Method for promoting accumulation of camptothecin by overexpressing tryptophan synthetase

Abstract

The invention belongs to the field of plant tissue culture, and particularly relates to a method for promoting accumulation of camptothecin by overexpressing tryptophan synthetase. The invention provides a method for promoting camptothecin accumulation of camptotheca acuminate cells, which comprises the following steps: constructing camptotheca acuminate cells overexpressing tryptophan synthetase genes, performing tissue culture amplification, and extracting camptothecin from the amplified camptotheca acuminate cells. The method for overexpressing the tryptophan synthetase gene is superior to a method for directly adding tryptophan into a culture medium, and the camptothecin yield can be increased by 2.33 times.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant tissue culture, and particularly relates to a method for promoting camptothecin accumulation by overexpressing tryptophan synthetase. BACKGROUND

[0002] Camptothecin is a plant anticancer drug, and its chemical formula is C 20 H 16 N2O4, light yellow needle-like crystal. It is extracted from the Camptotheca acuminata distributed in central and southern China and southwest China. Camptothecin mainly plays a role by inhibiting the activity of DNA topoisomerase. Camptothecin has good curative effect on gastrointestinal tract and head and neck cancer, but has the side effect of hematuria on a few patients; the anticancer activity of its derivative 10-hydroxy camptothecin is higher than that of camptothecin, and it also has obvious curative effect on liver cancer and head and neck cancer, and less side effect. Now two analogues of camptothecin have been approved and used for cancer chemotherapy, such as topotecan and irinotecan.

[0003] The content of camptothecin in camptothecin plants is low, less than 0.03%. Camptothecin can be synthesized by chemical synthesis. In 1969, the Wall team of the Triangle Institute in the United States first disclosed a synthesis method of camptothecin; E.J. Corey and his team of Harvard University (located in Cambridge, Massachusetts) also announced a convergent nine-step reaction full synthesis route. However, the chemical synthesis method has low yield. Chinese scientists found that there are three key rate-limiting enzymes in the synthesis pathway of camptothecin: G10H, SLS and TDC, and the content of intermediate products can be increased by 40 times by overexpressing the three enzymes in tobacco.

[0004] The biosynthesis of camptothecin starts from tryptophan, which is an amino acid that is decarboxylated in the plant body to form tryptamine. Tryptamine is combined with a terpenoid compound, geranyl pyrophosphate, to form strictosidine, which is catalyzed by strictosidine synthase. In Camptotheca acuminata, strictosidine undergoes three hydroxylation reactions to add hydroxyl groups at the 7th, 21st and 20th carbon sites, which are dominated by cytochrome P450 enzymes. The intermediate product after hydroxylation is modified by methylation through methylation enzyme to form a compound with a specific spatial structure. The key step in the last stage is the ring closure reaction, which is catalyzed by an oxidase to form a five-ring structure, completing the construction of the core skeleton of camptothecin. Therefore, increasing the intermediate of camptothecin or catalytic enzyme is a potential method for promoting the accumulation of camptothecin. CN101831456B discloses a method for increasing the content of camptothecin in Camptotheca acuminata callus by transferring aoc gene of Camptotheca acuminata. SUMMARY

[0005] The application aims to provide a method for promoting camptothecin production, overexpressing tryptophan synthetase, increasing the content of tryptophan, providing sufficient raw materials for synthesizing camptothecin, promoting the synthesis and accumulation of camptothecin, and using tissue culture technology to subculture camptothecin stem cells and produce camptothecin.

[0006] The application first provides a method for promoting camptothecin accumulation in camptothecin cells, comprising the following steps: constructing camptothecin cells overexpressing tryptophan synthetase genes and tissue culture expansion, and then extracting camptothecin from the expanded camptothecin cells, wherein the amino acid sequence of the tryptophan synthetase is as shown in SEQ ID NO: 1.

[0007] Further, the sequence of the tryptophan synthetase gene is as shown in SEQ ID NO: 2.

[0008] Further, the method comprises the following steps: (1) Constructing a plasmid overexpressing tryptophan synthetase genes, and using E. coli to amplify the plasmid; (2) Extracting the plasmid and electrotransforming Agrobacterium, and then using the Agrobacterium to infect camptothecin cells to obtain camptothecin cells overexpressing tryptophan synthetase genes; (3) Subculturing the infected camptothecin cells, and extracting camptothecin from the expanded camptothecin cells.

[0009] Further, the Agrobacterium is Agrobacterium GV3101.

[0010] Further, the camptothecin cells are camptothecin stem cells, which are obtained by culturing in an induction medium; the composition of the induction medium is: B5 medium, 2 mg / L 2,4-dichlorophenoxyacetic acid, 1 mg / L naphthaleneacetic acid, 30 g / L sucrose, and 0.2 g / L citric acid.

[0011] Further, the composition of the medium for subculture is: B5 medium, 2 mg / L 2,4-dichlorophenoxyacetic acid, 2 mg / L 6-BA, 30 g / L sucrose, 0.2 g / L citric acid, 0.2 g / L vitamin C, and optionally 0.5 g / L tryptophan.

[0012] Further, the extraction of camptothecin from camptothecin cells comprises: taking the camptothecin cells overexpressing tryptophan synthetase genes after subculture, separating, washing, and grinding, adding 1-4 times the volume of ethyl acetate to the grinding liquid, heating at 50-80°C for 1-2 hours, collecting the organic phase, and purifying camptothecin by high performance liquid chromatography.

[0013] The application also provides the use of the tryptophan synthetase gene for promoting camptothecin accumulation in camptothecin cells.

[0014] Compared with the prior art, the application has at least the following beneficial effects: 1、The present application relates to related enzymes and intermediates involved in the synthetic pathway of camptothecin, and constructs a camptotheca cell overexpressing tryptophan synthetase gene, increases the content of tryptophan in the camptotheca cell, and promotes the accumulation of camptothecin.

[0015] 2、The method of overexpressing tryptophan synthetase gene is superior to directly adding tryptophan in the culture medium, and can increase the yield of camptothecin by 2.33 times. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Changes in camptothecin yield of the camptotheca stem cell overexpressing tryptophan synthetase after 5 subcultures. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application will be further described in detail below in combination with specific implementation manners. It should be understood that the specific examples described in the following implementation manners of the present application are only exemplary illustrations of the specific implementation manners of the present application, and are intended to explain the present application, but do not constitute a limitation on the present application.

[0018] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. These ranges and values should be construed as approximately.

[0019] Example 1: Effect of adding tryptophan in the culture medium on the yield of camptothecin In this example, tryptophan is added in the subculture medium, and whether the intake of tryptophan by the camptotheca cell has an effect on the yield of camptothecin is evaluated.

[0020] The steps of preparing camptothecin by the camptotheca cell are as follows: ①Take camptotheca leaves, first soak them in a 75% alcohol solution for 30 seconds, then soak them in a 1% sodium hypochlorite solution for 10 minutes, and then clean them with sterile water. Under sterile conditions, cut the camptotheca leaves into small pieces of 0.5 mm, inoculate them on the induction medium, and induce them at 22°C in the dark for 5-10 days to obtain explants. The composition of the induction medium is: B5 medium, 2 mg / L 2,4-dichlorophenoxyacetic acid, 1 mg / L naphthaleneacetic acid, 30 g / L sucrose, and 0.2 g / L citric acid.

[0021] ②Separate callus from explants, shake to disperse to get Camptotheca acuminata cambium stem cells, transfer to subculture medium to suspend culture, culture in 25℃ culture room for 14 days. The composition of subculture medium: B5 medium, 2 mg / L 2,4-dichlorophenoxyacetic acid, 2 mg / L 6-BA, 30 g / L sucrose, 0.2 g / L citric acid, 0.2 g / L vitamin C (control group). The composition of subculture medium: B5 medium, 2 mg / L 2,4-dichlorophenoxyacetic acid, 2 mg / L 6-BA, 30 g / L sucrose, 0.2 g / L citric acid, 0.2 g / L vitamin C and 0.5 g / L tryptophan (tryptophan group). Subculture every 14 days, and a large number of Camptotheca acuminata stem cells are obtained by expansion.

[0022] ③Take Camptotheca acuminata stem cells subcultured for 14 days, separate, wash and grind, add 2 times volume of ethyl acetate to the grinding liquid, heat at 60℃ for 1h, repeat extraction for 3 times, collect the organic phase, filter and dry, then dissolve in 2:3 acetonitrile and water solution, detect by HPLC, the yield of camptothecin in the control group is 1.521 mg / L, and the yield of camptothecin in the tryptophan group is 1.848 mg / L, so providing tryptophan is beneficial to improve the yield of camptothecin. In addition to providing tryptophan in the culture medium, increasing the synthesis of tryptophan in Camptotheca acuminata cells is also a potential method.

[0023] Example 2: Construction of tryptophan synthetase gene overexpression vector This example constructs overexpression of tryptophan synthetase gene to promote the synthesis of tryptophan in Camptotheca acuminata cells. First, query the amino acid sequence of tryptophan synthetase of Camptotheca acuminata from NCBI data, as shown in SEQ ID NO: 1 (GenBank: AAB97526.1). According to the queried amino acid sequence, optimize the gene sequence, as shown in SEQ ID NO: 2.

[0024] Amino acid sequence of tryptophan synthetase (SEQ ID NO: 1): MAVYTNPACRTNTSAFPGPYRPYSNPSRFSFNLDKFRPRTSAIKVPSICCTIAREMEKERSEREPDVLQRPDSFGRFGKFGGKYVPETLMYALTELESAFRSLSGDQVFQKELDGILKDYVGRESPLYFAERLTLHYKRPNGEGPEIYLKREDLNHTGAHKINNAVAQALLAKRLGKKRIIAETGAGQHGVATATVCARFGLQCVIYMGAQDMERQALNVFRMRLLGAEVRAVHSGTATLKDATSEAIRDWVTNVESTHYILGSVAGPHPYPMMVREFHAVIGKETRKQALEKWGGKPDVLVACVGGGSNAMGLFHEFVDDKDVRMIGVEAAGFGLDSGKHAATLTKGEVGVLHGAMSYLLQDDDGQIIEPHSISAGLDYPGVGPEHSFLKDIGRAEYYCCTDEEALEAFKRLSRLEGIIPALETSHALAFLEKLCPTLPNGTKVVLNCSGRGDKDVHTAIKHLQV Gene sequence of tryptophan synthetase (SEQ ID NO: 2): According to the sequence of tryptophan synthetase gene, the multiple cloning site of overexpression vector pBI121 and the sequence near the multiple cloning site, restriction enzyme cutting sites (AscI and KpnI) are selected and primers are designed for PCR amplification. The PCR product is purified and recovered, and the overexpression vector pBI121 containing the target gene fragment and the overexpression vector pBI121 are double enzyme cut by restriction enzymes BamH1 and SmaI, respectively. The pBI121 vector and the target fragment obtained by enzyme cutting are recovered and connected by T4 ligase. The enzyme cutting reaction is carried out at 37°C, and the connection reaction is carried out at 4°C overnight. The connection product is transformed into the competent cells of Escherichia coli, and single colonies are picked and expanded for extracting plasmids. The positive plasmid is obtained by PCR, enzyme cutting and sequencing identification, which is the overexpression vector of tryptophan synthetase gene.

[0025] Example 3: Preparation of Camptotheca acuminata stem cells overexpressing tryptophan synthetase gene The preparation method comprises the following steps: ①The positive plasmid is electroporated into Agrobacterium GV3101 to obtain the transformed Agrobacterium GV3101, which is inoculated into YEB liquid medium for culture and standby.

[0026] ②The Camptotheca acuminata leaves are soaked in 75% alcohol solution for 30 seconds, then in 1% sodium hypochlorite solution for 10 minutes, and then washed with sterile water. Under sterile conditions, the Camptotheca acuminata leaves are cut into small pieces of 0.5 mm, inoculated on the induction medium, and induced to culture at 22°C in the dark for 5-10 days to obtain the explants. The induction medium comprises B5 medium, 2 mg / L 2,4-dichlorophenoxyacetic acid, 1 mg / L naphthalene acetic acid, 30 g / L sucrose and 0.2 g / L citric acid.

[0027] ③The callus is separated from the explants, shaken and dispersed to obtain Camptotheca acuminata cambium stem cells, which are transferred to the subculture medium for suspension culture in a 25°C culture room for 14 days. The subculture medium comprises B5 medium, 2 mg / L 2,4-dichlorophenoxyacetic acid, 2 mg / L 6-BA, 30 g / L sucrose, 0.2 g / L citric acid and 0.2 g / L vitamin C. The subculture is carried out once every 14 days, and a large amount of Camptotheca acuminata stem cells overexpressing tryptophan synthetase gene are obtained by amplification, which are the overexpression group.

[0028] ④The Camptotheca acuminata stem cells overexpressing tryptophan synthetase gene after subculture for 14 days are separated, washed, ground, and then 2 times the volume of ethyl acetate is added to the grinding liquid. The grinding liquid is heated at 60°C for 1 hour, and the extraction is repeated for 3 times. The organic phase is collected, filtered and dried, and then dissolved in a 2:3 solution of acetonitrile and water. The Camptotheca acuminata grinding liquid is detected by HPLC, and the yield of overexpression group camptothecin is 3.544 mg / L, which is 2.33 times of the control group in Example 1 and 1.92 times of the tryptophan group. It is superior to ordinary Camptotheca acuminata stem cells and culture medium added with tryptophan.

[0029] The Camptotheca acuminata stem cells overexpressing tryptophan synthetase gene were subcultured for 5 times to investigate the subculture stability of the overexpressed gene, and the results are shown in Table 4. Figure 1 The results show that the production of camptothecin can be steadily increased after 5 generations of culture.

[0030] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and do not constitute a limitation on the content of the present application. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A method for promoting camptothecin accumulation in a camptotheca acuminata cell, characterized by, The method comprises the following steps: constructing a Camptotheca acuminata cell overexpressing a tryptophan synthetase gene, and tissue culture expansion, and then extracting camptothecin from the expanded Camptotheca acuminata cell, wherein the amino acid sequence of the tryptophan synthetase is shown as SEQ ID NO:

1.

2. The method of claim 1, wherein, The sequence of the tryptophan synthetase gene is shown as SEQ ID NO:

2.

3. The method of claim 1, wherein, The method comprises the following steps: (1) constructing a plasmid overexpressing a tryptophan synthetase gene, and using Escherichia coli to amplify the plasmid; (2) extracting the plasmid and performing electroporation on Agrobacterium, and then using the Agrobacterium to infect Camptotheca acuminata cells to obtain Camptotheca acuminata cells overexpressing the tryptophan synthetase gene; (3) subculturing the infected Camptotheca acuminata cells, and extracting camptothecin from the expanded Camptotheca acuminata cells.

4. The method of claim 3, wherein, The Agrobacterium is Agrobacterium GV3101.

5. The method of claim 3, wherein, The Camptotheca acuminata cells are Camptotheca acuminata stem cells, which are obtained by culturing in an induction medium; the induction medium comprises B5 medium, 2 mg / L 2,4-dichlorophenoxyacetic acid, 1 mg / L naphthaleneacetic acid, 30 g / L sucrose, and 0.2 g / L citric acid.

6. The method of claim 3, wherein, The medium for subculture comprises B5 medium, 2 mg / L 2,4-dichlorophenoxyacetic acid, 2 mg / L 6-BA, 30 g / L sucrose, 0.2 g / L citric acid, and 0.2 g / L vitamin C.

7. The method of claim 6, wherein, The medium for subculture further comprises 0.5 g / L tryptophan.

8. The method of claim 3, wherein, The extraction of camptothecin from the Camptotheca acuminata cells comprises the following steps: taking the subcultured Camptotheca acuminata cells overexpressing the tryptophan synthetase gene, separating, washing, and grinding, adding 1-4 times the volume of ethyl acetate to the grinding liquid, heating at 50-80 ℃ for 1-2 h, collecting the organic phase, and purifying camptothecin by high-performance liquid chromatography.

Images