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Method for improving capacity of Streptomyces diastatochromogenes for synthesizing toyocamycin

A technology for producing Streptomyces chromogenes and toyomycin, which is applied in the field of microorganisms and achieves the effect of improving the production level

Inactive Publication Date: 2015-03-11
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

There is no report on the improvement of amylase chromogenic Streptomyces chromogenes by using ribosome engineering technology to improve the synthesis ability of toyocamycin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: the acquisition of amylase Streptomyces chromogenes mutant strain

[0019] Prepare the GYM solid plate. The GYM solid plate described in the present invention contains 10 μg / mL of erythromycin except for special instructions. Use a pipette to draw 100 μL of amylase chromogenic Streptomyces D spore suspension (1 × 10 6 cells / mL) on the GYM solid plate, spread evenly with a sterile applicator stick, culture in an incubator at 28°C for 7 days, pick a single colony to a new GYM solid plate (one single colony marks a plate), place Cultivate in an incubator at 28°C for 5 days, and the strains that can grow on the new GYM solid plate again are the amylase-chromogenic Streptomyces mutant strains. A total of 3 amylase-chromogenic Streptomyces mutant strains were obtained, numbered h1, respectively. h2, h3;

Embodiment 2

[0020] Embodiment 2: liquid fermentation of amylase Streptomyces chromogenic mutant strain

[0021] Fermentation culture: The fermentation medium contains 20 g of soluble starch per 1000 mL, 40 g of soybean powder, and NH 4 Cl 3 g, CaCO 3 5 g, MgSO 4 2 g, the balance is water; 60 mL of fermentation broth per 300 mL Erlenmeyer bottle, after preparation, sterilize at 121°C for 20 min, cool to 50°C, and pick a platinum cycloamylase chromogenic Streptomyces respectively under sterile conditions D. The spores of mutant strain h1, mutant strain h2 and mutant strain h3 were inoculated, fermented and cultured for 6 days at 28°C±1°C; the fermentation broth was centrifuged at 5000 r / min for 10 min, and the supernatant was used to determine the content of toyocamycin ;

Embodiment 3

[0022] Embodiment 3: the detection of toyocamycin

[0023] Method: A SHIMADZU C18 column (150 mm×4.6 mm, 5 μm) was used, methanol was used as mobile phase A, water was used as mobile phase B, and gradient elution was performed. The elution program was 0-15 min, 5%-37% A; 15-30 min, 37%-100% A; 30-40 min, 100%-5% A; flow rate 1.0 mL / min, detection wavelength 279 nm, column temperature 30 ℃.

[0024] The results showed that the contents of toyocamycin in the fermented broth of amylase Streptomyces chromogenes h1, h2, and h3 were 815.63 mg / L, 678.31 mg / L, and 436.75 mg / L, compared with the control 152.12 mg / L They were increased by 5.36, 4.46 and 2.87 times respectively, among which the amylase Streptomyces chromogenes mutant strain h1 had the strongest ability to synthesize toyocamycin, and the fermentation titer reached 815.63 mg / L.

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Abstract

The invention discloses a method for improving the capacity of Streptomyces diastatochromogenes for synthesizing toyocamycin and belongs to the technical field of microbes. According to the method, 10 mug / mL of erythromycin is added to a solid medium for culturing Streptomyces diastatochromogenes D, a Streptomyces diastatochromogenes mutant strain with higher resistance to the toyocamycin is obtained, and the capacity of the mutant strain for synthesizing the toyocamycin is higher than that of the Streptomyces diastatochromogenes D.

Description

technical field [0001] The present invention relates to the technical field of microbes, in particular to improving amylase chromogenic streptomyces ( Streptomyces diastatochromogenes ) method for synthesizing toyocamycin. 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 producing bacteria has not met the requirements of industrialization. Therefore, a major problem facing researchers is to improve the production levels of existing and developing agricultural antibiotic-producing bacteria, and to speed up the industrialization process of agricultural antibiotics. Conventional microbial mutation breeding is time-consuming, labor-intensive, and highly random. Genetic engineering can achieve directed evolution and overexpression of enzymes encoded by single genes, but the production of natural products such as antibiotics that require clustered gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/40C12R1/525
Inventor 俞晓平申屠旭萍王正亮郝培应刘光富许益鹏
Owner CHINA JILIANG UNIV
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