CN101486974B - Endogenetic fungus for producing paclitaxel - Google Patents
Endogenetic fungus for producing paclitaxel Download PDFInfo
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- CN101486974B CN101486974B CN2009100714320A CN200910071432A CN101486974B CN 101486974 B CN101486974 B CN 101486974B CN 2009100714320 A CN2009100714320 A CN 2009100714320A CN 200910071432 A CN200910071432 A CN 200910071432A CN 101486974 B CN101486974 B CN 101486974B
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Abstract
The invention discloses a taxol-producing endophytic fungus and relates to taxol-producing fungi. The invention provides a new taxol-producing endophytic fungus and increases the number of strains that can be used in microbial fermentation of taxol. The taxol-producing endophytic fungus HD86-9 belongs to aspergillus niger yew variants and is preserved in China Center for Type Culture Collection with the preservation date of December 25th, 2006 and the preservation number of CCTCC NO: 206137. The taxol-producing endophytic fungus HD86-9 belongs to a new aspergillus niger yew variant under the analysis of morphology, 18S rDNA sequence and ITS. The invention not only provides an additional taxol-producing endophytic fungus, but also establishes physical foundation for the genetic modification of taxol-producing microorganisms in future.
Description
Technical field
The present invention relates to a kind of paclitaxel produced fungi.
Malignant tumour is seriously threatening human beings'health, and cancer is the second largest cause of the death that is only second to cardio-cerebrovascular disorder in the present human diseases.
Taxol is the diterpene alkaloid class medicine with multiple antitumor curative effect that extracts from Chinese yew genus plants.Because Ramulus et folium taxi cuspidatae (having another name called Japanese yew) is a rare tree species; the speed of growth is slow; and its biological characteristics is had relatively high expectations to ecotope; the natural regeneration ability; so population quantity is very limited; belong to protective plant in imminent danger, resource is very limited, so Chinese scholars is all at the source new drugs of researching and developing taxol energetically.
Microbial fermentation production is the Perfected process that reduces cost, obtains in a large number taxol.Chemist Andrea Stierle of U.S. Meng Tana state university in 1993 and phytopathologist Gary Strobel are separated to a strain endogenetic fungus Taxomycesandreanae in the yewtree in park, country glacier, the Meng Tana northwestward, working once delivering of Stierle etc., 1,000,000 dollars of these researchs of supporting Meng Tana state universities of just bidding of U.S. cell clone company.Because the huge medical value of taxol causes the numerous and confused research that adds paclitaxel produced microorganism of scholar both domestic and external, but it is still very few to separate the bacterial strain that can be used for the taxol microbial fermentation that obtains at present, is difficult to satisfy the needs of market and research.
The objective of the invention is increases the bacterial strain quantity that can be used for the taxol microbial fermentation for a kind of new paclitaxel produced endogenetic fungus is provided.
The paclitaxel produced endogenetic fungus HD86-9 (Aspergillus niger var.taxi HD86-9) of the present invention belongs to aspergillus niger Japanese yew mutation (Aspergillusniger var.taxi D.P.Zhou, K.Zhao et W.X.Ping, var.nov), be preserved in Chinese typical culture collection center, preservation date is on December 25th, 2006, and preserving number is CCTCC NO:M 206137.
The paclitaxel produced endogenetic fungus HD86-9 of the present invention adopts existing conventional fermentation process and fermentation condition to carry out paclitaxel produced fermentation, and taxol output is 273.46 μ g/L in the fermented liquid.
It is that (Aspergillus niger var.taxi D.P.Zhou, K.Zhao et W.X.Ping var.nov), belong to novel species to the mutation of aspergillus niger Japanese yew that the paclitaxel produced endogenetic fungus HD86-9 of the present invention analyzes through morphology, 18S rDNA sequence and ITS.The present invention has not only increased a kind of paclitaxel produced endogenetic fungus, and is the genetic modification of paclitaxel produced microorganism in the future, has established basic substance.
The paclitaxel produced endogenetic fungus HD86-9 (aspergillus niger Japanese yew mutation HD86-9Aspergillusniger var.taxi HD86-9) of the present invention belongs to aspergillus niger Japanese yew mutation (Aspergillus niger var.taxi D.P.Zhou, K.Zhao et W.X.Ping, var.nov), be preserved in Chinese typical culture collection center (CCTCC), the preservation address is a Wuhan University, preservation date is on December 25th, 2006, and preserving number is CCTCC NO:M 206137.
Description of drawings
Fig. 1 is paclitaxel produced endogenetic fungus HD86-9 colonial morphology figure; Fig. 2 is the observation by light microscope figure (* 1000 times) of paclitaxel produced endogenetic fungus HD86-9 conidial head; Fig. 3 is the electron microscope observation figure of the endogenetic fungus HD86-9 conidial head of taxol; Fig. 4 is the paclitaxel produced conidial sem observation figure of endogenetic fungus HD86-9 (* 5000 times); Fig. 5 is the constructed phylogenetic tree of 18S rDNA of the close bacterial strain of including among paclitaxel produced endogenetic fungus HD86-9 and the Genbank; Fig. 6 is the constructed phylogenetic tree in ITS district (comprising 5.8S rDNA) of the close bacterial strain of including among paclitaxel produced endogenetic fungus HD86-9 and the Genbank.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the paclitaxel produced endogenetic fungus HD86-9 (Aspergillusniger var.taxi HD86-9) of present embodiment belongs to aspergillus niger Japanese yew mutation (Aspergillus niger var.taxi D.P.Zhou, K.Zhao et W.X.Ping, var.nov), be preserved in Chinese typical culture collection center, preservation date is on December 25th, 2006, and preserving number is CCTCC NO:M 206137.
Paclitaxel produced endogenetic fungus HD86-9 has high homology between various in 18S rDNA sequence alignment (ncbi database) and Aspergillus Aspergillus, homology is more than 98%, therefore confirms that paclitaxel produced endogenetic fungus HD86-9 is nearer with the sibship of Aspergillus Aspergillus on the evolution status.
The constructed phylogenetic tree of the 18S rDNA of the close bacterial strain of including among paclitaxel produced endogenetic fungus HD86-9 and the Genbank as shown in Figure 5.The constructed phylogenetic tree in the ITS district of the close bacterial strain of including among paclitaxel produced endogenetic fungus HD86-9 and the Genbank (comprising 5.8S rDNA) as shown in Figure 6.
Embodiment two: the difference of present embodiment and embodiment one is: paclitaxel produced endogenetic fungus HD86-9 is the endogenetic fungal bacterial strain in taxus chinensis in northeast (the Taxus cuspidata Sieb.et Zucc.) bark.Other is identical with embodiment one.
The paclitaxel produced endogenetic fungus HD86-9 of present embodiment separates from taxus chinensis in northeast (Taxus cuspidataSieb.et Zucc.) bark and obtains.The paclitaxel produced endogenetic fungus HD86-9 of present embodiment separates acquisition according to the following steps: one, that the taxus chinensis in northeast bark is first with aseptic water washing (removing the impurity such as dust on bark surface), be alcohol-pickled 3～10min of 75% again with mass concentration, use aseptic water washing (alcohol is removed in flushing) then, be cut into the taxus chinensis in northeast bark fritter that area is 0.5cm * 0.5cm with sterile scissors; Two, taxus chinensis in northeast bark fritter is planted respectively behind PDA substratum, Cha Shi (Czapek) substratum and Martin (Martin) nutrient agar flat board that constant temperature culture obviously grows mycelia around taxus chinensis in northeast bark fritter under 25 ℃ of conditions; Three, adopt most advanced and sophisticated mycelia picking method picking colony to be inoculated in the PDA solid medium, under 28 ℃ of constant temperatures, cultivated 3～5 days, the dull and stereotyped upward fast growth of selection, single bacterium colony that colony diameter is big, be inoculated in that improvement S-7 substratum lucifuge on 25 ℃, the constant temperature shaking table of 120r/min is cultivated, fermentation 12 days, adopt thin-layer chromatography and high performance liquid chromatography to carry out qualitative and quantitative analysis respectively afterwards, choose paclitaxel produced microorganism and obtain the paclitaxel produced endogenetic fungus HD86-9 of present embodiment.
The improvement S-7 substratum that uses in the paclitaxel produced endogenetic fungus HD86-9 sepn process of present embodiment is at people such as Sterle (Sterle A, Strobel G, Stierle D.Taxol and taxant production byTaxomyces andreanae, an endophttic fungus of pacific yew.Science, 1993,260 (9): 214-216) on the former S-7 medium base phenylalanine, tyrosine, linolic acid are increased to final concentration 1.5～5.0mg/L.
Embodiment three: present embodiment and embodiment one or twos' difference is: paclitaxel produced endogenetic fungus HD86-9 bacterium colony quality is the fine hair shape, radial rill is arranged that the bacterium colony reverse side is deep to tawny or chocolate for yellow, color ecto-entad add gradually; Paclitaxel produced endogenetic fungus HD86-9 bacterial strain has aerial hyphae, the conidium structure of paclitaxel produced endogenetic fungus HD86-9 is a large amount of, conidial head is carbon black look, sphere, and the conidial head diameter is 150～500 μ m, and the top capsule is 30～80 μ m for spherical or subsphaeroidal, diameter.Other is identical with embodiment one or two.
Split into several cylindrical-shaped structures after the conidial head of paclitaxel produced endogenetic fungus HD86-9 is old, diameter can reach 800 μ m or bigger.
Paclitaxel produced endogenetic fungus HD86-9 cultivated 5 days under Cha Shi yeast nutrient agar, 25 ℃ of conditions, and bacterium colony is smooth, diameter is 43～60mm.
The bacterium colony colonial morphology of paclitaxel produced endogenetic fungus HD86-9 is (PDA substratum) as shown in Figure 1, the conidial head of observing paclitaxel produced endogenetic fungus HD86-9 under opticmicroscope as shown in Figure 2, conidial head such as Fig. 3 of observing paclitaxel produced endogenetic fungus HD86-9 under electron microscope show, the conidium of observing paclitaxel produced endogenetic fungus HD86-9 under scanning electronic microscope as shown in Figure 4.
Embodiment four: the difference of present embodiment and embodiment three is: the top capsule all surfaces of paclitaxel produced endogenetic fungus HD86-9 can be educated, and conidial fructification is double-deck; Paclitaxel produced endogenetic fungus HD86-9 metulae is of a size of 12～35 μ m * 5～10 μ m, is brown when old; Paclitaxel produced endogenetic fungus HD86-9 bottle stalk is of a size of 8～12 μ m * 2～5 μ m; Conidium has irregular ridge projections for spherical, diameter are that 20～33 μ m, wall are coarse.Other step and parameter are identical with embodiment three.
The metulae of paclitaxel produced endogenetic fungus HD86-9 increases along with the increase of cell age in the certain hour scope, is generally 12～35 μ m * 5～10 μ m, and big person is 80 μ m.The conidium of paclitaxel produced endogenetic fungus HD86-9 is compared with aspergillus niger and enlarges markedly, and the conidium wall is obviously coarse.
Embodiment five: the paclitaxel produced endogenetic fungus HD86-9 of present embodiment prepares taxol according to the following steps: 1) thalline activation and seed culture: paclitaxel produced endogenetic fungus HD86-9 is inoculated in the PDA inclined-plane, 28 ℃ of activation 2～3 times; Bacterial classification after the activation is connected to PDA liquid nutrient medium (50mL/250mL triangular flask) then, and shaking table is cultivated (seed culture) under 28 ℃, 120r/min condition; 2) fermentation culture: seed culture fluid is changed in the S-7 substratum (the 200mL/500mL triangle shakes bottle) after the improvement with 3% (v/v) inoculum size, under 28 ℃, 120r/min condition, cultivate, establish 3 parallel samples for every group; 3) taxol extracts: shake flask fermentation is cultivated extractive fermentation liquid after 12 days, and filtrate and mycelium all reclaim, and at twice filtrate are extracted with isopyknic ethyl acetate, and the static 1h layering of separating funnel is dissolved with taxol in the upper organic phase, and lower floor's water discards; Grind mycelium simultaneously and add the ethyl acetate of 30mL, concussion extraction 30min filters, merge organic phase and remove ethyl acetate with Rotary Evaporators, treat remaining 5mL solution, solution is poured in the plate, and 50 ℃ of dryings are washed the taxol of the trace on plate surface repeatedly with a spot of acetonitrile, fixed molten to 2mL, preserve or detect for 4 ℃.
Taxol output is 273.46 μ g/L in the fermented liquid of the endogenetic fungus HD86-9 that present embodiment is paclitaxel produced.
Claims (1)
1. paclitaxel produced endogenetic fungus, it is characterized in that paclitaxel produced endogenetic fungus HD86-9 (Aspergillus niger var.taxi HD86-9) belongs to aspergillus niger Japanese yew mutation Aspergillus niger var.taxi D.P.Zhou, K.Zhao et W.X.Ping, var.nov, be preserved in Chinese typical culture collection center, preservation date is on December 25th, 2006, and preserving number is CCTCC NO:M 206137.
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| CN2009100714320A CN101486974B (en) | 2009-02-24 | 2009-02-24 | Endogenetic fungus for producing paclitaxel |
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