Mutagenesis and screening method of sterptomyces kasugaensis efficiently producing kasugamycin

A technology of Streptomyces aureus and kasugamycin, which is applied in the field of microorganisms, can solve the problems of limited increase in the fermentation yield of kasugamycin, and achieve the effects of increased potency, good genetic stability, and important economic value

Inactive Publication Date: 2019-04-12
陕西麦可罗生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] These conventional methods of mutation breeding have limited improvement in the fermentation yield of kasugamycin, so new transformation strategies are needed

Method used

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  • Mutagenesis and screening method of sterptomyces kasugaensis efficiently producing kasugamycin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Screening of Kasugamycin High-Producing Strains

[0056] 1. Preparation of Streptomyces aureus slant culture and slant spore counting

[0057] (1) Preparation of experimental materials

[0058] Streptomyces aureus ( Streptomyces microaureus ) ACCC 40060 is the starting strain.

[0059] Slant medium: 1.0% glucose, 0.5% maltose, 0.1% peptone, 0.1% sodium chloride, 5.0% bean cake powder, 0.1% calcium carbonate, 2.0% agar, the rest water, natural pH, sterilized at 121°C for 20 minutes, and set aside.

[0060] (2) Incline culture of Streptomyces aureus and slant spore count

[0061] Use an inoculation shovel to inoculate the starting strain on the slant medium and culture it at 28°C for 9 days to obtain the spore slant, then scrape off all the spores on the slant, and dilute it with sterilized normal saline for 10 6 times, spread it on a plate containing medium (same components as the slant medium), culture at 28°C for 2 days, count single colonies, and calcul...

Embodiment 2

[0079] Example 2 Counting of slant spores

[0080] Use an inoculation shovel to inoculate the starting strain on the slant medium and incubate at 28°C for 7-10 days to obtain the spore slant, then scrape off all the spores on the slant, and dilute it with sterilized normal saline for 10 3 -10 4 times, spread it on a plate containing medium (same components as the slant medium), culture at 25-30°C for 2 days, count single colonies, and calculate the total number of spores on the slant according to the dilution ratio. 9 indivual.

Embodiment 3

[0081] Example 3 Passage Stability Investigation of Kasugamycin High-Producing Strains

[0082]The kasugamycin high-yielding strain S3 preserved in Example 1 was transferred to the slant medium prepared in Example 1, and cultured in a 28°C constant temperature dry incubator in the dark for 9 days, and the slant strains were transferred to the slant medium again On, repeat the operation 5 times. Inoculate the slant strains obtained from these six cultures into the seed medium prepared in Example 1, culture them on a shaker at 28°C and 220 rpm / min for 40 h, and immediately Transfer to the fermentation medium, continue to ferment and cultivate for 140 h, and obtain fermentation liquid containing different concentrations of kasugamycin. The content of kasugamycin in the fermentation broth was determined by HPLC. According to the sequence of passage, the contents were: 5048 μg / mL, 5146 μg / mL, 5310 μg / mL, 5258 μg / mL, 5063 μg / mL, 5011 μg / mL. It can be seen from the experimental res...

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Abstract

The invention relates to a mutagenesis and screening method of sterptomyces kasugaensis efficiently producing kasugamycin, belonging to the technical field of microorganisms. The method utilizes a newatmospheric and room-temperature plasma (ARTP) breeding system to mutagenize a strain producing kasugamycin, and mainly includes the following steps: (1) slant culture of sterptomyces kasugaensis andcounting of slant spores; (2) preparation of spore suspension; (3) mutagenesis of sterptomyces kasugaensis by atmospheric and room-temperature plasma (ARTP); and (4) screening of a strain efficientlyproducing kasugamycin. The highest potency of shaking flask fermentation of sterptomyces kasugaensis screened by the method can reach 5091 [mu]g/mL, which is 130% higher than that of the original strain. The method has important economic values in industrial production.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a mutagenesis and screening method for Streptomyces aureus producing high-efficiency kasugamycin. Background technique [0002] Kasugamycin is produced by Streptomyces aureus ( Streptomyces kasugaensis ) produced secondary metabolites, belonging to aminoglycoside antibiotics. Kasugamycin is widely used in agriculture, and has significant control effects on rice blast, Chinese cabbage black rot, and cucumber wilt. [0003] Obtaining high-yield strains of antibiotics is the goal that enterprises have always pursued. The traditional strategies to increase the yield of kasugamycin-producing strains mainly include breeding methods such as optimization of fermentation medium, ultraviolet irradiation and natural separation. Such as Chinese patent application 201610817957.4 provides a kind of method that utilizes Streptomyces microaureus BJX007 to ferment and produce kasu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N13/00C12N1/20C12Q1/04C12R1/465
CPCC12N1/20C12N13/00C12Q1/04
Inventor 高嫚妮吴亮亮李昶志郑鹏飞杨宏勃潘忠成李蒲民
Owner 陕西麦可罗生物科技有限公司
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