A kind of streptomyces producing 5-ketomilbemycin and the method for producing 5-ketomilbemycin
A technology of milbemycin and streptomyces, which is applied in the field of producing 5-ketomilbemycin, and can solve problems such as shortening the production process of milbemycin
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Embodiment 1
[0139] Embodiment 1: the extraction of Streptomyces total DNA
[0140] Inoculate 50 μL of Streptomyces spore suspension in a cryopreserved tube into 30 mL of TSB medium (purchased from Bacto Tryptic SoyBroth.BD), culture at 28°C and 220 rpm for 48 hours, then centrifuge at 4000 rpm for 10 minutes in a 50 mL centrifuge tube, remove the supernatant, and precipitate After washing twice with 30 mL of sucrose-Tris buffer (in which the mass percent of sucrose is 10.3%, the molar-volume concentration of Tris-HCl is 10 mM, and the pH value is 8.0), it is 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, centrifug...
Embodiment 2
[0141] Embodiment 2: The construction of the recombinant vector that is used for knocking out Streptomyces milF gene
[0142] With pMD19 (Simple) as the backbone, its construction process is as follows figure 2 As shown, specifically:
[0143] 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;
[0144] 1. Preparation of PCR amplification reaction solution, in which PrimeSTAR kit was purchased from TaKaRa:
[0145]
[0146] 2. Divide into 2 tubes for PCR reaction, the procedure is:
[0147]
[0148] 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.
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Embodiment 3
[0158] Embodiment 3: The construction of the recombinant vector that is used for knocking out Streptomyces milF gene
[0159] With pBlueScript SK(+) as the skeleton, its construction process is as follows Figure 4 As shown, specifically:
[0160] 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:
[0161] 1. Preparation of PCR amplification reaction solution, in which PrimeSTAR kit was purchased from TaKaRa:
[0162]
[0163] 2. Divide into 2 tubes for PCR reaction, the procedure is:
[0164]
[0165] 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 sequence ...
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