52 ± 0.16 mg / L (data expressed as mean ± standard deviation, n=3). Quantitatively, this yield was 7.86 times that of the blank control group (3.50 ± 0.14 mg / L). This result unequivocally demonstrates the superior and unexpected effect of composition E in maximizing paclitaxel production, thus proving that adding, altering, or removing any component of the composite inducer of this application, and the content of each component, cannot achieve the technical effect of this application.

[0041] 2. Synergistic effect of key components and their concentration ranges The superior performance of Experiment E is attributed to the precise matching and synergy of its key components and their specific concentration ranges: The core inducing effect of methyl jasmonate: The composition contains a moderate concentration of 200 μmol / L methyl jasmonate, which acts as a crucial stress signal and potent activator of secondary metabolism, a prerequisite for initiating the paclitaxel biosynthetic pathway. The growth and signaling synergistic effect of indolebutyric acid: The high concentration of 25 μmol / L indolebutyric acid in the experiment ensured vigorous cell growth, providing sufficient precursors and biomass for paclitaxel synthesis. Simultaneously, it generated an unexpected positive interaction with the methyl jasmonate signaling pathway, jointly forming the metabolic basis for efficient synthesis. The synergistic effect of salicylic acid: The 0.5 mg / L salicylic acid in the composition further enhanced the above synergistic effect. As another important signaling molecule, it cross-talks with the methyl jasmonate pathway, potentially pushing the paclitaxel synthesis efficiency to its peak by regulating the expression of key enzyme genes.

[0042] 3. Significant advancements in the technical solution In summary, the specific inducer composition (Group E) provided by this invention, through the precise synergy of its components at specific concentrations, successfully increased the yield of paclitaxel in Taxus chinensis stem cell culture to nearly eight times the baseline level. This technical effect far exceeds the conventional levels expected by those skilled in the art when using single or simple mixtures of inducers, demonstrating outstanding substantive features and significant progress, and providing a technical solution with great industrial application value for the large-scale production of paclitaxel. Taxus chinensis embryonic stem cells prepared in the laboratory can be induced and cultured in large-capacity fermenters to obtain large quantities of paclitaxel powder, which can be used in health products and pharmaceuticals.

[0043] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples. Various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A complex inducer for promoting paclitaxel production from Taxus chinensis embryonic stem cells, characterized in that... The final concentration of the composite inducer in the culture medium during use is: methyl jasmonate 50-800 μmol / L, indolebutyric acid 12.5-50 μmol / L, and salicylic acid 0.1-0.5 mg / L.

2. The composite inducer according to claim 1, characterized in that, The final concentration of the composite inducer in the culture medium during use is: methyl jasmonate 150-400 μmol / L, indolebutyric acid 20-30 μmol / L, and salicylic acid 0.3-0.5 mg / L.

3. The composite inducer according to claim 2, characterized in that, The final concentration of the composite inducer in the culture medium during use is: methyl jasmonate 200 μmol / L, indolebutyric acid 25 μmol / L, and salicylic acid 0.5 mg / L.

4. The composite inducer according to claim 3, characterized in that, When using the composite inducer, arachidonic acid is added to the culture medium at a final concentration of 0.1-0.5 mg / L.

5. A method for increasing paclitaxel production in Taxus chinensis embryonic stem cells, characterized in that, The method includes adding an effective amount of the composite inducer as described in any one of claims 1 to 4 to the culture system during the exponential growth phase of yew embryonic stem cells.

6. The method according to claim 5, characterized in that, The composite inducer was added between 40 and 56 hours after the start of culture.

7. The method according to claim 5, characterized in that, After adding the aforementioned compound inducer, continue culturing for 3 to 7 days to harvest paclitaxel.

8. The method according to claim 5, characterized in that, Includes the following steps: Step 1: Determine the growth curve of Taxus chinensis embryonic stem cells: Divide the growth curve into five phases: lag phase, exponential growth phase, deceleration phase, quiescent phase, and death phase. Step 2: Resuscitate Taxus chinensis embryonic stem cells: Inoculate Taxus chinensis embryonic stem cells into MS liquid culture medium, seal with sealing film, and place in a shaking incubator for two days at 25℃ and 100-110 rpm. Step 3: Add compound inducer culture: At the 48th hour of Taxus chinensis embryonic stem cell culture, add methyl jasmonate (200 μmol / L), indolebutyric acid (25 μmol / L), and 0.5 μmol / L of compound inducer to the culture medium. mg / L salicylic acid was used to seal the culture medium, which was then cultured in a shaking incubator for 5 days. Step 4: Collection and processing of Taxus chinensis embryonic stem cells: The culture medium was transferred to a sterile collection bottle, balanced, and centrifuged. The supernatant was discarded, and the cells were resuspended in sterile deionized water and washed 2 to 3 times. The cells were collected and weighed wet, and then dried in an electric heating drying oven at 45-50℃. The dried cells were ground into powder and weighed dry. Step 5: The paclitaxel content was determined by high performance liquid chromatography.

9. A culture of Taxus chinensis embryonic stem cells, characterized in that, It is a paclitaxel-rich culture obtained by the method described in any one of claims 5 to 8.

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