New strain and method of producing pacilitaxel using said strain

A paclitaxel and variant technology, applied in the directions of using fungi, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of difficulty in industrial production, waste of land and manpower, low yield of paclitaxel, etc., and can meet clinical drug requirements. needs, solving process problems, and protecting the effect of natural resources

Active Publication Date: 2006-01-25
SUNGEN BIOSCIENCE CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are following explorations: (1) chemical semi-synthesis (Holton, R.A, Somoza, C., Kim, H-B., Liang, F., et al., J.Am.Chem.Soc., 1994,116 , 1597; 1994, 116, 1599; Nicolaou, R.C., Yang, z., Liu, J.J., et al., Nature, 1994, 367, 630; Danishefskky, S.J., Masters, et al., Angew.Chem, Int. Ed.Engl., 1995, 34, 1723; Wende, A., Badham NeilF., et al., J.Am.Chem.Soc., 1997, 119, 2755-2757): the United States in early 1996, has canceled the tree The bark extraction method is used to produce paclitaxel, and the chemical semi-synthesis method is used instead. This method is mainly to extract the paclitaxel intermediate 10-deacetyl1-baccatin (10-DAB) in the leaves and bark of yew, and then synthesize it through 2-4 steps of chemical reaction Paclitaxel, although it solves the problem of ecological protection, still needs a large number of plantations to support, which consumes a lot of land and manpower. From a long-term perspective, this method is still not advisable
(2) chemical synthesis (DenisJN, Greene AE, Guenard D, et al., J.Am.Chem.Soc., 1988,110,2917; Kingston DG, Chaudhary AG, Gunatilaka AAL, et al., Tetrahedron Lett. , 1994, 35, 4483; Kanazawa AM, Denis JN, GreeneAE, J.Chem.Soc.Chem Commun, 1994, 2591; SISTI, Nicholas, J., WO 96 / 40667; Zhang Hongjie, CN 1241565A): After more than 20 years Years of hard work, in 1994, the total synthesis of paclitaxel was successful, but the entire chemical reaction required more than 20 steps, the efficiency was too low, and the cost was high, so it did not have the meaning of industrial production
(3) Taxus plant cell tissue culture (Huazhong University of Science and Technology, CN1096820A; Minoru Seki, Chiharu Ohzora, Mayuko Takede, Biotechnology and Bioengineering, Vol.53, Pp.214-219, 1997): This method has a long cultivation cycle and industrial production tougher
(4) Paclitaxel produced by microbial fermentation (Andrea Stierle, Gary Strobel, WO93 / 21338; Nippon Steel Corporation, WO95 / 04154; Novopharm, WO 96 / 32490; Gary strobel, Xianshu Yang, et al., Microbiology, 1996, 142, 435-440; Jianfend Wang, Guiling Li, et al., FEMS Microbiology Letters, 2000, 193, 249-253): The production of paclitaxel by this method is not limited by the scarcity of natural resources and ecological protection, but the currently international patented strain A kind of ubiquitous shortcoming that paclitaxel productive rate is low, is not suitable for industrialized large-scale production

Method used

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  • New strain and method of producing pacilitaxel using said strain
  • New strain and method of producing pacilitaxel using said strain
  • New strain and method of producing pacilitaxel using said strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Isolation of taxol-producing microorganisms from yew tree samples

[0040] We collected 310 samples from the branches, bark, leaves and root bark of seven Yunnan yew (Taxus yunnanensis) trees more than 300 years old in the mixed coniferous and broad-leaved forest at an altitude of 2500-3000 meters in Laojun Mountain, Lijiang area, Yunnan Province. Each was cut into small pieces of 1cm×1cm size, sterilized with 50-90% alcohol for 5 minutes, homogenized with a homogenizer, and then spread on PDA medium, cultured at 25°C for 4-5 days, and the grown The hyphae were transferred to the slope, cultured at 20°C-35°C for 4 days, and 1cm 2 Bacteria were inoculated in the fermentation medium (250ml Erlenmeyer flask capacity 100ml, medium formula: glucose 2%, soybean cake powder 0.5%, peanut cake powder 0.2%, MgSO 4 : 0.01%, KH 2 PO 4 : 0.05%), 25 ℃, 300rpm in the shaker culture 16 days, filter with double-layer gauze, get wet thallus, wet thallus is dried in oven (60 ...

Embodiment 2

[0041] Example 2 Purification and identification of new strains producing paclitaxel

[0042] Several paclitaxel-producing strains obtained in Example 1 were separated and purified on PDA medium plates for three generations (20-35°C, 4-10 days) to obtain the ST-026 strain. Strain ST-026 grows rapidly on PDA medium and produces abundant spores, which are usually black or olive green. Conidiophores grow directly from the agar medium, and branch conidia chains grow on the side shoots of the hyphae. The spore chains consist of 50-70 spores, the initial spores are produced directly from the sporophore, or from 1-2 spores are produced on the soil surface, spores are rod-shaped, oval-elliptic, about 22-31(25)X 9-13(11)UM in size, with 4-5 transverse septa, 2-3 ratio septa or oblique diaphragm. The initially formed young spores are narrowly oval, with dense, fine verrucous protrusions, such dense protrusions that the internal diaphragm of the spores cannot be seen clearly under a 10...

Embodiment 3

[0044] Example 3 Seed culture

[0045]Inoculate the obtained CGMCC No.0899 freeze-dried bacteria of Example 2 into a 250ml Erlenmeyer flask with 100ml seed culture medium (see Table 1), at 20~30°C, 100-300rpm shaker culture for 24-28 hours, to be ready Inoculated strains.

[0046] Element

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PUM

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Abstract

The invention relates to a new fungus that could produce taxales mellow-Alternaria alternate var.monosporus ST-026-R CGMCC No.0899. The invention also discloses the method to produce taxales mellow that includes the following steps: cultivating CGMCC 0899 in culture medium to produce and gather taxales mellow in the strain cell and the culture medium. Thus, the taxales mellow could be recycle and purify in the cell and culture medium. It could solve the technology problem of taxales mellow and protect natural resource.

Description

technical field [0001] The invention relates to a new bacterial strain and a method for producing paclitaxel by fermenting the bacterial strain. Background technique [0002] Paclitaxel (Paclitaxel, chemical name 5β, 20-epoxy-1,2α, 4,7β, 10β, 13α-hexahydroxytaxane-11-en-9-one-4,10-diacetate-2 - Benzoate-13[(2'R,3'S)-N-benzoyl-3-phenylisoserine ester]) is one of the best anticancer drugs in the world today, FDA (US Food and Drug Administration Bureau) has been approved for the treatment of ovarian cancer, breast cancer, non-small cell lung cancer and AIDS-related kaposi's sarcoma, with significant efficacy. Due to the unique pharmacological mechanism of paclitaxel (that is, promoting the aggregation of tubulin during cancer cell division, stabilizing microtubules, and finally causing cancer cell apoptosis), it can be used for polycystic kidney disease (Cutoclonal pharmaceuticals company in the United States), vascular restenosis (USA Boston Scientific Company) senile dement...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12P17/00
CPCC12N1/38C12P17/02C12R1/645C12N1/14C12N1/145C12R2001/645
Inventor 陈杰鹏邓士谨段丽丽王荣华赵素兰邱雪莲韦少明陈沛泽程首先郑卓君陈红敏李洁
Owner SUNGEN BIOSCIENCE CO LTD
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