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Screening method for breeding Streptomyces roseosporus strains producing Daptomycin and obtained strains

A technology of Streptomyces roseospora and daptomycin, which is applied in the field of strain selection and breeding, can solve the problems of non-directional mutation, heavy screening workload of target strains, and affecting the efficiency of mutagenesis breeding, etc., so as to reduce production costs , speed up the process of strain selection and breeding, and the effect of great promotion value

Inactive Publication Date: 2015-06-17
SHANGHAI INST OF PHARMA IND CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mutations produced by such a screening method are random and non-directional, resulting in a large screening workload for the target strain, which directly affects the work efficiency of mutagenesis breeding.

Method used

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  • Screening method for breeding Streptomyces roseosporus strains producing Daptomycin and obtained strains
  • Screening method for breeding Streptomyces roseosporus strains producing Daptomycin and obtained strains
  • Screening method for breeding Streptomyces roseosporus strains producing Daptomycin and obtained strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Streptomycin Screening

[0021]Wild-type Streptomyces roseosporus NRRL11379 obtained from the Culture Collection of the United States Department of Agriculture and preserved in cryopreservation tubes was spread on Gaoshi No. 1 slant and cultured at 28°C for 5-6 days. Wash the spores with sterile water, and spread them on Gaoshi No. 1 plates containing streptomycin at various concentrations of 0.5-2mg / L (its composition is: soluble starch 2.0%, NaCl 0.05%, KNO 3 0.1%, K 2 HPO 4 ·3H 2 O 0.05%, MgSO 4 ·7H 2 O 0.05%, FeSO 4 ·7H 2 O 0.001%, agar 2.0%; said percentages are mass volume percentages), cultured at 28°C for 5 days. Then randomly select the single colony grown on the plate and transfer it to the primary seed medium. The composition and mass content of the primary seed medium are: 30g of TSB, 35g of maltodextrin, dissolved in 1L of distilled water, and the initial pH is 7.0. Then shake culture at 30° C. and 200 rpm shaker for 25 hours. Then, the...

Embodiment 2

[0030] Example 2 Daptomycin screening

[0031] The treatment steps of the wild-type Streptomyces roseospore before coating are the same as in Example 1. Then, the wild-type Streptomyces roseospora were coated on Gaoshi No. 1 plates containing daptomycin at various concentrations of 0.5-3 mg / L, and cultured at 28° C. for 5 days. Then the single colony grown on the plate was transferred to primary seed medium. The steps of seed culture and fermentation culture are the same as in Example 1. The detection and calculation of the fermentation unit of daptomycin in the fermentation broth are also the same as in Example 1.

[0032] The results are shown in Table 2. It can be seen from the results in the table that the more daptomycin-tolerant strains obtained from the plate, the higher the fermentation unit of daptomycin. The positive rate of this screening method is 15-25%, while the positive rate of traditional random selection is less than 1%.

[0033] Table 2. Effect of daptom...

Embodiment 3

[0036] Example 3 Screening of inhibition zones

[0037] The treatment steps of the wild-type Streptomyces roseospore before coating are the same as in Example 1. Then the wild-type Streptomyces roseospora was spread on Gao's No. 1 plate and cultured at 28°C for 5 days. Then streak the single colony grown on the plate on the plate of Gao Shi No. 1, and culture at 28°C for 5 days. After the bacterial lawn grows evenly on the plate, use a sterile puncher (aperture diameter is about 5mm) to punch holes on the bacterial lawn, and then put the agar circle in the hole on the surface of the coated Staphylococcus aureus bacterial solution. LB tablet. Incubate at 28°C for 1 day, and screen by comparing the diameter of the inhibition zone. Transfer the corresponding strain of the strain with larger inhibition zone diameter to the primary seed medium. The steps of seed culture and fermentation culture are the same as in Example 1. The detection and calculation of the fermentation uni...

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Abstract

The invention discloses a screening method for breeding Streptomyces roseosporus strains producing Daptomycin and the obtained strains. The screening method comprises the following steps of: 1) coating bacteria liquid of wild Streptomyces roseosporus or mutant strains thereof subjected to mutagenesis on a solid panel containing streptomycin and / or Daptomycin, and culturing to obtain streptomycin and / or Daptomycin tolerant Streptomyces roseosporus; and 2) respectively selecting a single colony of the streptomycin and / or Daptomycin tolerant Streptomyces roseosporus for shake flask fermentation, detecting the Daptomycin content in fermented materials, and selecting strains with the Daptomycin content higher than that of starting strains. The screening method has the advantages of directivity, intuition, simpleness, quickness, high efficiency, great reduction of workload, and acceleration of strain breeding process. The Daptomycin fermentation unit of the screened Streptomyces roseosporus strains is obviously improved, which can be improved by more than 5 times to the maximum.

Description

technical field [0001] The invention belongs to the technical field of strain breeding, and in particular relates to a screening method for breeding Streptomyces roseospora strains producing daptomycin and the obtained strains. Background technique [0002] With the widespread use of antibiotics in clinical practice, the problem of bacterial resistance is becoming more and more serious. Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) are showing an alarming upward trend. In the 1980s, MRSA infection accounted for 20% to 50% of Staphylococcus aureus infections. The US Centers for Disease Control and Prevention reported that from 1989 to 1998, the number of nosocomial infections with vancomycin-resistant enterococci rose from 0.3% to 21.2%. Because MRSA has the characteristics of strong toxicity, multiple high drug resistance, and fast transmission, once infection occurs, it will spread rapidly in the hospital and the disease is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/04C12N1/20C12R1/465
Inventor 陈少欣廖艳艳王岩
Owner SHANGHAI INST OF PHARMA IND CO LTD
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