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Process for selecting cultivating high yield strain of datomycin by laser inducing rose sporestreptomycete

A technology of Streptomyces roseospora and daptomycin, which is applied in the field of breeding daptomycin high-yielding mutants by means of laser mutagenesis, can solve unseen problems and achieve the effects of simple equipment, easy method and safe operation.

Inactive Publication Date: 2006-06-28
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no relevant patents and literature reports on the application of laser in the breeding of daptomycin-producing strains.

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0013] At room temperature, take Streptomyces roseospora slant and make 10 with sterile water 4 ~10 5 spores / ml of spore suspension, draw 0.2ml and place it in a sterile quartz tank with a total volume of 3.5ml (10×10×35mm), and carry out He-Ne laser (λ=632.8nm, irradiation distance 20cm, laser beam diameter 10mm) ) for 5 minutes of irradiation, with an irradiation power of 10mW. Take 0.1ml of the irradiated suspension and dilute it by 100-1000 times, spread it on Gaoshi No. 1 medium plate, cultivate it at 30°C for 10 days, pick a single strain with good growth and inoculate it in 30ml of liquid fermentation medium (composition For: glucose 0.75g / L, dextrin 3g / L, casein 1g / L, peanut powder 0.5g / L, L-asparagine 0.1g / L, K 2 SO 4 0.5g / L, pH 7.5) in 250ml shake flasks, at 30°C, 200rpm shaker culture 110h, HPLC method detection of daptomycin production in the fermentation broth, selection of genetically stable mutants with high yield of daptomycin. The positive mutation rate o...

Embodiment 2

[0015] At room temperature, take Streptomyces roseospora slant and make 10 with sterile water 4 ~10 5 spores / ml of spore suspension, draw 0.2ml and place it in a sterile quartz tank with a total volume of 3.5ml (10×10×35mm), and carry out He-Ne laser (λ=632.8nm, irradiation distance 20cm, laser beam diameter 10mm) ) for 25 minutes of irradiation, with an irradiation power of 30mW. Take 0.1ml of the irradiated suspension and dilute it by 100-1000 times, spread it on Gaoshi No. 1 medium plate, cultivate it at 30°C for 10 days, pick a single strain with good growth and inoculate it in 30ml of liquid fermentation medium (composition For: glucose 0.75g / L, dextrin 3g / L, casein 1g / L, peanut powder 0.5g / L, L-asparagine 0.1g / L, K 2 SO 4 0.5g / L, pH 7.5) in 250ml shake flasks, at 30°C, 200rpm shaker culture 110h, HPLC method detection of daptomycin production in the fermentation broth, selection of genetically stable mutants with high yield of daptomycin. The positive mutation rate ...

Embodiment 3

[0017] At room temperature, take Streptomyces roseospora slant and make 10 with sterile water 4 ~10 5 spores / ml of spore suspension, draw 0.2ml and place it in a sterile quartz tank with a total volume of 3.5ml (10×10×35mm), and carry out He-Ne laser (λ=632.8nm, irradiation distance 20cm, laser beam diameter 10mm) ) for 30 minutes of irradiation, with an irradiation power of 25mW. Take 0.1ml of the irradiated suspension and dilute it by 100-1000 times, spread it on Gaoshi No. 1 medium plate, cultivate it at 30°C for 10 days, pick a single strain with good growth and inoculate it in 30ml of liquid fermentation medium (composition For: glucose 0.75g / L, dextrin 3g / L, casein 1g / L, peanut powder 0.5g / L, L-asparagine 0.1g / L, K 2 SO 4 0.5g / L, pH 7.5) in 250ml shake flasks, at 30°C, 200rpm shaker culture 110h, HPLC method detection of daptomycin production in the fermentation broth, selection of genetically stable mutants with high yield of daptomycin. The positive mutation rate ...

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PUM

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Abstract

The invention discloses a method used laser mutagenic streptomyces roseosporus to breed daptomycin high yield strain breeding. It belongs to strain mutagenesis breeding technology. The method includes the following steps: forming spore suspension; loading in aseptic quartz groove; irradiating by He-Ne laser for certain time; coating on Gao-NO.1 culture medium flat after diluting by aseptic water to culture pure streptomyces roseosporus; selecting out pure strain; and transferring it to liquid fermentation medium to culture high yield strain. The invention has the advantages of simple device, practical method, and safe operation. Its mutagenesis effect is far better than traditional physical chemistry mutagenesis method. Compared with original strain, the fermentation unit of the formed streptomyces roseosporus can rise by 0.5-3.3 times.

Description

technical field [0001] The invention belongs to the technical field of microbial mutagenesis, in particular to the technology of breeding daptomycin high-yield mutant strains by means of laser mutagenesis. Background technique [0002] The resistance of pathogenic bacteria to antibiotics is one of the severe challenges facing our country and even the world. Finding antibiotics that can effectively inhibit drug-resistant bacteria is the fundamental way to solve this problem. Vancomycin was once recognized as the last line of defense against Gram-positive bacteria. However, more and more bacteria resistant to this drug have been found in my country and even in the world. It is imminent to develop new and efficient new generation antibiotics. [0003] "Streptomyces roseosporus" (Latin name "Streptomyces roseosporus") is a Streptomyces genus actinomycetes, the fermentation product of which can extract daptomycin (English name "daptomycin"), a decane I...

Claims

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Application Information

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IPC IPC(8): C12N13/00C12N1/20C12R1/465
Inventor 闻建平卢文玉范晶华冯伟财音青格乐
Owner TIANJIN UNIV
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