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Genetic engineering bacterium capable of promoting biological synthesis of medermycin and application thereof

A technology of genetically engineered bacteria and medamycin, applied in the field of genetic breeding, can solve the problems of low antibiotics, high antibiotic-producing bacteria breeding bottlenecks, and restrict antibiotic development, etc., and achieve the effect of high-efficiency expression

Inactive Publication Date: 2012-09-12
HUAZHONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Streptomyces can produce many useful antibiotics, but the level of antibiotics produced by most natural strains is very low, which limits the further development of antibiotics, so the breeding of antibiotic high-yielding bacteria has always been a bottleneck in the antibiotic industry

Method used

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  • Genetic engineering bacterium capable of promoting biological synthesis of medermycin and application thereof
  • Genetic engineering bacterium capable of promoting biological synthesis of medermycin and application thereof
  • Genetic engineering bacterium capable of promoting biological synthesis of medermycin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Solid fermentation of engineering bacteria AM-7161 / pHSL56.2:

[0045] Eight recombinant strains AM-7161 / pHSL56.2 that may contain the plasmid pHSL56.2 were identified. They were cultured on solid R4 medium at 30°C for 5 days until sporulation. The spores were collected and stored in 20% glycerol for cryopreservation. Get engineering bacterium AM-7161 / pHSL56.2 and wild bacterium AM-7161 spore suspension and spread on 20ml solid R4 medium respectively (R4 medium formula sees content of the invention, do not add in the medium when cultivating wild bacterium AM-7161 thiostrepton, because the wild bacteria AM-7161 has no thiostrepton resistance); placed in static culture at 30°C for 3-5 days, it can be seen that due to the high expression of ribosome recycling factors, the U.S. The production of daxamycin was significantly increased (secreted into the culture medium, showing reddish brown) ( image 3 ); collect the solid culture (together with the culture medium), cut into ...

Embodiment 2

[0047] Liquid fermentation of engineering bacteria AM-7161 / pHSL56.2:

[0048] Draw 50 μl of spore suspension of engineering bacteria AM-7161 / pHSL56.2 and inoculate it in 5mL seed medium (see the contents of the invention for the medium formulation), cultivate it for 2 days at 200 rpm and 30°C, and then press 1:100 Ratio Transfer the seed culture into the liquid R4 hormone-producing medium (see the content of the invention for the medium formula, add 12.5 μg / ml thiostrepton as an inducer), and cultivate 5- 6 days. Transfer the fermentation broth into a 50ml centrifuge tube, centrifuge at 3000rpm at room temperature for 10min, and collect the supernatant. Adjust the pH of the supernatant to neutral, extract three times with ethyl acetate, mix the extracts, add water to extract once (wash), filter with anhydrous sodium sulfate powder to remove water, filter with filter paper, remove ethyl acetate by rotary evaporation, and finally in dissolved in ethyl acetate.

[0049]The col...

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Abstract

The invention provides a genetic engineering bacterium capable of promoting the synthesis of antibiotic medermycin and a method for producing the medermycin by using the same. The method comprises the steps that by taking deoxyribonucleic acid (DNA) of a genome of Streptomyces nashvillensis AM-7161 as a formwork and utilizing degenerate primers to conduct polymerase chain reaction (PCR) amplification, a new complete sequence of genes of ribosome recycling factors (RRFs) of the Streptomyces is obtained; the genes are downstream placed on an efficient promoter PtipA of the Streptomyces, so that a plasmid pHSL56.2 capable of efficently expressed in the Streptomyces is constructed; the pHSL56.2 is led into the host cell Streptomyces AM71-61, and then an engineering bacterium AM71-61 / pHSL56.2 (with a preservation number of CCTCCNo:M2012093) is obtained; and when the engineering bacterium AM71-61 / pHSL56.2 is used to conduct solid fermentation and liquid fermentation, the accumulation of the antibiotic medermycin can be effectively promoted. The efficient expression plasmid pHSL56.2 of the genetic engineering bacterium can also be directly led into a Streptomyces strain cell for producing other antibiotics of a benzoisochromanequinones family or other aromatic polyketide antibiotics, so as to obtain corresponding antibiotic high-yield engineering bacteria. By utilizing the method, the antibiotic high-yield bacteria can be genetically bred, so that the synthetic capability of the antibiotics is enhanced.

Description

Technical field: [0001] The present invention relates to a genetic breeding method for improving the production of anti-tumor antibiotic Midamycin, especially a kind of cloning of ribosome recycling factor (ribosome recycling factor, RRF) from Streptomyces nasvierii and its use in antibiotic Midamycin. A method for breeding daxamycin high-yielding bacteria. The Streptomyces ribosome recycling factor can promote the accumulation of metamycin and greatly improve the biosynthesis efficiency of antibiotics. Background technique: [0002] Streptomyces can produce many useful antibiotics, but the level of antibiotics produced by most natural strains is very low, which limits the further development of antibiotics, so the breeding of antibiotic high-yielding bacteria has always been a bottleneck in the antibiotic industry. At present, there are many methods for breeding antibiotic high-yielding bacteria, including traditional physical and chemical mutagenesis, protoplast fusion, m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/76C12P17/16C12R1/465
Inventor 李爱英万娟王慧利
Owner HUAZHONG NORMAL UNIV
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