Aspergillus fumigatus strain TMS-26 for producing taxol

Abstract

The invnetion provides aspergillus fumigatus strain TMS-26 for producing taxol. The strain can produce the taxol under the condition of liquid fermentation. The strain is collected in China Center For Type Culture Collection (Wuhan University) on June 15, 2014, and the collection number is CCTCC M2014258. The taxol yield of the strain under the conditions of PDA liquid culture medium, 28 DEG C, 60r / min and dark culture for 10 to 12d can reach 696.24[mu]g / L to the maximum degree. The novel strain provided by the invention provides good study basis for the breeding of good strains in industrial large-scale production, and the application prospects are wide.

Description

technical field

[0001] The invention provides a strain capable of producing paclitaxel, which belongs to the field of applied microbiology. Background technique

[0002] Paclitaxel (Taxol, trade name Paclitaxel), molecular formula C 47 h 51 NO 14 , is a natural secondary metabolite isolated and purified from the gymnosperm Taxus genus. Studies have found that paclitaxel can act on cell microtubules (Microtubule), leading to abnormal or cessation of cell mitosis, and can prevent cancer cell proliferation. This unique mechanism of action makes paclitaxel have excellent anticancer effects. Moreover, paclitaxel is a pure natural drug with high efficiency, low toxicity and broad spectrum. Once it comes out, it has become the world's largest-selling natural anti-cancer drug, especially for ovarian cancer, uterine cancer and breast cancer, which have a high incidence of cancer. The curative effect is unique.

[0003] At present, there are five production methods of paclitaxel...

Examples

Embodiment 1

[0012] Screening of taxol-producing endophytic fungi from Taxus mandia

[0013] (1) culture medium

[0014] PDA solid medium: 200g potato, 20g glucose, 15g agar, 1000mL tap water, natural pH.

[0015] PDA liquid medium: 200g potato, 20g glucose, 1000mL tap water, natural pH.

[0016] All the above mediums were autoclaved at 121°C for 30 minutes before use.

[0017] (2) Isolation and purification of bacterial strains

[0018] The explant material in this experiment comes from the 9-year-old Mandia yew introduced and cultivated in the Expo Garden of Northwest A&F University. Rinse the collected yew roots, stems, leaves and bark in running tap water for 3 to 5 minutes to remove dust and impurities, then rinse with sterile water three times, transfer to a clean bench, and use Blot the surface moisture with sterile filter paper. Then carry out surface disinfection treatment of explants, first soak with 75% ethanol for 30s, rinse with sterile water for 3 to 5 times after comple...

Embodiment 2

[0021] Screening of paclitaxel-producing strains

[0022] (1) Strain liquid fermentation

[0023] Activate the preserved strains. After the activation is completed, use a sterile scalpel to cut out 0.5×0.5cm uniformly grown bacterial blocks with culture medium in a clean workbench, and then insert them into 100mL triangles containing 50mL PDA liquid medium. In the bottle, seal it with parafilm, and do three parallels for each strain. After the inoculation, the inoculated Erlenmeyer flask was placed in a constant temperature oscillator for fermentation and culture. The culture conditions were 28°C, 160r / min and dark culture for 10-12 days. During the culture process, the growth of each strain was observed and recorded every day. When the fermentation liquid in the bottle was relatively viscous, the color deepened, and the mycelial balls began to crack, the fermentation was stopped, and it was taken out for the next experiment.

[0024] (2) Extraction of paclitaxel in fermenta...

Embodiment 3

[0037] Identification of strains

[0038] Observe the colony morphology of the high-yield strain on the PDA plate, and observe the hyphae and spore morphology of the strain with an optical microscope and a scanning electron microscope, and compare the morphological characteristics of the high-yield strain obtained in the "Handbook of Fungal Identification" written by Mr. Wei Jingchao, The preliminary classification and identification results of high-yield strains were obtained. Then PCR amplifies the 18SrDNA sequence and the ITS sequence of the high-yielding bacterial strain obtained and analyzes it. The basic process is as follows:

[0039] The total genomic DNA of the strain was extracted by CTAB method, and the general primers for filamentous fungi were:

[0040] 18SrDNA sequence:

[0041] 18S-NS1: 5'-GTAGTCATATGCTTGTCTC-3',

[0042] 18S-NS8: 5'-TCCGCAGGTTCACCTACGGA-3'.

[0043] ITS sequence:

[0044] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3',

[0045] ITS4: 5'-TCCTCCGCTTATTGAT...

technical field

[0001] The invention provides a strain capable of producing paclitaxel, which belongs to the field of applied microbiology. Background technique

[0002] Paclitaxel (Taxol, trade name Paclitaxel), molecular formula C 47 h 51 NO 14 , is a natural secondary metabolite isolated and purified from the gymnosperm Taxus genus. Studies have found that paclitaxel can act on cell microtubules (Microtubule), leading to abnormal or cessation of cell mitosis, and can prevent cancer cell proliferation. This unique mechanism of action makes paclitaxel have excellent anticancer effects. Moreover, paclitaxel is a pure natural drug with high efficiency, low toxicity and broad spectrum. Once it comes out, it has become the world's largest-selling natural anti-cancer drug, especially for ovarian cancer, uterine cancer and breast cancer, which have a high incidence of cancer. The curative effect is unique.

[0003] At present, there are five production methods of paclitaxel...

Examples

Embodiment 1

[0012] Screening of taxol-producing endophytic fungi from Taxus mandia

[0013] (1) culture medium

[0014] PDA solid medium: 200g potato, 20g glucose, 15g agar, 1000mL tap water, natural pH.

[0015] PDA liquid medium: 200g potato, 20g glucose, 1000mL tap water, natural pH.

[0016] All the above mediums were autoclaved at 121°C for 30 minutes before use.

[0017] (2) Isolation and purification of bacterial strains

[0018] The explant material in this experiment comes from the 9-year-old Mandia yew introduced and cultivated in the Expo Garden of Northwest A&F University. Rinse the collected yew roots, stems, leaves and bark in running tap water for 3 to 5 minutes to remove dust and impurities, then rinse with sterile water three times, transfer to a clean bench, and use Blot the surface moisture with sterile filter paper. Then carry out surface disinfection treatment of explants, first soak with 75% ethanol for 30s, rinse with sterile water for 3 to 5 times after comple...

Embodiment 2

[0021] Screening of paclitaxel-producing strains

[0022] (1) Strain liquid fermentation

[0023] Activate the preserved strains. After the activation is completed, use a sterile scalpel to cut out 0.5×0.5cm uniformly grown bacterial blocks with culture medium in a clean workbench, and then insert them into 100mL triangles containing 50mL PDA liquid medium. In the bottle, seal it with parafilm, and do three parallels for each strain. After the inoculation, the inoculated Erlenmeyer flask was placed in a constant temperature oscillator for fermentation and culture. The culture conditions were 28°C, 160r / min and dark culture for 10-12 days. During the culture process, the growth of each strain was observed and recorded every day. When the fermentation liquid in the bottle was relatively viscous, the color deepened, and the mycelial balls began to crack, the fermentation was stopped, and it was taken out for the next experiment.

[0024] (2) Extraction of paclitaxel in fermenta...

Embodiment 3

[0037] Identification of strains

[0038] Observe the colony morphology of the high-yield strain on the PDA plate, and observe the hyphae and spore morphology of the strain with an optical microscope and a scanning electron microscope, and compare the morphological characteristics of the high-yield strain obtained in the "Handbook of Fungal Identification" written by Mr. Wei Jingchao, The preliminary classification and identification results of high-yield strains were obtained. Then PCR amplifies the 18SrDNA sequence and the ITS sequence of the high-yielding bacterial strain obtained and analyzes it. The basic process is as follows:

[0039] The total genomic DNA of the strain was extracted by CTAB method, and the general primers for filamentous fungi were:

[0040] 18SrDNA sequence:

[0041] 18S-NS1: 5'-GTAGTCATATGCTTGTCTC-3',

[0042] 18S-NS8: 5'-TCCGCAGGTTCACCTACGGA-3'.

[0043] ITS sequence:

[0044] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3',

[0045] ITS4: 5'-TCCTCCGCTTATTGAT...

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