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Method for breeding high-yield ascomycin strain by performing femtosecond laser mutagenesis on streptomyces hygroscopicus ascomycota subspecies and culture medium preparation

A technology of culture medium preparation and mold ascus, which is applied in the field of microorganisms, can solve problems such as gaps, and achieve the effects of small focus size, short pulse time, and large instantaneous power

Inactive Publication Date: 2011-11-23
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In 2007, Indian scholar Parveen et al. applied for patent WO2007 / 029082 and reported that the unit of ascomycin reached 350-400 mg / L, but domestic research on ascomycin is still in its infancy, and the fermentation unit is generally 150-200 mg / L, which is comparable to Compared with foreign fermentation titers, there is a big gap
Tianjin University Wen Jianping research group utilizes femtosecond laser mutagenesis Rhizopus oryzae (Wen Jianping, Yu Shouzhi. Method for breeding fumaric acid high-yield strains by femtosecond laser mutagenesis Rhizopus oryzae, CN102061294A), and achieved very good results. However, there are no relevant patents and literature reports on the application of femtosecond laser in the breeding of ascomycin-producing strains

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] At room temperature, take ascomycin (FK-520) mature spore slope and add appropriate amount of sterile water to make 10 6 -10 7 Single spore suspension per ml, 0.2ml suspension is divided into sterile 1.5ml centrifuge tubes, and femtosecond laser (center wavelength 800nm, pulse width 150fs, frequency 76MHz) is irradiated for 10min, and the irradiation power 10mW. The irradiated spore suspension was diluted 10 times with sterile water. 3 -10 4 After doubling, pipette 0.1ml on a solid plate medium (soy flour 15g / L, mannitol 15g / L, agar 20g / L, pH 7.0) and culture for 10 days in the dark for 28 days, and select a well-growing single strain to inoculate Slope medium (soy flour 20g / L, mannitol 15g / L, agar 20g / L, pH 7.0) 28 ℃ culture for 10 days, dig out fresh spore slant on 40ml seed medium (starch 8g / L, glucose 35g / L, peptone 6g / L, yeast powder 7g / L, calcium carbonate 1.5g / L, initial pH 7.0) 250ml shake flask, 28 ℃, 200rpm shaker for 48h, after 10% (v / v ) Inoculated into 40ml...

Embodiment 2

[0013] At room temperature, take the mature spore slope and add appropriate amount of sterile water to make 10 6 -10 7 Single spore suspension per ml, 0.2 ml of suspension was divided into sterile 1.5 ml centrifuge tubes, and femtosecond laser (center wavelength 800nm, pulse width 150fs, frequency 76MHz) was irradiated for 8min, and the irradiation power 15mW. The irradiated spore suspension was diluted 10 times with sterile water. 3 -10 4 After doubling, draw 0.1ml and spread it on a solid plate medium (soy flour 25g / L, mannitol 15g / L, agar 20g / L, pH7.0) and cultivate for 10 days at 28°C in the dark, and select a well-growing single strain for inoculation Cultivate for 10 days at 28°C on the slant medium (soy flour 15g / L, mannitol 22g / L, agar 22g / L, pH 7.0), dig out the fresh spore slant and place it on 40ml seed medium (starch 10g / L, Glucose 32g / L, peptone 7g / L, yeast powder 7g / L, calcium carbonate 3g / L, initial pH 7.0) 250ml shake flask, 28 ℃, 200 rpm shaker for 48 hours, af...

Embodiment 3

[0015] At room temperature, take the mature spore slope and add appropriate amount of sterile water to make 10 6 -10 7 Single spore suspension per ml, 0.2ml of suspension was divided into sterile 1.5ml centrifuge tubes, and femtosecond laser (center wavelength 800nm, pulse width 150fs, frequency 76MHz) was irradiated for 6min, and the irradiation power 20mW. The irradiated spore suspension was diluted 10 times with sterile water. 3 -10 4 After doubling, pipette 0.1ml on a solid plate medium (soy flour 20g / L, mannitol 20g / L, agar 20g / L, pH 7.0) and cultivate in the dark at 28°C for 10 days, and select a well-growing single strain for inoculation Cultivate the slant medium (22g / L soy flour, 22g / L mannitol, 20g / L agar, pH7.0) at 28°C for 10 days, dig out the fresh spore slant and place it on a 40ml seed medium (starch 10g / L, Glucose 30g / L, peptone 6g / L, yeast powder 6g / L, calcium carbonate 2g / L, initial pH 7.0) 250ml shake flask, 28 ℃, 200 rpm shaker for 48 hours, after 10% (v / v ...

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Abstract

The invention discloses a method for breeding an high-yield ascomycin strain by performing femtosecond laser mutagenesis on streptomyces hygroscopicus ascomycota subspecies and a culture medium preparation method; the method comprises the following steps: at room temperature, mixing mature slant spores of the streptomyces hygroscopicus ascomycota subspecies (ATCC14891) and sterile water to obtain monospore suspension containing 106-107 spores per milliliter; irradiating the spore suspension for 1-10 min by using the titanium sapphire femtosecond laser with centre wavelength of 800 nm, pulse width of 150 fs and frequency of 76 MHz under a irradiation power of 10-30m W; properly diluting the spore suspension, coating on a solid panel to culture, then screening by using a shake flask to obtain a high-yield ascomycin mutation strain. The method provided by the invention has the advantages that the used femtosecond laser mutagenesis method is feasible and safe to operate; the mutation effect is better than that of the traditional physical and chemical mutagenesis method, and the femtosecond laser mutagenesis method has great popularization value in breeding of microorganism pharmaceutical strains; by using the femtosecond laser irradiation to perform the mutagenesis, the high-yield ascomycin mutation strain can be bred. The positive mutation rate of the mutation strain is 5-30%, and fermenting unit is improved by 10-60% in comparison with that of an original strain.

Description

Technical field [0001] The invention belongs to the technical field of microorganisms, and particularly relates to a method for breeding ascomycin high-yielding strains by femtosecond laser mutagenesis of Streptomyces hygroscopicus subspecies ascus and preparation of culture media. Background technique [0002] Macrolide immunosuppressants are a new group of immunosuppressants with a macrolide structure. Studies in vivo and in vitro have proven to have obvious immunosuppressive effects, including ascomycin (FK-520) and its derivative. Ascomycin was isolated from the soil containing Streptomyces hygroscopicus No.KK317 by Fujisawa Pharmaceutical Company of Japan. It is an ethyl analog of the immunosuppressant tacrolimus (FK-506), which can be used to treat autoimmune diseases, skin diseases and prevent organ transplant rejection. In addition, a series of studies have shown that ascomycin also has anti-malaria, neuroprotection and regeneration, and anti-spasm activities. [0003] A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N13/00C12N15/01
Inventor 闻建平齐海山
Owner TIANJIN UNIV
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