Method utilizing fosfomycin to carry out breeding of daptomycin high-yield bacterial strains

A technology of daptomycin and high-yielding strains, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve unseen problems and achieve the effects of simple equipment, good effect, and feasible method

Inactive Publication Date: 2015-02-11
JIANSGU INST OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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Method used

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Embodiment 1

[0012] The slant spore of embodiment 1 parent strain is made 10 with aseptic water 5 -10 7 spores / ml monospore suspension, 2ml of the spore suspension was divided into glass dishes, treated under 15 watts of ultraviolet light for 0.5 minutes, and then the radiation-treated spore suspension was diluted to 10 with sterile water -3 -10 -5 Afterwards, spread it on a culture plate and cultivate it at 26°C for 7 days, and use a sterile bamboo stick to pick out the grown colony and inoculate it on a screening plate containing 1000 μg / ml of fosfomycin (composed of 3 g of yeast powder, malt leaching Powder 9g, glucose 3g, fosfomycin 1g, agar 20g, deionized water 1L, PH natural), pick the colony sensitive to this concentration of fosfomycin, cultivate it on the slant medium for 6 days, inoculate it in a 30ml liquid fermentation medium 250ml shake flask of culture medium (composed of soluble starch 8g, yeast powder 5g, dextrin 10g, casein 5g, tryptone 6g, K 2 SO 4 3g, 1g of L-asparag...

Embodiment 2

[0014] The inclined spores of the parent plant were made with sterile water 10 5 -10 7 spores / ml monospore suspension, 2ml of the spore suspension was divided into glass dishes, treated under 15 watts of ultraviolet light for 0.6 minutes, and then the spore suspension treated with radiation was diluted to 10 with sterile water -3 -10 -5 Afterwards, spread it on a culture plate and cultivate it at 26°C for 7 days, and use a sterile bamboo stick to pick out the grown colony and inoculate it on a screening plate containing 800 μg / ml of fosfomycin (composed of yeast powder 3g, malt Extract powder 9g, glucose 3g, fosfomycin 0.8g, agar 20g, deionized water 1L, PH natural), pick the colonies sensitive to this concentration of fosfomycin, culture it on the slant medium for 6 days, inoculate in 30ml 250ml shake flask of liquid fermentation medium (composed of soluble starch 8g, yeast powder 5g, dextrin 10g, casein 5g, tryptone 6g, K 2 SO 4 3g, 1g of L-asparagine, dissolved in 1L of...

Embodiment 3

[0016] The inclined spores of the parent plant were made with sterile water 10 5 -10 7 spores / ml single spore suspension, 2ml of the spore suspension was divided into glass dishes, treated under 15 watts of ultraviolet light for 1.0 minutes, and then the irradiated spore suspension was diluted to 10 with sterile water -3 -10 -5 Afterwards, spread it on a culture plate and cultivate it at 26°C for 7 days, and use a sterile bamboo stick to pick out the grown colony and inoculate it on a screening plate containing 600 μg / ml of fosfomycin (composition: 3 g of yeast powder, malt leaching powder 9g, glucose 3g, fosfomycin 0.6g, agar 20g, deionized water 1L, PH natural), pick the colony sensitive to this concentration of fosfomycin, cultivate it on the slant medium for 6 days, and inoculate it in a liquid containing 30ml 250ml shake flask of fermentation medium (composed of soluble starch 8g, yeast powder 5g, dextrin 10g, casein 5g, tryptone 6g, K 2 SO 4 3g, 1g of L-asparagine, d...

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Abstract

The invention discloses a method for obtaining daptomycin high-yield bacterial strains through screening streptomyces roseosporus, which is sensitive to fosfomycin, and belongs to the technology of bacterial strain breeding. The method comprises the following steps: dissolving spores cultured in a slope in sterile water to prepare a spore suspension, subjecting the spore suspension to a UV treatment, painting the suspension on a separated culture medium, picking the colonies growing on the culture medium by a sterile bamboo stick, then inoculating the colonies on solid screening culture mediums with different fosfomycin concentrations, selecting colonies that are sensitive to fosfomycin in different concentrations, inoculating the selected colonies on a slope culture medium to culture the colonies, then transferring the colonies to a liquid culture medium to carry out liquid culture in the end, and finally picking out the high-yield bacterial strains with stable genetic characters.

Description

technical field [0001] The invention belongs to the technical field of microbial strain screening, in particular to the technology of using fosfomycin to screen high-yield strains of daptomycin. Background technique [0002] With the wide application of antibiotics in clinical practice, the problem of bacterial drug resistance is becoming more and more serious, which has aroused widespread concern. Vancomycin has been used as the last line of defense against many serious Gram-positive bacterial infections for a long time, but with frequent clinical application, more and more drug-resistant strains, the current clinical application of vancomycin is not very ideal. Therefore, it is of great significance to develop novel antibiotics against drug-resistant bacteria. [0003] Daptomycin (Cubicin) is a cyclolipopeptide antibiotic originally researched by Lily (Lily) and developed by Cubist Pharmaceuticals. In September 2003, antibiotics as a new structural class were first liste...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N13/00C12R1/465
CPCC12N13/00C12N1/20
Inventor 沈依群张晓来陆茂林钱国芬许春燕司竑飞杨晓伟
Owner JIANSGU INST OF MICROBIOLOGY
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