Method for improving output of tetraene macrolide antibiotics

A macrolide and antibiotic technology, applied in the field of microorganisms, to achieve a reliable effect of the production level

Inactive Publication Date: 2015-04-22
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Using ribosome engineering technology to improve the amylase Streptomyces chromogenes to increase the synthetic ability of tetraenyl macrolide antibiotics (tetramycin P, tetramycin B and tetramycin A) has not been reported

Method used

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  • Method for improving output of tetraene macrolide antibiotics

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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 100 μg / mL paromomycin unless otherwise specified, and 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 5 amylase-chromogenic Streptomyces mutant strains were obtained, numbered P31, respectively. P32, P33, P34, P35;

Embodiment 2

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

[0021] Fermentation culture: The fermentation medium is GYM liquid medium, the components and concentrations are glucose 4 g / L, Dried yeast extract 4 g / L, Dried Malt extract-S 10 g / L, N-Z-Amine A 1 g / L, NaCl 2 g / L, OB solution 600 μL, adjust the pH to 7.3 with 2 mol / L NaCl solution after constant volume; the OB solution configuration method: weigh MgSO4 7H2O 25g, CuSO4 5H2O 2.5g, FeSO4 7H2O 3.75g, MnSO4·5H2O 1.8g, CaCl2·2H2O 17.5g, ZnSO4·7H2O 4.5g, add 500 mL water, shake well before use; each 300 mL Erlenmeyer bottle contains 60 mL fermentation broth, after preparation, sterilize at 121°C for 20 min, and leave to cool to 50°C, under aseptic conditions, pick a platinum cycloamylase chromogenous Streptomyces D, mutant strains P31, P32, P33, P34, P35 spores to inoculate, 28°C ± 1°C, fermentation culture for 6 days; Centrifuge at 5000 r / min for 10 min, and the supernatant is used for the...

Embodiment 3

[0022] Embodiment 3: the detection of tetraene macrolide antibiotics

[0023] Method: A SHIMADZU C18 chromatographic column (150 mm×4.6 mm, 5 μm) was used, methanol was used as mobile phase A, water was used as mobile phase B, gradient elution, elution program was 0-30 min, 65%-80% A; 30-50 min, 80%-100% A; 50-60 min, 100%-65% A; flow rate 1.0 mL / min, detection wavelength 305 nm, column temperature 30 ℃.

[0024] Table 1 Ability of amylase Streptomyces chromogenes D and mutant strains to synthesize tetraene macrolide antibiotics

[0025]

[0026] The results showed that the content of tetraene macrolide antibiotics in the fermentation liquid of amylase Streptomyces chromogenes mutant strains P31, P32, P33, P34, and P35 was higher than that of the control amylase chromogenes Streptomyces D, in which amylase chromogenes Streptomyces mutant strain P31 had the strongest ability to synthesize tetraene macrolide antibiotics, and the fermentation titers of tetramycin P, tetrac...

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Abstract

The invention discloses a method for improving output of tetraene macrolide antibiotics and belongs to the technical field of microorganisms. According to the method, 100mu g/mL paromomycin is added in a solid medium which is used for culturing Streptomyces diastatochromogenes D to obtain a Streptomyces diastatochromogenes mutant strain which has relatively good resistance to the paromomycin; the capacity of synthesizing the tetraene macrolide antibiotics of the mutant strain is stronger 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 tetraene macrolide antibiotics. 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 re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/08C12R1/525
Inventor 申屠旭萍俞晓平汤谷刘楠楠郝培应许益鹏王正亮刘光富
Owner CHINA JILIANG UNIV
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