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Streptomycete generating 5-keto-milbemycins and method for producing 5-keto-milbemycins

A technology of mirbemycin and streptomyces, which is applied in the field of producing 5-keto mirbemycin and can solve the problems of shortening the production process of mirbemycin and the like

Active Publication Date: 2014-05-14
ZHEJIANG HISUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Obviously, the bacterial strain that can directly produce 5-ketomilbemycin will shorten the production process of milbemycin, but there is no report on the strain that directly produces 5-ketomilbemycin

Method used

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  • Streptomycete generating 5-keto-milbemycins and method for producing 5-keto-milbemycins
  • Streptomycete generating 5-keto-milbemycins and method for producing 5-keto-milbemycins
  • Streptomycete generating 5-keto-milbemycins and method for producing 5-keto-milbemycins

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] Embodiment 1: the extraction of Streptomyces total DNA

[0141] Inoculate 50 μL of Streptomyces spore suspension in cryopreserved tubes into 30 mL of TSB medium (purchased from Bacto Tryptic Soy Broth.BD), culture at 28°C and 220 rpm for 48 hours, centrifuge at 4000 rpm for 10 minutes in a 50 mL centrifuge tube, and remove the supernatant. The precipitate was washed twice with 30 mL of sucrose-Tris buffer (in which the mass percentage of sucrose was 10.3%, the molar-volume concentration of Tris-HCl was 10 mM, and the pH value was 8.0), and then suspended in 5 mL of sucrose-Tris buffer. Add 20 μL of lysozyme solution with a mass-volume solubility of 100 mg / mL, and bathe in water at 37° C. for 2 h. Add 500 μL of 10% SDS solution by mass percentage, and gently invert until it is basically clear. Add phenol-chloroform-isoamyl alcohol (the volume ratio of phenol-chloroform-isoamyl alcohol is 25:24:1 (pH value is 8.0)) solution 5mL, after gentle inversion several times, cent...

Embodiment 2

[0142] Embodiment 2: The construction of the recombinant vector that is used for knocking out Streptomyces milF gene

[0143] With pMD19 (Simple) as the backbone, its construction process is as follows figure 2 As shown, specifically:

[0144] a): using the Streptomyces total DNA obtained in Example 1 as a template, using an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 17, and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 18, through amplifying to obtain the first nucleotide fragment;

[0145] 1. Preparation of PCR amplification reaction solution, in which PrimeSTAR kit was purchased from TaKaRa:

[0146]

[0147] 2. Divide into 2 tubes for PCR reaction, the procedure is:

[0148]

[0149] After agarose gel electrophoresis, the PCR product was recovered with a DNA gel recovery kit (purchased from Shanghai Huasun Biotechnology Co., Ltd.) to obtain the first nucleotide fragment, whose sequence is shown in SEQ ID NO:36.

[0...

Embodiment 3

[0159] Embodiment 3: The construction of the recombinant vector that is used for knocking out Streptomyces milF gene

[0160] With pBlueScript SK(+) as the skeleton, its construction process is as follows Figure 4 As shown, specifically:

[0161] a) Using the total Streptomyces DNA as a template, using an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 20, and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 21, PCR amplifies to obtain SEQ The second nucleotide fragment of the nucleotide sequence shown in ID NO:24:

[0162] 1. Preparation of PCR amplification reaction solution, in which PrimeSTAR kit was purchased from TaKaRa:

[0163]

[0164] 2. Divide into 2 tubes for PCR reaction, the procedure is:

[0165]

[0166] After agarose gel electrophoresis, the PCR product was recovered with a DNA gel recovery kit (purchased from Shanghai Huasun Biotechnology Co., Ltd.) to obtain a second nucleotide fragment, the nucleotide sequenc...

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Abstract

The invention relates to the technical field of gene recombination, and particularly relates to streptomycete generating 5-keto-milbemycins and a method for producing 5-keto-milbemycins. The invention provides a recombinant vector for knocking out the milF gene of streptomycete, including the homologous fragment of the milF gene, wherein the sequence of the fragment is obtained by losing 105-767 nucleotides from the nucleotides from the sites 158-924 in the nucleotide sequence shown by SEQ ID No.2. The invention also provides an establishment method of the recombinant vector, a method for knocking out the milF gene in streptomycete by use of the recombinant vector and streptomycete without milF gene. The streptomycete can be directly fermented to generate 5-keto-milbemycins, so that the synthesis process of milbemycin oxime is simplified, and the pollution caused by the traditional chemical synthesis method is avoided.

Description

technical field [0001] The invention relates to the technical field of gene recombination, in particular to a streptomyces producing 5-ketomilbemycin and a method for producing 5-ketomilbemycin. Background technique [0002] Milbemycin is a macrolide anthelmintic drug, which was discovered by Sankyo Co., Ltd. in Japan in 1967. After years of improvement, it was officially launched in 1986 under the trade name of milbemycin oxime. Milbemycin is an oxime derivative of Milbemycin A3 and Milbemycin A4. Milbemycin is effective in controlling and preventing most common parasitic diseases. It is usually used to prevent heartworm disease, control dog and cat diseases caused by nematodes and hookworms, and whipworm disease in dogs. Due to the high insecticidal activity and low toxicity of milbe oxime, LD 50 It is more than 2000 times of the clinically recommended dosage, and at the same time, dogs sensitive to abamectins have less toxicity, so it has a good market prospect. [000...

Claims

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Application Information

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IPC IPC(8): C12N15/76C12N15/66C12N1/21C12P17/18C12R1/465
Inventor 黄隽徐赛珍周敏
Owner ZHEJIANG HISUN PHARMA CO LTD
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