CN101386819B - Saprophytic fungus for producing paclitaxel and method for producing paclitaxel using thereof - Google Patents
Saprophytic fungus for producing paclitaxel and method for producing paclitaxel using thereof Download PDFInfo
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- CN101386819B CN101386819B CN2008101525001A CN200810152500A CN101386819B CN 101386819 B CN101386819 B CN 101386819B CN 2008101525001 A CN2008101525001 A CN 2008101525001A CN 200810152500 A CN200810152500 A CN 200810152500A CN 101386819 B CN101386819 B CN 101386819B
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Abstract
The invention relates to a saprophytic fungus Pestalotiopsis microspora NK17 (with a culture preserving number of CGMCC2694) capable of producing taxol, which belongs to the technical field of bioengineering. The strain is a saprophytic fungus which is found in Pestalotiopsis for the first time and can produce the taxol by the fermentation method, and the product is the taxol which is the most efficient anticancer medicine. The invention also relates to a method for producing the taxol by application of the strain, which comprises the following steps: culturing the strain in a culture medium, producing and collecting the taxol in cells of the strains and the culture medium, and reclaiming and purifying the taxol in the cells and the culture mediu. The method has high taxol yield, low production cost and short fermentation period, can completely solve the problem of producing the taxol, and not only protect natural resources but also can meet the market demand.
Description
[technical field]: the invention belongs to biotechnology and biological pharmacy technical field.Be specifically related to the saprophytic fungus plan dish stey that a strain can be paclitaxel produced, and use the method that this strain fermentation is produced taxol.
[background technology]: taxol (paclitaxel, trade(brand)name Taxol) is a kind of natural cancer therapy drug of complexity, belong to diterpene alkaloid, its basic structure by Baccatine III (baccatin III) be connected its 13 carbon on a phenylalanine derivative constitute.
The early stage strategy of industrial production taxol, be to adopt the method for from the Chinese yew wood raw material, directly extracting, because content of taxol is extremely low, the 0.0001-0.008% that has only the trees dry weight, the taxol water-soluble is poor in addition, mean yield is about 0.015%, produces 1 kilogram of taxol, generally needs 7000 kilograms, is equivalent to the raw materials of 2000-2500 Ramulus et folium taxi cuspidatae trees.With 1992 be example, Bristol-Myers-Squibb (BMS) company has consumed 1,200,000 kilograms Ramulus et folium taxi cuspidatae for clinical trial provides 16 kilograms taxol.Someone estimates, treats the Chinese yew that an ovarian cancer patients needs 6 age of tree 60-100.Along with the expansion of this pharmaceutical applications scope, required Ramulus et folium taxi cuspidatae quantity will be very considerable, and this also brings serious threat to taxus resource, may cause the havoc of ecotope.
The complete synthesis taxol of chemical method has under lab been obtained success in 1994.But the taxol chiral radicals is more, complex synthetic route, and cost height, productive rate are low, can't be applied to industrial production so far.In recent years, Chinese scholars has been carried out a large amount of taxus callus and has been cultivated and cell suspension culture research, the yew cell system of cultivating is above 10, wherein major part has confirmed to produce taxol, but isolated culture is produced taxol and is realized that the key issue of suitability for industrialized production is difficult to break through, and for example: the culture biomass is little, and taxol yield is lower, production cycle is long, and needs to keep cell growth and stablizing of production characteristic or the like.
After Christen utilized vegetable cell suspension culture technology to produce taxol first, research had attracted numerous laboratory follow-ups, and the research of this respect has also obtained certain progress.For example: ESC Agenetics company announces that in the taxol symposial second time that NCI in 1992 presides over they are 2-5 times of content in the bark with cell culture method products therefrom content of taxol.The sweet tired far away discovery taxusyunnanensis cell increment in fermentor tank that waits of China reaches 12g/L, and content of taxol is 0.119%, is about 12 times of content in the bearing tree bark, for cultivating 40 times of plant.But there are some problems equally in this technology, and as the brownization problem of culturing cell, though the report of practical application is not seen in the test that has this technology to enter the bio-reactor amplification stage, the possibility of successful Application is less in a short time.
The current production technology that more generally adopts of international drugmaker then is biochemical semi-synthesis method.The molecular biology research that taxus chinensis in northeast (Taxus cuspidate) taxol is generated shows; taxol synthesizes and is broadly divided into three main biosynthesizing stages: 1) taxane-ring baccatin III parent nucleus intermediate is synthetic; 2) C-13 position side chain intermediate is synthetic; 3) acylation reaction of taxane-ring and side chain forms complete taxol molecule.Baccatin III and C-13 position side chain intermediate are independently synthetic, can stable existence, and water-soluble better.According to these characteristics, utilize biological enzyme that the Ramulus et folium taxi cuspidatae raw material is handled, make baccatin III and C-13 position side chain intermediate obtain to a certain degree enrichment, extract these more diffluent precursors then, utilize enzyme process or chemical method at last the synthetic finished product taxol of these precursors.Though semi-synthesis method has improved taxol output, there is no difference in essence with the way of direct extraction taxol, still need to consume a large amount of Ramulus et folium taxi cuspidatae trees, BMS company has opened up 3,000 ten thousand strain Ramulus et folium taxi cuspidatae plantations for this reason.
Because the market supply of taxol is limited by the restriction of raw material sources and technology of preparing two aspects, makes that the medicine high price is expensive.For this reason, new better raw material and method are being sought always by pharmaceutical industry and academia, replace existing production technology, with the output that improves taxol, satisfy clinical demand.The most significant progress that obtained in surplus past ten year is to have found to produce taxols with more symbiotic fungies of naked sub-xylophyta such as Ramulus et folium taxi cuspidatae, and its chemical structure and biological activity and Ramulus et folium taxi cuspidatae paclitaxel produced identical.
1993, Stierle etc. are separated to 1 strain endosymbiosis fungi-An Delie Japanese yew bacterium (Taxomycesandreanae) from the phloem of Pacific Ocean Ramulus et folium taxi cuspidatae, through cultivating, adopt methods such as mass spectrum, chromatogram (TLC/HPLC) and radio chemistry mark, find to have taxol in the fermented liquid of this bacterium, although output is lower, every liter of fermented liquid contains taxol 24-50ng.Confirm also that simultaneously taxol structure and character are just the same in these mycetogenetic taxols and the Ramulus et folium taxi cuspidatae.Subsequently, many people are separated to different paclitaxel produced endosymbiosis fungies.China has also found multiple paclitaxel produced endosymbiosis fungi within the border.According to statistics, the generation taxol fungal species of having reported surpasses 30 kinds both at home and abroad, and these fungi overwhelming majority are ascomycetes or imperfect fungi, and all are the endosymbiosis fungies.Because the content of taxol is very low in these fungus fermentation products at present, can not reach the level that industrial fermentation is produced, therefore can not satisfy the demand in market far away.
In sum, the subject matter that existing taxol production technology exists is: (1) adopts the method for directly extracting from the Chinese yew wood raw material, because content of taxol is extremely low, a large amount of felling of Ramulus et folium taxi cuspidatae have caused the havoc of ecotope.(2) the complete synthesis taxol of chemical method because the taxol chiral radicals is more, complex synthetic route, cost height, productive rate are low, can't be applied to industrial production so far.(3) biochemical semi-synthetic taxol though improved taxol output, there is no in essence difference with the method for direct extraction taxol, still needs to consume a large amount of Ramulus et folium taxi cuspidatae trees.(4) vegetable cell suspension culture technology is produced taxol, and the culture biomass is little, and taxol yield is lower, and the production cycle is long, and brownization easily takes place culturing cell, and needs to keep the stable of cell growth and production characteristic.(5) utilize the endosymbiosis fungi fermentation to produce taxol, the content of taxol is very low in the tunning, can not reach the level that industrial fermentation is produced, and therefore can not satisfy the demand in market far away.
Based on taxol production and demands status, world market is badly in need of the novel method of energy suitability for industrialized production taxol.
[summary of the invention]: the objective of the invention is to overcome the prior art above shortcomings, the new bacterial strain that a strain can be paclitaxel produced is provided, and utilize this bacterial strain to produce the method for taxol.
The paclitaxel produced new bacterial strain of energy provided by the invention is a strain saprophytic fungus plan dish stey Pestalotiopsis microspora NK-17 (culture presevation CGMCC2694).Bacterial strain of the present invention is for finding first both at home and abroad.Therefore producing taxol with the method for this kind strain fermentation is that one of approach of application prospect is arranged most.
The present invention finds and has cultivated a kind of saprophytic fungus through a large amount of for many years further investigations, and this bacterium can produce taxol, and the inventor works out the method for utilizing this bacterial classification to come the fermentative production taxol through a large amount of experiments simultaneously.
This bacterium taxol output is higher, and fermentation condition is controlled easily, and culture cycle is short, is the dominant bacteria that a strain industrial fermentation is produced taxol.
The present invention separates obtaining this microorganism by the method from the nature separate microorganism.This microorganism is cultivated, and preparation fermentation crude extract uses thin-layer chromatography (TLC), high performance liquid chromatography (HPLC), and LC-MS mass spectroscopy (LC-MS) and nucleus magnetic resonance methods such as (NMR) are analyzed tunning and are identified.Utilize morphology and molecular biology method that this microorganism has been carried out the kind evaluation, it is accredited as the plan Pestalotia, called after NK-17.
The invention provides from the nature separation and obtain this method of microorganism.
The present invention utilizes morphology and molecular biology method that this microorganism has been carried out the kind evaluation.
The invention provides the preparation method of this method for culturing microbes and fermentation crude extract.
The invention provides the method that this microbial fermentation product is analyzed and identified, prove that this microorganism can produce taxol.
With embodiment separation method, kind authentication method, the cultural method of this microorganism among the present invention, the preparation method and the contents such as tunning analysis and authentication method of fermentation crude extract are described below.
Advantage of the present invention and positively effect:
The invention provides the saprophytic fungus NK-17 that a strain can be paclitaxel produced.
Microorganism provided by the invention is the paclitaxel produced saprophytic fungus of finding both at home and abroad of first strain, belongs to and intends Pestalotia, compares with known paclitaxel produced endosymbiosis fungi, has novelty and practicality.This microbial fermentation cycle is short, the output height.The known paclitaxel produced endosymbiosis fungi fermentation cycle is long, generally is 2-3 weeks, and taxol yields poorly, and zymotechnique is difficult to grasp.This just means, might realize the large scale fermentation production of taxol with bacterial strain of the present invention.
Compare with other taxol production method, as the method for from the Chinese yew wood raw material, directly extracting, the method of the complete synthesis and biochemical semi-synthetic taxol of chemical method, vegetable cell suspension culture technology is produced the method for taxol, and microorganism provided by the invention can pass through fermentation culture direct production taxol, its production cost is low, can not damage ecological resources, zymotechnique is easy to grasp, and follow-up purification procedures is simple, therefore the productive rate height all has distinct comparability on some at this.
[description of drawings]:
Fig. 1 is the conidium form of bacterial strain NK-17 provided by the invention;
Fig. 2 is high performance liquid chromatography (HPLC) analytical procedure purifying and identifies contained taxol in the NK-17 tunning, Fig. 2-1, taxol standard substance (12.847min), Fig. 2-2, testing sample (12.840min);
Fig. 3 is that liquid-matter coupling (LC-MS) analytical procedure is identified contained taxol in the NK-17 tunning;
Fig. 4 is that nucleus magnetic resonance (NMR) analytical procedure is identified contained taxol in the NK-17 tunning, Fig. 4-1, taxol standard substance, Fig. 4-2, testing sample.
The paclitaxel produced new bacterial strain of energy provided by the invention is a strain saprophytic fungus plan dish stey, be preserved in Chinese common micro-organisms culture presevation administrative center on October 8th, 2008, preservation address: No. 3 Institute of Microorganism, Academia Sinica in A, DaTun Road, Chaoyang District, BeiJing City, culture presevation CGMCC2694, classification name: Pestalotiopsis microspora NK17.
[embodiment]:
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to " molecular cloning laboratory manual " that the people showed (New York:Cold Spring Harbor Laboratory Press such as normal condition such as Sambrook, 1989) condition described in, or the condition of advising according to manufacturer.
The separation method of embodiment 1, microorganism strains NK-17 provided by the invention
The useless cloth that derives from place, south China city is cut into 1 * 1cm fritter; It is that 75% ethanol soaks and carried out sterilization and disinfection in 3 minutes that this sample is put into percent by volume; Be inoculated in PDA flat board (every plate 3-4 piece sample) after cleaning with sterilized water, cultivate after 3-4 days for 28 ℃ and observe; When treating tentatively to grow bacterium colony, colony inoculation is in the PDA slant culture preferably to select growing way, and purifying is preserved.The PDA culture medium prescription (/L): potato 200g, sucrose 20g, agar 20g (adding in the solid medium).
The kind of embodiment 2, microorganism strains NK-17 provided by the invention is identified
(1) morphological method is identified
With the NK-17 strain culturing about 30 days, treat that bacterium colony grows conidium after, carry out microscopy.Show by colonial morphology and conidial electron micrograph (experimental result is seen Fig. 1), this microorganism to intend Pestalotia (Pestalotiopsis) morphology on very similar.
(2) molecular biology method is identified
By design primer UK4F (5 '-cyggttgatcctgccrg-3 ') and UREV (5 '-gytaccttgttacgactt-3 '), we use PCR method that the 18SrDNA of this microorganism is increased, and have obtained the fragment of a long 1631bp.Compare by sequence among check order back and the Genbank, find to have reached 99% (sequence results is seen sequence table) with the similarity of Pestalotiopsis microspora 18S rDNA.
According to the result of embodiment 2, we are that Pestalotiopsis belongs to called after NK-17 with this microorganism identification.
The cultural method of embodiment 3, microorganism NK-17 provided by the invention
The preparation of substratum is prepared by method related among the embodiment 1.In the solid culture process, culture temperature is 28 ℃, obtains conidium as need, needs cultured continuously about 30 days; In the liquid culture process, culture temperature is 28 ℃, rotating speed 200rpm, obtain taxol in a large number as need, incubation time need reach about 15 days, because the product taxol is stable between pH4-8, therefore need monitor pH during the fermentation, it is within this scope.
Embodiment 4, microorganism NK-17 tunning pre-treatment process provided by the invention
Tunning is carried out suction filtration, make mycelia separate with fermented liquid; With gained mycelia lyophilize behind the suction filtration, used the methyl alcohol lixiviate 1-2 days after the liquid nitrogen grinding smudge cells; With leach liquor with carry out suction filtration once more after before fermented liquid mixes, disgorging, and regulate pH to 7.0; Slightly carried product after using rotatory evaporator that gained filtrate of last step is concentrated, carried out next step analysis and evaluation.
The analysis and the evaluation of contained taxol in embodiment 5, the microorganism NK-17 tunning provided by the invention
The tunning of NK-17 analyzed with following method and identified:
(1) thin-layer chromatography (TLC)
Use the exhibition coating systems of methylene dichloride: ethyl acetate=6:1, product is slightly carried in NK-17 fermentation to launch, on the GF254 silica-gel plate, can show tangible dim spot by UV-irradiation, by comparing with the taxol standard substance, find that both have identical Rf value, prove that both are same substance.
(2) high performance liquid chromatography (HPLC)
(4.6 * 250mm, Kromasil), chromatographic condition: detect wavelength 227nm, flow velocity 1mL/min, column temperature are room temperatures, and moving phase is methyl alcohol: water=7:3 to select analysis mode C-18 ODS chromatographic column.Show by experiment, contain the material (experimental result see Fig. 2) identical in the tunning with taxol standard substance retention time.(1. taxol standard substance, 12.847min; 2. NK17 tunning, 12.840min.).According to going out peak area, can calculate the content of taxol in the tunning.
(3) mass spectrum (LC-MS)
Used among the embodiment and carried out liquid-matter coupling through the sample behind the HPLC purifying and detect on a small quantity.Experimental result shows, the main component molecular weight [M+Na] in the sample
+=876, can draw [M]=853 thus, identical with the molecular weight of taxol standard substance, can illustrate that both are same substance (experimental result is seen Fig. 3).
(4) nucleus magnetic resonance (NMR)
Used among the embodiment and carried out magnetic resonance detection in a large number through the sample behind the HPLC purifying.Show that by experiment testing sample has identical response signal with the taxol standard substance, confirm that from molecular structure both are same substance (experimental result is seen Fig. 4).
Sequence table
＜110〉Nankai University
＜120〉the paclitaxel produced saprophytic fungus of a strain and use the method that this bacterial strain is produced taxol
<160>1
<210>1
<211>1631
<212>DNA
＜213〉plan dish stey (Pestalotiopsis microspora NK17)
<220>
<221>gene
<222>(1)...(1631)
<400>1
Claims (8)
1. the paclitaxel produced saprophytic fungus of a strain, its classification called after is intended Pestalotia (Pestalotiopsis microspora) NK17, culture presevation number: CGMCC2694.
2. method of using the described saprophytic fungus of claim 1 to produce taxol, it is characterized in that: in substratum, cultivate the described saprophytic fungus of claim 1 so that produce in described strain cell and in the described substratum and the gathering taxol, and in described cell and the substratum, reclaim and purification of paclitaxel.
3. method according to claim 2 is characterized in that described substratum is the PDA substratum of nature pH, and it consists of g/L: potato 180-220g, sucrose 20g, solid add the 20g agar powder.
4. method according to claim 2 is characterized in that described cultivation is under aerobic conditions to carry out, and temperature is 25-30 ℃, and the fermentation initial pH value is PDA substratum nature pH.
5. method according to claim 2 is characterized in that described bacterial strain fermentation culture in 500mL-1 L triangular flask, and vaccination ways adopts the vaccination ways of block mycelia.
6. method according to claim 2 is characterized in that the step of described recovery and purification of paclitaxel comprises:
(1) isolates fermentation thalline and fermented supernatant fluid from the gained nutrient solution;
(2) with the thalline lyophilize, liquid nitrogen grinding is used the methyl alcohol lixiviate; With leach liquor with carry out suction filtration, disgorging after fermented supernatant fluid mixes; Use rotatory evaporator that gained filtrate is concentrated, slightly carried product;
(3) (2) step gained is slightly carried the method purifying of product with chromatogram purification, get the product taxol.
7. method according to claim 6 is characterized in that, uses saturated Na in (2) step
2CO
3It is 6.0-8.0 that solution is regulated the gained pH value of filtrate.
8. method according to claim 6 is characterized in that: in the described chromatogram purification method of (3) step, use one or more chromatographic column, filler is C18, and moving phase is methyl alcohol/H
2O.
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| CN102191182B (en) * | 2010-11-09 | 2013-03-13 | 浙江省农业科学院 | Method for identifying pathogen of paroxysmal foliage wilting disease in myrica rubra |
| CN104017840A (en) * | 2014-06-27 | 2014-09-03 | 南开大学 | Method for producing pestalotiollide A by Pestalotiopsis microspora liquid fermentation |
| CN107034145B (en) * | 2016-12-30 | 2020-07-28 | 广东海洋大学 | Desmopsis virescens CAMT66351 and application thereof |
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| 戴水莲等.PDA培养基中加入青霉素、链霉素的抗菌作用试验简报.中国食用菌.2007,26(4),53-54. * |
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