CN103146614A - Camptotheca endophytic bacterium LY214 for producing camptothecin and application thereof - Google Patents

Abstract

The invention relates to a Camptotheca acuminata endophytic bacterium isolated and purified from Camptotheca acuminata and its application. The endophytic bacterium LY214 involved in the present invention is identified as Paenibacillus polymyxa by morphology and molecular biology. On the 19th, deposit number CGMCC 6841. The notable feature of the present invention is that the high-performance liquid chromatography, ultraviolet spectrum and mass spectrometry analysis show that the camptothecin, an antitumor active ingredient, can be produced by the camptothecin endophytic bacterium strain LY214 through liquid fermentation. The invention discloses for the first time the isolation and cultivation of endophytic bacteria producing camptothecin from camptothecin, and provides a new way for the preparation of camptothecin.

Description

A camptothecin-producing endophytic bacterium-LY214 and its application

technical field

The invention relates to the isolation, cultivation and application of a camphor tree endophytic bacterium, belonging to the field of medical biotechnology.

Background technique

Camptothecin (CPT) is a terpenoid indole alkaloid formed by the condensation of tryptamine and split loganin. It was first isolated from the bark of the unique plant Camptotheca acuminata in my country, and was later found in many plants. Such as Ervatamiaheyneana (Ervatamiaheyneana) in the Oleander family, Nothapodytes foetida (Nothapodytes foetida) in the Tea Cornaceae family, and Ophiorrhiza pumila in the Rubiaceae family. Camptothecin has a unique anti-tumor mechanism, and exerts anti-tumor activity by inhibiting topoisomerase I. Camptothecin series derivatives are effective against pancreatic cancer, prostate cancer, breast cancer, lung cancer, ovarian cancer, colon cancer, cervical cancer, gastric cancer and blood tumors, and are the main varieties in the global anti-tumor drug market.

The main sources of camptothecin are 1) extraction from plants; 2) chemical synthesis; 3) plant cell culture. Extracting camptothecin from plants is the main method currently used. However, the content of camptothecin in plants is low (generally about 0.1-0.2%, and the content in fresh leaves is slightly higher, reaching 0.4-0.5%), and its extraction, separation, purification, and preparation processes are cumbersome and difficult; In addition, the growth cycle of camptothecin-producing plants is long, and the accumulation process of camptothecin is slow. my country is the main producer and exporter of camptothecin, and simply extracting it from plants will inevitably cause huge damage to plant resources and the ecological environment. In addition, the use of a large amount of organic solvents in the extraction process leads to large environmental pollution and cumbersome steps. The chemical total synthesis of camptothecin is synthesized by different ring-building methods, such as split method, asymmetric oxidation method, etc. Most methods have shortcomings such as too long route and low total yield, so it is difficult to realize industrialization. The use of plant cell culture to produce high value-added plant secondary metabolites (such as camptothecin and its derivatives) was once considered a promising alternative, but due to the slow growth of plant cells and the heterogeneity of the culture process Factors such as severe globalization, unstable output, and production costs have made it difficult to realize industrialized production. In short, the sources of camptothecin are limited and cannot meet the market demand at all (the annual demand for camptothecin in the international market is 3000kg, but the global annual output of camptothecin is only 600kg), and it is necessary to discover and develop alternative resources and technologies.

Plant endophytes refer to those fungi or bacteria that live in various tissues and organs of healthy plants or in the intercellular space during a certain stage or all stages of their life history. In view of the fact that some endophytes can promote the synthesis of secondary metabolites of medicinal plants and produce the same or similar medicinal active ingredients as the host, no matter from the perspective of ecology or economy, using plant endophytes as resources to find and symbiotic The same or similar natural active substances of plants, especially anti-tumor natural medicines will be an inexhaustible resource. In addition, microorganisms grow rapidly, are easy to cultivate, and the cost is relatively low. At the same time, microorganisms are easy to undergo directional transformation through biotechnology such as genetic engineering, so they have good development prospects.

So far, there have been studies on the isolation of endogenous camptothecin and its analogues from plants of different genera such as camptothecin, Apodytes dimidiata E.Mey.ex Arn, etc. Related reports on fungi (see Table 1), these studies show that it is possible to use microbial fermentation to produce camptothecin, and the use of plant endophyte fermentation to produce camptothecin is undoubtedly a green way to solve the source of camptothecin raw materials.

Contents of the invention

The object of the present invention is to provide for the first time a camptothecin endophytic bacterium Paenibacillus polymyxa LY214 isolated and cultivated from camptothecin, and its application, that is, for the preparation of camptothecin.

For achieving the purpose of the present invention, the technical scheme that the present invention adopts is as follows:

The invention provides a camphor tree endophytic bacterium named Paenibacillus polymyxaLY214. The endophytic bacterium Paenibacillus polymyxa LY214 of the present invention is an endophytic bacterium isolated from the bark of Campylodontaceae plant. After morphological and molecular biological identification, the bacteria belonged to Paenibacillus polymyxa. The bacterium has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures (Address: Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with a preservation date of November 19, 2012 and a preservation number of CGMCC 6841.

The 16S rDNA base sequence of the above-mentioned endophytic bacterium Paenibacillus polymyxa LY214 isolated from Campylodon japonica is shown in SEQ ID NO.1 as follows:

AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGGGTTGATTAGAAGCTTGCTTCTAATCAACCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCCACAAGACAGGGATAACTACCGGAAACGGTAGCTAATACCCGATACATCCTTTTCCTGCATGGGAGAAGGAGGAAAGACGGAGCAATCTGTCACTTGTGGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAACGTCTTGTAGAGTAACTGCTACAAGAGTGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTTTTTAAGTCTGGTGTTTAATCCCGAGGCTCAACTTCGGGTCGCACTGGAAACTGGGGAGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGATATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGGCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTTAACACATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACAT CCCTCTGACCGGTCTAGAGATAGACCTTTCCTTCGGGACAGAGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGCTTAGTTGCCAGCAGGTCAAGCTGGGCACTCTAAGCAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCGGTACAACGGGAAGCGAAATCGCGAGGTGGAGCCAATCCTAGAAAAGCCGGTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTACAACACCCGAAGTCGGTGGGGTAACCCGCAAGGGAGCCAGCCGCCGAAGGTGGGGTAGATGATTGGGGTGAAGTCGTAACAAGGTAACC。

The sequencing results were submitted to NCBI (http://blast.ncbi.nlm.nih.gov/Blast) for comparison. Based on the BLAST results and morphological characteristics, it was concluded that the strain belonged to Paenibacillus polymyxa polymyxa).

The present invention also provides a method for isolating Paenibacillus polymyxa LY214 from camptophylla, the method comprising:

Cut the plant material of Campylodon chinensis into sections according to the routine operation, clean and disinfect, inoculate it into 5% water agar solid medium, and cultivate it at a constant temperature of 28°C±2°C for 2 to 3 weeks. Continue culturing on the Sabouraud agar solid medium, and finally obtain the endophytic bacterium Paenibacillus polymyxa LY214 after purification. Wherein, in this step, according to the general operation method of inoculation, the camphor tree plant material through cutting, cleaning and disinfection can be cut off before inoculation, and the new cut surface can be inoculated into 5% water agar solid medium.

Wherein, the plant material of Campylodon chinensis is root or bark or fruit or leaves or branches of Campylomania japonica.

The present invention also provides an application of the above-mentioned Paenibacillus polymyxa LY214 in preparing camptothecin.

Specifically, in the above-mentioned use of Paenibacillus polymyxa (Paenibacillus polymyxa) LY214 to prepare camptothecin, wherein the method for preparing camptothecin from Paenibacillus polymyxa (Paenibacillus polymyxa) LY214 includes Paenibacillus polymyxa (Paenibacillus polymyxa) ) LY214 strain fermentation culture step and camptothecin extraction step.

Wherein, in the step of fermentation and cultivation, inoculate Paenibacillus polymyxa LY214 strain into Sabouraud agar liquid medium, shaker 160±20rpm, 28±2°C, ferment and cultivate for 8-15 days to obtain the fermentation mixture ;

In the camptothecin extraction step, the fermentation mixture was centrifuged at 4000 rpm for 15 minutes, filtered, and the filtrate and mycelium were collected respectively; the filtrate was extracted with a mixed solvent of chloroform-methanol (V/V=4:1), and the organic phase Concentrate to dryness under reduced pressure at 40°C to obtain the fermentation broth extract; the mycelium is ground and broken, soaked in methanol overnight, extracted with an equal volume of chloroform-methanol (V/V=4:1) mixed solvent, and the organic phase is decompressed at 40°C Concentrate to obtain mycelium extract; add chloroform-methanol (V/V=4:1) to dissolve the fermentation broth extract or mycelium extract, and filter through a needle filter to obtain a crude camptothecin extract.

In addition, in the above-mentioned method of preparing camptothecin by using Paenibacillus polymyxa LY214, according to routine operations, if necessary, Paenibacillus polymyxa LY214 strain fermentation can be carried out before the Paenibacillus polymyxa LY214 strain fermentation step. Preparation steps of Paenibacillus polymyxa LY214 strain seed solution. Wherein the seed solution preparation step includes strain activation and seed cultivation. Among them, in the activation of strains, the activation medium is Sabouraud solid medium, 28±2°C, and the culture time is 5-7d; in seed cultivation, the medium is Sabouraud liquid medium, 28±2°C, shaker 160±20rpm Cultivate for 2-3 days.

The component analysis of the fermentation broth extract or mycelium extract obtained by Paenibacillus polymyxa LY214 through fermentation culture showed that camptothecin mainly existed in the fermentation broth extract, and only traces of camptothecin were present in the mycelium extract. amount of camptothecin.

Beneficial effects of the present invention:

(1) A new camptothecin endophyte, Paenibacillus polymyxa LY214, was isolated from camptothecin plants, providing a new microorganism for the development of new drug sources for camptothecin; (2) Paenibacillus polymyxa Paenibacillus polymyxa LY214 has the characteristic of producing camptothecin, and can be applied to the preparation of camptothecin; (3) Compared with the prior art, the object of the present invention is bacteria, which has the advantages of rapid growth and reproduction, easy cultivation and fermentation (see table 1); (4) The isolation method of Paenibacillus polymyxa LY214 is simple and easy to control; (5) The method of obtaining camptothecin from Paenibacillus polymyxa LY214 is simple and easy to control.

The comparison of table 1 camptophytic endophytic bacteria of the present invention and existing reports

Description of drawings

Figure 1 is the HPLC-DAD diagram of the camptothecin standard (I) and the fermentation extract (II) of the camptothecin-producing endophytic bacterium Paenibacillus polymyxa LY214 (Paenibacillus polymyxa LY214), where Rectamine;

Figure 2 is the ultraviolet diagram of camptothecin standard (I) and camptothecin (II) produced by endophytic bacteria Paenibacillus polymyxa LY214 (Paenibacillus polymyxa LY214);

Figure 3 is the mass spectrum of camptothecin standard (I) and camptothecin produced by endophytic bacteria Paenibacillus polymyxa LY214 (Paenibacillus polymyxa LY214);

Figure 4 is the SBA solid culture (A) and SBA liquid culture (B) diagrams of camptothecin-producing endophytic bacteria Paenibacillus polymyxa LY214 (Paenibacillus polymyxa LY214);

Fig. 5 is a phylogenetic tree diagram of the camptothecin-producing endophytic bacterium Paenibacillus polymyxa LY214 (Paenibacillus polymyxa LY214).

Detailed ways

The preferred embodiments of the present invention will be further described below in conjunction with the accompanying drawings.

Example 1: Isolation and screening of camptothecin-producing endophytes from camptothecin

Isolation of endophytic bacterial strain Paenibacillus polymyxa LY214 from Campylodon japonica.

1. Test material

1.1 Plant material:

Select the organs or tissues of philodendron that grow normally and healthy as plant materials, such as tree roots, bark, fruits, leaves, branches and other parts.

The collected plant materials were cut into sections, cleaned and disinfected according to routine operations. In this embodiment, tap water is used to thoroughly rinse to remove dust and other sundries, and the plant material is cut into small pieces of 10mm×10mm with a sterile scalpel, and soaked in 75% ethanol for 5 minutes, 10% sodium hypochlorite solution for 5 minutes, and 1% Triton. 5min, rinse with sterile water 5 times, 1min each time, and finally use sterile filter paper to absorb the surface moisture of each tissue part of Campylocarpus chinensis, and set aside.

1.2 Medium:

5% water agar solid medium: the agar content is 5% (W/W), prepared with water, without adding any nutrients.

Sabouraud agar solid medium (SBA solid medium): peptone 10g/l, glucose 40g/l, pH5.6, autoclaved at 121°C for 30min.

2. Plant material inoculation and cultivation

The prepared plant material was inoculated and cultivated according to conventional aseptic operation methods.

Cut the prepared camphor tree plant material along the central axis, inoculate the new cut surface on 5% water agar solid medium, and cultivate at a constant temperature of 28°C±2°C for 2 to 3 weeks; after the new cut surface of the plant material grows hyphae , pick the hyphae and move them to the SBA medium to continue culturing at a constant temperature of 28°C±2°C; finally, the endophytic bacterial strain Paenibacillus polymyxa LY214 was obtained after purification.

The strain Paenibacillus polymyxa LY214 was preserved on SBA solid slant medium.

Embodiment two

Camptothecin was obtained from the camptothecin endophytic bacteria strain Paenibacillus polymyxa LY214.

1. Culture medium and extraction solvent

Culture medium: Sabouraud liquid medium (SBA liquid medium), the components are the same as the SBA solid medium in Example 1, but no agar is added.

Extraction solvent: chloroform-methanol mixed solvent (V/V=4:1)

2. Extraction method of camptothecin

2.1 Fermentation culture

The bacterial strain Paenibacillus polymyxa LY214 preserved in Example 1 was prepared according to conventional methods to obtain seed liquid. Inoculate 50ml of seed solution into 500ml of SBA liquid medium, culture at 28±2°C, shaker 160±20rpm, for 8-15 days, and obtain the fermentation mixture.

2.2 Preparation of fermented extract

The fermentation mixture was centrifuged at 4000rpm for 15min, filtered, and the filtrate and mycelia were collected respectively.

The obtained filtrate was extracted with an equal volume of chloroform-methanol mixed solvent, and the organic phase was concentrated under reduced pressure at 40°C to obtain a fermentation broth extract; a certain amount of extraction solvent was added to dissolve, and filtered through a needle filter to obtain a crude camptothecin extract.

The obtained mycelium was dried, ground and crushed, soaked in methanol overnight, extracted with an equal volume of chloroform-methanol mixed solvent, and the organic phase was concentrated under reduced pressure at 40°C to obtain the mycelium extract; a certain amount of extraction solvent was added to dissolve, and filtered through a needle filter to obtain the mycelium extract. Tree alkaloid crude extract.

Embodiment Three

Camptothecin was obtained by using the camptothecin endophytic bacterial strain Paenibacillus polymyxa LY214, which is the same as Example 2 and will not be repeated. The difference is that the seed liquid preparation step is added before the fermentation culture, including strain activation and seed culture.

Activation of strains: the bacterial strain Paenibacillus polymyxa LY214 preserved in Example 1 was inoculated on SBA solid medium for activation, and cultured for 5-7 days to obtain activated strains;

Seed culture: Inoculate the activated strain into 50ml SBA liquid medium, culture at 28±2°C, shaker 160±20rpm, for 2-3 days to obtain the seed solution.

Test example one

HPLC-DAD detection of the fermentation product of strain Paenibacillus polymyxa LY214.

The fermentation broth extract and the mycelia extract obtained in Example 2 or Example 3 were respectively detected by HPLC-DAD.

HPLC-DAD analysis and detection conditions: Altima column C18 column (5μm, 250mm×4.6mm), mobile phase A: methanol, mobile phase B: water + 0.1% formic acid, column temperature: 35°C, flow rate: 1ml/min; Sample volume: 20 μl; detector: diode array (DAD); detection wavelength: 254nm, 266nm, 373nm; gradient elution program as shown in Table 2:

Table 2 HPLC-DAD gradient elution conditions

time (min) 0 12 30 39 45 Mobile phase A% 5 50 80 98 98 Mobile phase B% 95 50 20 2 2

Under the above conditions, the retention time of camptothecin standard was 25.39min; the retention time of camptothecin in the fermentation broth extract of endophytic strain Paenibacillus polymyxa LY214 was 25.38min. Figure 1 shows the HPLC-DAD diagram of camptothecin standard (I) and Paenibacillus polymyxa LY214 strain fermentation broth extract (II); Figure 2 shows the camptothecin standard (I) and Paenibacillus polymyxa LY214 fermentation broth The UV spectrum of camptothecin (II) produced by the extract (II). The results of HPLC-DAD showed that only traces of camptothecin were contained in the mycelia extract.

Test example two

HPLC-DAD-MS confirmation of camptothecin-producing endophytic strain Paenibacillus polymyxa LY214.

The fermentation broth extract verified by Test Example 1 containing the same retention time and ultraviolet spectrum as the camptothecin standard product was analyzed by liquid chromatography-mass spectrometry, and the product was confirmed to be camptothecin. Figure 3 shows the HPLC-MS images of camptothecin standard (I) and camptothecin (II) produced by the endophytic strain Paenibacillus polymyxa LY214.

Test example three

Taxonomic identification of the camptothecin-producing endophytic fungal strain Paenibacillus polymyxa LY214, part of the morphological identification.

The endophytic strain Paenibacillus polymyxa LY214 was inoculated on SBA agar medium, cultured at 28±2°C, and the colony morphology was observed. The strain Paenibacillus polymyxa LY214 grew rapidly on SBA medium, with thin colonies, translucent to opaque, irregular amoeba-like edges, slightly raised in the middle, and colonies adhered to the surface of the medium (Figure 4A);

The endogenous strain Paenibacillus polymyxa LY214 was inoculated into Sabouraud liquid medium, cultured on a shaker at 28±2°C, 160±20rpm, and the colony morphology was observed. The characteristics of liquid culture are that the bacterial liquid is clear after 1-2 days of culture, and the amount of bacteria increases after 3-4 days of culture, and the culture liquid is turbid. (Fig. 4B).

Test example four

Taxonomic identification of camptothecin-producing endophytic bacterial strain Paenibacillus polymyxa LY214, part of molecular biology identification.

Prepare the fermentation broth of bacterial strain Paenibacillus polymyxa LY214 as described in Example 2 or 3, centrifuge the fermentation mixture for 15 min at 4000 rpm, filter, and collect the mycelium; wash the mycelium twice with sterilized deionized water, and centrifuge to remove the supernatant Genomic DNA of strain Paenibacillus polymyxaLY214 was extracted according to the operating instructions of the bacterial genomic DNA extraction kit (E.Z.N.A.TM Bacterial DNA Kit); the quality and concentration of genomic DNA were detected by UV-1100D ultraviolet spectrophotometer (MAPADA, China), OD260/280 1.9, DNA quality is good.

The obtained strain Paenibacillus polymyxa LY214 genomic DNA was used as a template, 5'-AGAGTTTGATCCTGGCTCAG-3' was used as a forward primer (SEQ ID NO.2), and 5'-GGTTACCTTGTTACGACTT-3' was used as a reverse primer (SEQ ID NO.3); PCR reaction system and reaction conditions: 25 μl 2×Master Mix, 20 μl ddH ₂ O, 2 μl each of forward and reverse primers, 1 μl DNA template; the reaction conditions are listed in the kit as 94°C, 3 minutes of denaturation, and then cycle. The cycle parameters are: 94°C, 30sec; 55°C, 30sec; 65°C, 1min; after 35 cycles, extend at 65°C for 5min for PCR amplification to obtain 16S rDNA amplification products.

The PCR product was separated by electrophoresis on 1% agarose gel, and an obvious target amplified fragment appeared around 1500bp, indicating that 16S rDNA had been successfully amplified from Paenibacillus polymyxa LY214 strain. Agarose gel recovery kit (DP209, Tiangen, China) was used to recover and purify the target fragment; using pGM-T cloning kit (Tiangen, China), the recovered target fragment was combined with pGM-T vector according to the instruction Ligation; transform the ligation product into E.coli DH5α; perform blue-white screening; select a single clone and culture it in liquid LB medium with ampicillin resistance at 37°C and 180rpm overnight; extract the plasmid, detect it by gel electrophoresis, and PCR The correct fragments were verified and sent to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing. The base sequence results are shown in SEQ ID NO.1.

The 16S rDNA sequence of strain LY214 was compared with BLAST analysis in the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast) for homology, and the 16S rDNA sequence of the strain was compared with the sequence obtained by homology Sequences with high similarity have a similarity of more than 99%. The phylogenetic tree was constructed using the neighbor-joining method in the MEGA4.0 software, as shown in Figure 5. Therefore, the taxonomic status of Paenibacillus polymyxa LY214 strain was determined, and the endophytic bacterium Paenibacillus polymyxa LY214 was identified as Paenibacillus polymyxa.

Claims (10)

1. An endophytic bacterium isolated from Campylodontia involucrata, characterized in that the classification of the endophytic bacterium is named Paenibacillus polymyxa LY214, belongs to Paenibacillus polymyxa, and has been preserved in China Microorganism Culture Preservation Management Committee Center for General Microbiology, deposit number CGMCC6841. 2. The endophytic bacterium according to claim 1, characterized in that, the endophytic bacterium is characterized in that the 16S rDNA base sequence 1517bp of the endophytic bacterium Paenibacillus polymyxa LY214, as shown in SEQ ID NO.1. 3. a kind of method that obtains endophytic bacterium Paenibacillus polymyxa LY214 from isolation from Davidiaceae plant camptophyllum as claimed in claim 1, is characterized in that, the method comprises: Cut the plant material of Campylodon chinensis into sections according to the routine operation, clean and disinfect, inoculate it into 5% water agar solid medium, and cultivate it at a constant temperature of 28°C±2°C for 2 to 3 weeks. Continue culturing on the Sabouraud agar solid medium, and finally obtain the endophytic bacterium Paenibacillus polymyxa LY214 after purification. 4. The method according to claim 3, characterized in that, the plant material of Campylodon chinensis is root or bark or fruit or leaves or branches of Campylodont japonica. 5. the application of Paenibacillus polymyxa LY214 according to claim 1 in the preparation of camptothecin. 6. The application according to claim 5, characterized in that, the method of obtaining camptothecin by using the endophytic bacterium Paenibacillus polymyxa LY214 of the Camptotheca involucrata family plant comprises the fermentation of the Paenibacillus polymyxa LY214 bacterial strain Cultivation step and camptothecin crude extract step. 7. according to the described application of claim 5 or 6, it is characterized in that, in the fermentation culture step in the described method, polymyxa Paenibacillus polymyxa LY214 bacterial strain is inoculated to Sabouraud agar liquid medium, shaker 160 ± 20rpm, 28±2°C, ferment for 8-15 days to obtain the fermentation mixture; In the camptothecin extraction step in the method, the fermentation mixture is centrifuged at 4000rpm for 15min, filtered, and the filtrate and mycelia are collected respectively; the filtrate is extracted with a chloroform-methanol mixed solvent of V/V=4:1 , the organic phase was concentrated to dryness under reduced pressure at 40°C to obtain a fermentation broth extract; the mycelium was ground and broken, soaked in methanol overnight, extracted with an equal volume of V/V=4:1 chloroform-methanol mixed solvent, and the organic phase was 40 Concentrate under reduced pressure at ℃ to obtain mycelium extract; add chloroform-methanol with V/V=4:1 to dissolve the fermentation broth extract or mycelium extract, and filter through a needle filter to obtain a crude camptothecin extract. 8. The method according to claim 7, characterized in that, according to routine operations, the Paenibacillus polymyxa LY214 strain seed liquid preparation step can be performed before the Paenibacillus polymyxa LY214 strain fermentation step. 9. The method according to claim 8, characterized in that, said Paenibacillus polymyxa LY214 strain seed solution preparation step comprises strain activation and seed cultivation. 10. The method according to claim 9, characterized in that, in the activation of the strains, the activation medium is Sabouraud solid medium at 28±2°C, and the cultivation time is 5-7d; during seed cultivation, the medium is It is Sabouraud liquid medium, cultured at 28±2°C, shaker 160±20rpm for 2-3 days.

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