Method for increasing yield of toyocamycin through screening of united drug-resistant mutant strains

A technology of toyocamycin and mutant strains, which is applied in the field of microorganisms to achieve the effect of increasing production

Inactive Publication Date: 2016-02-24
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Amylase Streptomyces chromogenes ( Streptomyces diastatochromogenes ) D can synthesize toyocamycin, and there is no report on the improvement of amylase chromogenous Streptomyces chromogenes by using combined drug resistance mutation screening method to improve the synthesis ability of toyocamycin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Acquisition of low-concentration streptomycin-resistant mutants (first round of screening of drug-resistant mutants)

[0024] A GYM solid plate with a final streptomycin concentration of 100 μg / mL was prepared, that is, the streptomycin concentration in the GYM solid plate was 100 μg / mL. Pipette 1000 μL of amylase Streptomyces chromogenes D spore suspension (1×10 6 colonies / mL) on the GYM solid plate, spread evenly with a sterile spreader bar, incubate at 28°C for 7 days, and pick a single colony onto a new GYM solid plate (one colony) containing 100 μg / mL streptomycin. A single colony was drawn on a plate), placed in a 28°C incubator for 5 days, and the strain that could grow again on the new GYM solid plate was a low-concentration streptomycin-resistant mutant. A total of 8 low-concentration streptomycin-resistant mutant strains were obtained, numbered f11, f12, f13, f14, f15, f16, f17 and f18, and these 8 mutant strains were subjected to liquid treatment ...

Embodiment 2

[0025] Example 2: Obtainment of high-concentration streptomycin-resistant mutants (second round of screening of drug-resistant mutants)

[0026] A GYM solid plate with a final streptomycin concentration of 2000 μg / mL was prepared, that is, the streptomycin concentration in the GYM solid plate was 2000 μg / mL. Pipette 1000 μL of low-concentration streptomycin-resistant mutant f11 spore suspension (1 × 10 12 colonies / mL) on the GYM solid plate, spread evenly with a sterile spreader bar, incubate at 28°C for 10 to 15 days, and pick a single colony to a new GYM solid plate containing 2000 μg / mL streptomycin. (A single colony is drawn on a plate), and placed in a 28°C incubator for 7 to 10 days. The strain that can grow again on the new GYM solid plate is a high-concentration streptomycin-resistant mutant. A total of 3 high-concentration streptomycin-resistant mutant strains were obtained, numbered s223, s225, and s228. The three mutant strains were subjected to liquid fermentation...

Embodiment 3

[0027] Example 3: Acquisition of Paromomycin-resistant mutants (the third round of screening of drug-resistant mutants)

[0028] A GYM solid plate with a final concentration of paromomycin of 50 μg / mL was prepared, that is, the concentration of paromomycin in the GYM solid plate was 50 μg / mL. Pipette 1000 μL of high concentration streptomycin-resistant mutant s228 spore suspension (1×10 12colonies / mL) on the GYM solid plate, spread evenly with a sterile coating rod, and culture in a 28°C incubator for 10 to 15 days. Pick a single colony to a new GYM solid containing 50 μg / mL paromomycin. Plate (a single colony is drawn on a plate), placed in a 28°C incubator for 7 to 10 days, and the strain that can grow again on the new GYM solid plate is the paromomycin-resistant mutant. A total of 2 paromomycin-resistant mutants were obtained, numbered t3141 and t3145;

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Abstract

The invention discloses a method for increasing yield of toyocamycin through screening of united drug-resistant mutant strains and belongs to the field of microbial technologies. According to the method, Streptomyces diastatochromogenes D mutant strains generating drug resistance to low-concentration streptomycin, high-concentration streptomycin and paromomycin are obtained through screening of the united drug-resistant mutant strains, and the toyocamycin productivity of the obtained mutant strains is remarkably improved when compared with that of Streptomyces diastatochromogenes D.

Description

technical field [0001] The present invention relates to the technical field of microorganisms, in particular to a method for improving toyomycin production by screening combined drug-resistant mutant strains. Background technique [0002] At present, many self-developed new agricultural antibiotics are only in the laboratory stage, and the key reason is that the production level of the production bacteria does not meet the requirements of industrialization. Therefore, a major problem facing researchers at present is to improve the toxin-producing level of existing and developing agricultural antibiotic-producing bacteria, and to speed up the industrialization process of agricultural antibiotics. Conventional microbial mutagenesis breeding is time-consuming, labor-intensive, and highly random. Through genetic engineering, the directed evolution and overexpression of enzymes encoded by a single gene can be achieved, but the yield of natural products such as antibiotics that ne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P19/40C12R1/525
CPCC12P19/40C12N1/205C12R2001/525
Inventor 申屠旭萍许益鹏俞晓平汤谷刘楠楠
Owner CHINA JILIANG UNIV
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