CN106967623B

CN106967623B - Aspergillus niger for producing taxane compound baccatin III and application thereof

Info

Publication number: CN106967623B
Application number: CN201710354776.7A
Authority: CN
Inventors: 曹军卫
Original assignee: Wuhan Donghu University
Current assignee: Wuhan Donghu University
Priority date: 2017-05-17
Filing date: 2017-05-17
Publication date: 2019-12-13
Anticipated expiration: 2037-05-17
Also published as: CN106967623A

Abstract

The invention discloses an endophyte which is separated and screened from tissues of torreya grandis of Taxaceae and produces taxane compound baccatin III, and the endophyte is named as Aspergillus niger BP12 +/-3 through classification identification, and the preservation number is CCTCC NO: M2016319. The strain has the activity of resisting staphylococcus aureus and escherichia coli, the content of a crude extract of a taxol analogue in fermentation liquor can reach 120mg/L, wherein the relative percentage content of a taxane compound baccatin III is 10.5%, the yield is 12.6mg/L, and is higher than the yield disclosed in the prior art; the yield of baccatin III can be increased by 129.84% by using L-phenylalanine as precursor. The CCK-8 reagent method is adopted to prove that the fermentation liquor extract of the Aspergillus niger BP12 +/-3 has the inhibition effect on Hela tumor cells, and the inhibition rate can reach 62.91%.

Description

Aspergillus niger for producing taxane compound baccatin III and application thereof

Technical Field

the invention belongs to the technical field of microorganisms, and particularly relates to an endophyte separated from torreya grandis, named as Aspergillus niger BP12 +/-3, which has the characteristic of high yield of taxane compound baccatin III, and application of Aspergillus niger BP12 +/-3 in preparation of a medicine for treating tumor cells.

Background

Paclitaxel (taxol), the international common name Paclitaxel, is a diterpenoid compound isolated and extracted from the plant taxus pacifica by american scientists in the sixties of the last century, and through ii-iii clinical verification, the diterpenoid compound has good antitumor effect, especially has special efficacy on ovarian cancer, uterine cancer, breast cancer and the like with high incidence rate of cancers, and also has certain curative effect on lung cancer, colorectal cancer, melanoma, head and neck cancer, lymphoma, brain tumor, which is a significant discovery in the anticancer field in recent years, and has been approved by the united states Food and Drug Administration (FDA) as a new drug for treating metastatic ovarian cancer in 1992, and has been on the market.

Paclitaxel is white crystalline powder, insoluble in water, and easily soluble in organic solvents such as chloroform and acetone. Molecular formula C₄₇H_5lNO_l4the molecular weight is 853.92, and the melting point is 213-216 deg.C (Wani MC, Taylor HL, Wall ME, et al plant antioxidant agents, VI. the isolation and structure of taxol, a novel antioxidant and antioxidant agent from Taxusbrevefolia [ J]J Am Chem Soc,1971,93: 2325-2327.). Research shows that the antitumor active substances discovered at present are mainly taxane diterpenoid compounds, which are up to 7 types and have more than 100 skeletons. This provides a greater field of development for expanding the anti-tumor range, semisynthetic new drugs, and avoiding drug resistance.

Paclitaxel is a complex secondary metabolite in Taxus and is the only drug currently known that promotes microtubule polymerization and stabilizes polymerized microtubules. Isotopic tracing indicates that paclitaxel binds only to polymerized microtubules and does not undergo a dimeric reaction with unpolymerized tubulin. Cells exposed to paclitaxel accumulate large numbers of microtubules within the cell, which interfere with various functions of the cell, particularly arresting cell division at mitosis, and blocking normal cell division. Thus, paclitaxel inhibits the growth of tumor cells.

The determination shows that the content of the taxol in the barks of the taxus plants is about 0.003-0.069 percent, about 2000 trees need to be cut every 1kg of taxol is extracted, even if all natural resources are harvested, the short-term requirement can be met, and irreparable loss is caused to the living environment and biodiversity of forests and human beings. Therefore, the raw material guarantee of the paclitaxel becomes a key factor for the success of the medicine to market. The separation of taxol endophytic fungi is a new way for effectively solving the problem of taxol resources developed in recent years.

The term Endophyte (Endophyte) was first proposed by De Bary (De Bary a,1866, morpholino and Physiologie derPilze, flechen and myxomyceten. engelman, Leipzig, 1-316), referring to microorganisms present in plant tissues to distinguish between those epiphytes (epiphytes) living on the surface of plants. Petrini (Petrini O, Fungal endings of trees leaves. in: Andrews J HHirano and S sets. microbiological Ecology of leaves. New York Springer-Verlag.1991,179-197.) defines endophytes as bacteria that live within plant tissue at a certain period of their life history without causing significant disease symptoms to the plant tissue, and this definition includes those saprophytes that are superficial at a certain period of their life history, Latent pathogens (Latent pathogens) and mycorrhizal bacteria that are temporarily harmless to the host.

In 1993, Strobel et al isolated and screened an endophytic fungus (Taxomyces andreanae) from Taxus brevifolia (T. brevifolia), and found that it was able to synthesize taxol at the time of in vitro culture with a yield of 24-50. mu.g/L. While Strobel believes that the endophytic fungus synthesizing paclitaxel may be involved in the action of fungi against root invasion, while better mimicking the chemical environment of the host, and thus may have an advantage in interspecific competition (Stierle A, Stierle D, Strobel G, et a1.,1994, Endophytix fungi of pacific year (Taxus-brevifo 1ia) as source of taxol, taxanes, and other pharmacophores acquispossium servers.557: 64-77.). This is a completely new finding and is undoubtedly pioneering. Thereafter, scientists isolated a number of endophytic fungi capable of synthesizing paclitaxel in varying yields.

The host of the taxol-producing strain is commonly found in Taxus (Taxus) and Taxus australiana (Austotaxus) of the family Taxaceae (Taxaceae) as a gymnosperm. The former is widely distributed in continental europe and north america, such as the three provinces of northeast china, the central region, Yunnan, Guizhou, Sichuan, Tibet, and the United states and Canada; the plants are distributed in cold zone, warm zone and subtropical zone, have less forest formation and slow growth. The latter are seen only in the southern hemisphere, in very small numbers.

Therefore, any organism with substance and energy exchange with taxus plants has the possibility of genetic gene transfer of taxol secondary metabolites, especially the most possible epiphytic fungi. Research shows that the taxol content in the taxus chinensis has no obvious correlation with the species of the taxus chinensis, but is closely related with the ecological environment of the producing area of the taxus chinensis. Therefore, more taxol-producing bacteria have to be isolated from other plants having the same environmental requirements as those of Taxus chinensis, and the study of endophytic fungi in the trunk and lateral branches, bark, phloem and subcutaneous parts, roots, mature stems, fruits and leaves of Taxus chinensis has been reported. The content of taxol or analogues produced by wild fungi is generally low, and the yield of the taxol or analogues does not meet the requirement of industrial production.

to date, taxol-producing endophytic fungal strains have been isolated mainly from plants of the genus Taxus, an endangered species, while other plants of the genus Taxus, the family Taxaceae, have been ignored. Because research shows that taxanes compounds are found in the plants of the genus torreya of the family taxaceae, but the content is extremely low, the content of taxol is lower than 0.003%, and the medicinal development value is not large (the officinal beauty, the current research situation and development of torreya grandis in Qiu spring, 2013, 29 (8): 844-848). The strains which are separated from Cephalotaxus hainanensis Li by Liuyan and the like and produce secondary metabolites mainly produce possible antitumor substances which are Cephalotaxus hainanensis endophytic fungi CH1307 identification and antitumor activity research of Cephalotaxus hainanensis endophytic fungi (Liuyan and the like) [ D ] Hainan university, 2012.Jianchuanjie ] Hainan endophytic fungi antitumor activity strain screening [ D ]. Hainan university, 2014.)

the invention separates out an endophytic fungus from Torreyawaresii (Torreyafargesii, Taxaceae) tissue in geological forest park of Shennongjia country; screening out strains which produce secondary metabolites of paclitaxel or analogues through high performance liquid chromatography analysis and anti-tumor activity analysis; and qualitatively detecting by using a liquid chromatography-mass spectrometer (HPLC-MS) to judge that the taxane compound generated by the strain is Baccatin III.

the method for producing paclitaxel in large amount by microbial fermentation is expected to improve the current situations of high price and short supply and demand of paclitaxel. Even if the yield is equal to or slightly lower than the content of the taxol in the plant, the fermentation scale can be artificially controlled due to the short fermentation period (10-15 days) of the fungi, and the production capacity of the plant material is greatly superior to that of the plant material.

Disclosure of Invention

the invention aims to provide an endophyte Aspergillus niger BP12 +/-3 separated from torreya grandis, and the strain has the characteristic of high yield of taxane compound baccatin III.

Another objective of the invention is to provide a culture method of Aspergillus niger BP12 + -3, wherein the yield of baccatin III can be increased by 129.84% by adding precursor L-phenylalanine into the culture medium.

The invention also aims to provide the application of the Aspergillus niger BP12 +/-3 in preparing the medicine for treating the tumor cells, and experiments prove that the Aspergillus niger BP12 +/-3 fermentation liquid extract has obvious inhibition effect on Hela tumor cells, and the inhibition effect on cell proliferation is enhanced along with the increase of the concentration of paclitaxel.

In order to achieve the purpose, the invention is realized by the following technical scheme:

The separation and screening of the Aspergillus niger BP12 +/-3 comprises the following steps:

1. Respectively washing roots, stems, leaves and barks of torreya grandis with sterile water, drying with sterile absorbent paper, sequentially sterilizing with 75% (v/v) alcohol for 5min and 2% (v/v) hypochlorous acid for 8min, washing with sterile water, cutting the collected roots, stems and barks into small segments with a sterile knife, placing the materials of each part in a PDA solid culture medium plate, culturing at 28 deg.C, and inoculating fine mycelia on the edges of the section of the inoculum in the plate, and purifying to obtain pure culture;

2. Inoculating the purified fungus to PDA plate by hypha tip inoculation method, and culturing at 28 deg.C. Recording colony sizes, colors, hypha compactness, whether the colony edges are regular, colony growth speed, pigment production and other colony forms;

3. the antibacterial activity of the endophytic fungi is measured by an agar block method: LB culture medium for bacteria (Staphylococcus aureus, Escherichia coli) to be testedActivating, coating on LB solid medium surface, inoculating endophyte with agar medium, culturing at 37 deg.C for 1-2 days, observing the inhibition zone around the agar block, and obtaining a strain with anti-Staphylococcus aureus (G)⁺) And Escherichia coli (G)^-) The active strain is marked as BP12 plus or minus 3;

4. Observing the colony color of the strain to be dark black through the plate culture characteristic, wherein the reverse color of the colony is yellow to yellow brown, the colony texture is velvet and has radioactive grooves, and the meristematic spore head is loose and radial, and is in a top capsule spherical shape with the diameter of 50-80 mu m, a peduncle double layer, a peduncle base (4-6 mu m) x (2-3 mu m), a conidium spherical shape with the diameter of 3-4.5 mu m is identified as Aspergillus niger (Aspergillus niger) and is marked as BP12 +/-3 under the microscope observation;

5. Activating a strain Aspergillus niger BP12 +/-3 in a PDA liquid culture medium at 180rpm and 28 ℃ for 3 days to serve as a seed solution, inoculating the seed solution into the PDA liquid culture medium at an inoculation amount of 5% (v/v), and culturing at 180rpm and 28 ℃ for 7-9 days to obtain a fermentation broth;

6. After fermentation, collecting filtrate, adding 1/3 volume of ethyl acetate into the filtrate, performing countercurrent extraction for 3 times, collecting upper layer organic phase, and removing organic solvent in a rotary evaporator to obtain fat-soluble extract with content of about 120 mg/L;

7. High Performance Liquid Chromatography (HPLC) finds that the fat-soluble extract contains components with retention time basically consistent with that of a paclitaxel standard product, the retention time is 12.08min, and is similar to that of the paclitaxel standard product, which indicates that the fat-soluble extract contains paclitaxel or analogues with relative percentage content of 10.5%;

8. The structure of the taxol analogue in the metabolic extract is determined by liquid chromatography-mass spectrometry (HPLC-MS), and the parent ion Da is 609.200, the daughter ion Da is 549.200, 367.000 and 427.100 respectively, the source voltage is 146, the collision energy is 34, 40 and 40 respectively, and the taxol analogue is completely consistent with a taxane compound baccatin III standard product, so that the taxol analogue generated by Aspergillus niger (Aspergillus niger) BP12 +/-3 is judged to be baccatin III.

Aspergillus niger BP12 + -3 separated from the tissue of torreya grandis is preserved in China Center for Type Culture Collection (CCTCC) with the preservation address of Wuhan university in China, the preservation date is 2016, 6 and 8 days, and the preservation number is CCTCC NO. M2016319, and the Aspergillus niger BP12 + -3 is characterized in that the Aspergillus niger has the characteristics of high yield of taxane compound baccatin III, the content of baccatin III in fermentation liquor reaches 12.6mg/L, and the Aspergillus niger is resistant to staphylococcus aureus and escherichia coli.

The culture method of the Aspergillus niger BP12 +/-3 high-yield taxane compound baccatin III comprises the following steps: the precursor L-phenylalanine is added into the PDA culture medium to increase the yield of the baccatin III, and experiments show that the yield of the baccatin III of the Aspergillus niger BP12 +/-3 can be increased by 129.84 percent by adding 3.0mg/L of the L-phenylalanine.

application of aspergillus niger BP12 +/-3 in treating anti-tumor cells: the inhibition effect of the fermentation broth extract of Aspergillus niger BP12 +/-3 on Hela cells is measured by a CCK-8 method, the result shows that the inhibition effect on cell proliferation is enhanced along with the increase of the concentration of the fermentation broth extract, and the inhibition rate reaches 62.91% when the concentration of the fermentation broth extract is 11.55 mu g/mL.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. The invention relates to an endophytic fungus separated from the tissue of torreya grandis (taxaceae, torreya), which is another example of a plant endophytic fungus separated from plants except taxus to have the capability of producing paclitaxel or analogues, which greatly expands the resource range of obtaining the strains producing paclitaxel or analogues and is beneficial to more widely utilizing microbial resources.

2. researches report that the taxol producing capacity of some plant endophytes is unstable and often needs the existence of precursor substances, the plant endophyte Aspergillus niger BP12 +/-3 can produce taxol analogues very stably, the crude extract content of the taxol analogues in fermentation liquor can reach 120mg/L, wherein the relative percentage content of taxane compounds baccatin III is 10.5%, and the yield of the baccatin III is about 12.6mg/L, which is higher than that disclosed in the prior art; the yield of baccatin III can be increased by 129.84% by using L-phenylalanine as precursor.

3. The inhibition effect of Aspergillus niger BP12 +/-3 on Hela tumor cells is detected by a CCK-8 reagent method, which shows that the taxol analog of the Aspergillus niger BP12 +/-3 fermentation liquor has obvious inhibition effect on the tumor cells, the inhibition effect on cell proliferation is enhanced along with the increase of the concentration of taxol, when the concentration of the medicine is 0.1155 mu g/mL, the inhibition rate is 29.82%, when the concentration is increased to 1.155 mu g/mL, the inhibition rate reaches 40.27%, and when the concentration is increased to 11.55 mu g/mL, the inhibition rate reaches 62.91%.

4. And (4) carrying out qualitative detection by using a liquid chromatography-mass spectrometer, and judging that the taxane compound generated by the strain is Baccatin III. This provides a greater field of development for expanding the anti-tumor range, semisynthetic new drugs, avoiding drug resistance, etc.

5. The method for producing taxol or the like by fermenting the endophyte of the invention can be used for producing taxol or the like in a short time and in a large scale without being limited by resources, environment, conditions, equipment and the like.

Drawings

FIG. 1 shows the lawn of Aspergillus niger BP12 + -3, the colony color of strain BP12 + -3 is dark black, the reverse color of the colony is yellow to yellow brown, the colony texture is velvet, and has radioactive furrows.

FIG. 2 is a microphotograph of Aspergillus niger BP12 + -3, wherein the conidial head is loose and radial, and has a top sac spherical shape with a diameter of 50-80 μm, a petiole bilayer, a peduncle base (4-6 μm) × (2-3 μm), and a conidial spherical shape with a diameter of 3-4.5 μm.

FIG. 3 is a chromatogram of a paclitaxel standard, with the peak indicated by the arrow being the paclitaxel standard.

FIG. 4 is a chromatogram of paclitaxel analogs from metabolic extracts of Aspergillus niger BP12 + -3, with the peak indicated by the arrow representing the paclitaxel analog.

Detailed Description

example 1: separation of endophyte of torreya grandis

1. Pretreatment of torreya grandis tree material

Collecting roots, stems, leaves and barks of torreya grandis in a geological forest park of Shennongjia nationality of Hubei province, washing with sterile water, and drying with absorbent paper; respectively sterilizing with 75% (v/v) alcohol for 5min and 2% (v/v) hypochlorous acid for 8min, and washing with sterile water; the harvested roots, stems and bark were cut into approximately 2-3cm pieces and the leaves cut from the middle into sections.

2. Separation of endophyte of torreya grandis

Placing the materials of each pretreated part in a PDA solid culture medium plate, and culturing at 28 ℃, wherein the PDA culture medium comprises the following components in parts by weight: 200g of potato, 20g of glucose and KH₂PO₄ 1.0g，MgSO₄·7H₂0.5g of O, 1000mL of water, natural pH and agar (15g/L) as a PDA solid culture medium.

3. purification of endophyte of torreya grandis

when fine hyphae grow on the edge of the section of the inoculum in the plate, the hyphae are picked and inoculated into a newly prepared PDA solid plate, and the plate is purified for several times on a PDA slant culture medium until a pure culture is obtained.

example 2: detection of bacteriostatic activity of endophyte BP12 +/-3

1. Activating bacteria to be tested (staphylococcus aureus and escherichia coli) by using an LB liquid culture medium, and respectively coating the bacteria to be tested on the surface of the LB solid culture medium;

2. The purified endophyte agar medium block obtained in the example 1 is cut off, respectively placed and inoculated in a staphylococcus aureus and escherichia coli plate, cultured for 1-2 days at 37 ℃, the inhibition zone around the agar block is observed, and a strain (G) with staphylococcus aureus resistance is obtained by screening through the method⁺) And Escherichia coli (G)^-) The active strain was recorded as BP 12. + -.3.

Example 3: identification of endophytic fungus BP12 + -3

Observed by the plate culture characteristics (figure 1), the colony color of the strain is dark black, the reverse color of the colony is yellow to yellow brown, the colony texture is velvet, and the strain has radioactive furrows; observed under microscope (FIG. 2), conidial heads are loose and radial, apical sac spherical, diameter 50-80 μm, petiole bilayer, peduncle base (4-6 μm) × (2-3 μm), conidial spherical, diameter 3-4.5 μm. According to the 'fifth Aspergillus of Chinese mycolog and related sexual type' (compiled by Zizu, China mycolog (fifth) Aspergillus and related sexual type [ M ]. Beijing: scientific Press, 1997, 5-10.), the strain can be preliminarily identified as Aspergillus niger BP12 + -3.

Example 4: extraction of liposoluble matter from Aspergillus niger BP12 +/-3 fermenting liquid

1. the endophyte Aspergillus niger BP12 + -3 obtained in example 2 is activated in PDA liquid culture medium (activation condition: 28 ℃, 180rpm, culture for 5-7 days) to be used as seed liquid;

2. inoculating into PDA liquid culture medium according to the inoculation amount of 5% (v/v), culturing at 28 deg.C and 180rpm for 7-10 days;

3. Filtering and collecting filtrate, adding 1/3 volume of ethyl acetate into the filtrate, carrying out countercurrent extraction for 3 times, and collecting an upper-layer organic phase;

4. Removing organic solvent at 35 deg.C in rotary evaporator, dissolving the substance adhered to the wall with methanol, volatilizing, drying, and measuring the content of the crude extract of the fermentation liquid by gravimetric method; wherein the content of crude extract of endophyte BP12 + -3 fermentation liquor is about 120mg/L on average.

example 5: quantitative determination of Aspergillus niger BP12 + -3 metabolic active substance containing taxol analogue by High Performance Liquid Chromatography (HPLC)

1. Fat-soluble substances and mobile phase treatment obtained in example 4: centrifuging the concentrated product obtained in example 4 at 13200r/min for 2min, sucking the supernatant, filtering through a filter membrane with the pore diameter of 0.22 mu m to remove impurities, and then ultrasonically exhausting the gas together with a prepared mobile phase (methanol/water is 65/35, v/v) for 3min and 10-15 min respectively;

2. The chromatographic conditions are as follows: column chromatography, ODS (C18) column 4.6X 250mm, 5 nm; column temperature, normal temperature; sample and mobile phase, methanol/water 65/35 (v/v); flow rate, 1.0 mL/min; sample size, 20 μ L; ultraviolet detection wavelength is 227 nm;

3. the Rt (retention time) of paclitaxel standard (purchased from Sigma) was measured to be 13-14 min (FIG. 3);

4. A component similar to Rt (retention time) of paclitaxel standard was found in the lipid-soluble extract prepared in example 4 (fig. 4), the peak time was 12.8min, and endophyte BP12 ± 3 metabolically active substance was identified as paclitaxel analogue;

5. And measuring the relative peak area of the taxol analogue in the BP12 +/-3 metabolic extract to be 29445.61% and the relative percentage content to be 10.5% according to the chromatogram by using HPLC Wufeng liquid chromatography workstation software.

example 6: inhibition effect of Aspergillus niger BP12 + -3 fermentation liquor extract on Hela tumor cells

1. Spreading Hela cells (Hualianke biotechnology, Inc.) in logarithmic growth phase, and culturing the cells at 100 μ L/well (1-5 × 10)³Individual cells/well) were seeded in 96-well plates at 37 ℃ in 5% CO₂Culturing for 24h in an incubator;

2. the paclitaxel analogue-containing Aspergillus niger BP12 + -3 fermentation broth extract prepared in example 4 was prepared at different concentrations (Table 1), and 10. mu.L of the extract was added to the sample wells (1-5 × 10)³Individual cells/well), 37 ℃, 5% CO₂culturing for 24h in an incubator;

3. Taking out the culture plate, removing the supernatant, adding 90 μ L of fresh culture solution (DMEM culture solution containing 10% fetal calf serum) and CCK-810 μ L into each well, and culturing for 4 hr under the same condition;

4. Reading the absorbance at 450nm with a microplate reader (A)₄₅₀value), cell growth inhibition rate was calculated, and cell growth inhibition rate was calculated: hela cell Inhibition Ratio (IR) ═ control well a value-sample well a value)/control well a value × 100% (table 1).

through the experiment, the Aspergillus niger BP12 +/-3 fermentation liquid crude extract has obvious inhibition effect on Hela tumor cells, and the inhibition effect and the extract concentration are in positive correlation; when the concentration of the drug is 0.1155 mug/mL, the inhibition rate is 29.82%, when the concentration is increased to 1.155 mug/mL, the inhibition rate reaches 40.27%, and when the concentration is increased to 11.55 mug/mL, the inhibition rate reaches 62.91%.

TABLE 1 inhibition of Hela cells by Aspergillus niger BP12 + -3 fermentation broth extract

example 7: determination of paclitaxel analogue in BP12 + -3 fermentation broth as baccatin III by liquid chromatography-mass spectrometry (HPLC-MS)

1. Liquid chromatography

The instrument model is as follows: shimadzu LC-30A UPLC

(1) Sample and mobile phase treatment: centrifuging the fat-soluble concentrated product prepared in example 4 at 13200r/min for 2min, sucking supernatant, filtering through a filter membrane with the pore diameter of 0.22 mu m to remove impurities, and then ultrasonically exhausting the mixture together with a prepared mobile phase (acetonitrile/water is 65/35, v/v) for 3min and 10-15 min respectively;

(2) The chromatographic conditions are as follows: column chromatography, ODS (C18) column 4.6X 250mm, 5 nm; column temperature, normal temperature; sample and mobile phase, methanol/water 65/35 (v/v); flow rate, 1.0 mL/min; sample size, 20 μ L; ultraviolet detection wavelength is 227 nm;

2. mass spectrometer model AB sciex 4500QQQ (triple quadrupole liquid mass spectrometer)

further structural identification of the paclitaxel analogs extracted in example 4 and preliminarily identified in example 5 was performed by using a liquid chromatograph-mass spectrometer (table 2), which indicates that the parent ion Da is 609.200, the daughter ions Da are 549.200, 367.000 and 427.100, the source voltage is 146, the collision energies are 34, 40 and 40, respectively, and the paclitaxel analogs are completely consistent with the taxane baccatin iii standard. Thus, taxol analogs produced by endophyte BP12 ± 3 were characterized as baccatin iii.

TABLE 2 Mass Spectrometry parameters for baccatin III standards and BP12 + -3 Taxol Compounds

Q1 Mass(Da)	Q3 Mass(Da)	DP	CE	Dwell(msec)
					609.200	549.200	146	34	50
609.200	367.000	146	40	50
					609.200	427.100	146	40	50

Example 8: influence of different precursor substances on generation of taxane compound baccatin III from Aspergillus niger BP12 + -3

1. 3 precursor solutions of sodium benzoate, sodium acetate and L-phenylalanine are prepared and sterilized for 20min under 0.08 MPa;

2. Inoculating Aspergillus niger BP12 + -3 into a PDA liquid culture medium, fermenting and culturing at 28 deg.C and 180rpm for the 5 th day, adding 3 precursors into the fermentation culture medium respectively, wherein one bottle is not added with any precursor as a control, the addition amounts of the precursors are 3.0mg/L of L-phenylalanine, 3.5g/L of sodium acetate and 32.0mg/L of sodium benzoate respectively;

3. Adding sucrose solution into each fermentation medium, wherein the final concentration is 5.0 g/L;

4. After culturing to the 8 th day, the crude product of Aspergillus niger BP12 + -3 metabolism was extracted by the method described in example 4, and the taxane compound baccatin III in the metabolic extract was qualitatively and quantitatively detected by High Performance Liquid Chromatography (HPLC), and the growth rate of the taxane compound baccatin III was calculated, and the results are shown in Table 3. The yield of baccatin III was increased most with an increase of 129.84% using L-phenylalanine as precursor. The result shows that the L-phenylalanine can be used as an effective precursor substance for culturing Aspergillus niger BP12 +/-3 and efficiently producing the taxane compound baccatin III.

TABLE 3 Effect of different precursors on the production of the taxane compound baccatin III by Aspergillus niger BP12 + -3

Claims

1. An endophyte producing taxane compound baccatin III, which is characterized in that: the endophyte is Aspergillus niger BP12 +/-3, and the preservation number is CCTCC NO: m2016319.

2. The method for culturing the endophyte producing the taxane baccatin III according to claim 1, wherein the culture comprises: adding L-phenylalanine into PDA culture medium.

3. The use of the endophyte of claim 1 in the preparation of a medicament for the treatment of a tumor cell, wherein the tumor cell is a cervical cancer cell.