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Method for breeding selection of high yield strain of Wanlongmycin by using ion in low energy to induce mutation of gray variant streptomycete

A low-energy ion, Wanlongmycin technology, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., to achieve great promotion value and good effect of mutagenesis

Inactive Publication Date: 2007-12-26
PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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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 breeding of Wanlongmycin strains produced by implanting low-energy ion beams.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] At room temperature 25°C, take the slant spores of strain GAAS2507 and make 10 6 spore suspension per mL, spread 0.1mL of spore liquid evenly in a sterile plate, and dry it in a sterile state to form a layer of bacteria film. Use a nitrogen ion beam with an energy of 20KeV, and the dose is 7.8× 10 14 ions / cm 2 Under the conditions of pulse injection, then wash the mutagenized pellicle with 1.0mL sterile water, dilute 10 3 After doubling, smear it on Gaoshi No. 1 medium plate containing 0.6 μg / mL streptomycin, culture at 28°C for 10 days, and select a single strain with good growth (a single strain with large colony diameter and large spore production) to inoculate In a 500mL shake flask filled with 100mL liquid fermentation medium, cultivate in a shaker at 28°C and 160rpm for 120h, detect the yield of vantermycin in the fermentation broth by HPLC, and select genetically stable mutants with high yield of vantermycin for mutagenesis of Streptomyces varitans. The posit...

Embodiment 2

[0018] At room temperature 25°C, take the slant spores of strain GAAS2507 and make 10 7 spore suspension per mL, spread 0.2mL of spore liquid evenly in a sterile plate, and dry it in a sterile state to form a layer of bacteria film. Use a nitrogen ion beam with an energy of 20KeV and a dose of 13× 10 14 ions / cm 2 Under the conditions of pulse injection, and then washed with 0.5mL sterile water to wash the mutagenized pellicle, dilute 10 5 After doubling, smear it on the Gaoshi No. 1 medium plate containing 0.8 μg / mL streptomycin, culture at 30°C for 8 days, and select a single strain with good growth (a single strain with large colony diameter and large sporulation production) to inoculate In a 500mL shake flask filled with 100mL liquid fermentation medium, culture in a shaker at 30°C and 180rpm for 112h, and detect the yield of Wanlongmycin in the fermentation broth by HPLC, and select the genetically stable mutant strain with high yield of Wanlongmycin for mutagenesis of ...

Embodiment 3

[0021] At room temperature 25°C, take the slant spores of strain GAAS2507 and make 10 8 spore suspension per mL, spread 0.3mL of spore liquid evenly in a sterile plate, and dry it in a sterile state to form a layer of bacteria film. Use a nitrogen ion beam with an energy of 20KeV and a dose of 18.2× 10 14 ions / cm 2 Pulse injection was performed under the condition of 6 After doubling, smear it on Gaoshi No. 1 medium plate containing 1 μg / mL streptomycin, culture it at 32°C for 7 days, and select a single strain with good growth (a single strain with large colony diameter and large spore production) to inoculate in In a 500mL shake flask filled with 100mL of liquid fermentation medium, culture at 32°C and 200rpm shaker for 96h, detect the yield of Wanlongmycin in the fermentation broth by HPLC, and select the genetically stable mutant strain with high yield of Wanlongmycin for mutagenesis Streptomyces mutans griseus. The positive mutation rate of the mutant strain was 7%-9%...

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Abstract

This invention discloses low-energy ion implantation method for mutagenizing Streptomyces griseovariablis to obtain high-yield wanlongmycin-producing bacterium. The method comprises: spreading Streptomyces griseovariablis spore suspension in a sterile culture dish at room temperature, drying in sterile condition, performing ion implantation with nitrogen ion beam, washing with sterile water, diluting, spreading in Gause's medium No.1 containing streptomycin, culturing, selecting well grown single strains, inoculating into a liquid culture medium, and culturing to obtain Streptomyces griseovariablis with high genetic stabilityand high wanlongmycin yield. The method has such advantages as simple process, high safety and good mutagenic effect. The fermentation efficiency of the obtained bacterial strain is 20-90% higher than the original strain.

Description

technical field [0001] The invention belongs to the technical field of microbial mutagenesis, and in particular relates to a method for mutagenizing Streptomyces griseus mutans by using low-energy ion mutagenesis means to select and breed a high-yielding mutant strain of Wanlongmycin. Background technique [0002] The resistance of plant pathogenic bacteria to agricultural antibiotics is one of the severe challenges facing the world, and finding agricultural antibiotics that can effectively inhibit drug-resistant bacteria is the fundamental way to solve this problem. [0003] "Streptomyces griseovariabilis" (Latin name "Streptomyces griseovariabilis") is an actinomycete of the genus Streptomyces. Its fermentation product can extract Wanlongmycin (English name "Wanlongmycin"), a cyclic lipopeptide antibiotic belonging to the family of quinoxaline antibiotics (Quinoxaline antbiotics), which is a cyclic ester composed of 10 amino acid residues ( Decapeptide, two unique quinoxa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12N15/01C12R1/465
Inventor 林壁润沈会芳张晓勇谢双大潘群英高向阳
Owner PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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