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An endophytic fungus with high yield of paclitaxel and method for producing paclitaxel using the strain

A technology of endophytic fungi and paclitaxel, which is applied in the field of bioengineering and biopharmaceuticals to achieve the effects of easy fermentation process, simple separation and purification steps, and high yield

Active Publication Date: 2016-04-06
HUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the problem in the prior art that taxus-producing endophytic fungi with a wider species distribution range and higher paclitaxel yield still need to be screened from Taxus chinensis, the present invention has carried out a large amount of screening and research work on Taxus chinensis to obtain New endophytic fungi with high taxol production

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  • An endophytic fungus with high yield of paclitaxel and method for producing paclitaxel using the strain
  • An endophytic fungus with high yield of paclitaxel and method for producing paclitaxel using the strain
  • An endophytic fungus with high yield of paclitaxel and method for producing paclitaxel using the strain

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Embodiment 1

[0028] Example 1: Isolation of taxol-producing endophytic fungi

[0029] In this embodiment, the taxol-producing endophytic fungus J11 strain is isolated from the stem of Taxus chinensis var. mairei. The paclitaxel-producing J11 strain of the present embodiment is isolated and obtained according to the following steps: 1) Rinse the stems of Taxus chinensis with sterile water (to remove impurities such as dust on the surface of the bark), then soak them in 75% ethanol for 3 to 10 minutes, and then Rinse with sterile water (wash away ethanol), and then cut into small pieces with an area of ​​0.5cm × 0.5cm with sterile scissors; ~5 samples), put them in a 25-28°C incubator and cultivate them until colonies grow obviously around the small pieces; 3) Pick colonies with better growth and inoculate them on PDA solid medium, and cultivate them at a constant temperature of 28°C , purification, preservation.

[0030] The above PDA (potatodextroseagar) medium was prepared according to ...

Embodiment 2

[0032] Embodiment 2: the species identification of microbial bacterial strain J11

[0033] For the identification of paclitaxel-producing endophytic fungi, since the identification of fungal microorganisms needs to be based on sexual spores as the main basis for judgment, it is difficult in practice, especially for endophytic fungi that are difficult to produce sexual spores. It seems untenable. With the continuous development and improvement of modern molecular biology and its experimental methods, DNA barcodes are also used internationally as the main means and methods for identifying fungi. Ribosomal ITS (internal transcribed spacer) sequence and ribosomal LSU (large subunitrRNAgene) sequence as molecular tags (barcodes) are summarized by scientists after a large number of long-term experiments. Compared with other molecular tags, these two molecular tags are more reliable, and have universal significance for the molecular identification of fungi and endophytic fungi. The...

Embodiment 3

[0043] Embodiment 3: Identify whether the J11 strain produces paclitaxel

[0044] Thin layer chromatography (TCL) uses the Rf value and high performance liquid chromatography (HPCL) uses the retention time (retention time) as a method to identify paclitaxel-producing endophytic fungi, but due to the large error range, it has been gradually eliminated , only as a preliminary basis for judging paclitaxel-producing endophytic fungi, not as a final identification method. Although the nuclear magnetic resonance (nuclearmagneticresonancespectroscopy, NMR) method has reliable identification results and can identify the chemical results of paclitaxel, it involves the separation and purification of high-purity paclitaxel from the fermentation broth, such as the purification of high-purity paclitaxel by preparative liquid chromatography. It is complex and requires high experimental conditions. High resolution mass spectrometry (high resolution mass spectroscopy) has higher sensitivity ...

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Abstract

The invention discloses an endophytic fungus strain, which is capable of producing paclitaxel and is separated from young stems of taxus chinensis. The endophytic fungus strain is named as Botryosphaeria dothidea J11, is preserved in the China center for type culture collection and has an accession number of CCTCC NO: M2013591. The invention also relates to a method for producing paclitaxel by the preserved endophytic fungus strain. The method comprises the following steps of culturing the preserved endophytic fungus strain by a culture medium so that paclitaxel is produced and gathered in strain hyphae, recovering the paclitaxel in the cells and carrying out purification. The endophytic fungus strain provides a novel strain for microbial fermentation production of paclitaxel. Through the endophytic fungus strain, a paclitaxel fermentation production period is short and a cost is low. A HPLC analysis detection result shows that through the shake flask fermentation of the endophytic fungus strain, a paclitaxel yield has 617.6 microgrammes per liter. The endophytic fungus strain has a potential for use in industrial production.

Description

technical field [0001] The invention relates to a paclitaxel-producing fungus, in particular to a paclitaxel-producing fungus viticola and a method for producing paclitaxel by fermenting the strain, belonging to the technical fields of bioengineering and biopharmaceuticals. Background technique [0002] Paclitaxel (trade name: Taxol) is the most sold plant-derived anticancer drug in the world. Paclitaxel is a diterpenoid compound with complex structure, which is a natural product of Taxus genus. In 1963, American scientists Wall and Wani et al. isolated paclitaxel from the bark of Pacific short-leaved yew (Taxus brevifolia Nutt.). In 1971, Wall and Wani et al. identified the structure of this natural active product. Studies have confirmed that paclitaxel inhibits the proliferation of tumor cells by inhibiting the depolymerization of mitotic spindles. Because paclitaxel has a clear tumor suppressor mechanism, and can grow and proliferate tumor cells at extremely low concent...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12P17/02C07D305/14C12R1/645
Inventor 刘明志段中岗吕镇城
Owner HUIZHOU UNIV
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