Milbemycins producing recombinant streptomyces as well as preparation method and application thereof

A technology of mirbemycin and streptomyces, which is applied in the field of molecular biology and can solve problems such as being difficult to remove

Active Publication Date: 2017-05-31
ZHEJIANG HISUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there is also a class of milbemycin analogs α9 and α10 ( figure 1 ) is difficult to completely remove during the extraction and purification of Milbemycins (Takiguchi Y et al.1980, Milbemycins, a new family of macrolide antibiotics: fermentation, isolation and physico-chemical properties. J Antibiot (Tokyo). 1980 Oct; 33 (10 ):1120-7)
At present, the synthetic pathways of milbemycin analogs α9 and α10 in Streptomyces have not been identified in the field, so there is no relevant report on reducing or removing milbemycin analogs α9 and α10 by modifying related pathways

Method used

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  • Milbemycins producing recombinant streptomyces as well as preparation method and application thereof
  • Milbemycins producing recombinant streptomyces as well as preparation method and application thereof
  • Milbemycins producing recombinant streptomyces as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Extraction of total DNA from Streptomyces

[0088] Take 50μL of Streptomyces HS023 cryopreservation tube spore suspension and inoculate it in 30mL TSB medium (purchased from Bacto TrypticSoy Broth. BD). After incubating at 28°C and 220rpm for 48h, centrifuge at 4000rpm for 10min in a 50mL centrifuge tube and remove the supernatant. The precipitate was washed twice with 30 mL of sucrose-Tris buffer (the mass percentage of sucrose was 10.3%, the molar-volume concentration of Tris-HCl was 10 mM, and the pH was 8.0), and then suspended in 5 mL of sucrose-Tris buffer. Add 20μL of lysozyme solution with a mass-volume solubility of 100mg / mL and water bath at 37°C for 2h. Add 500 μL of 10% SDS solution and gently invert until it is almost clear. Add 5 mL of phenol-chloroform-isoamyl alcohol (the volume ratio of phenol-chloroform-isoamyl alcohol is 25:24:1 (pH value is 8.0)). After gently inverting several times, centrifuge at 4000 rpm for 10 minutes. Take 4 mL of the u...

Embodiment 2

[0089] Example 2: Construction of Milbemycin-producing Strain Genomic Library

[0090] Apply the cosmid carrier SuperCos 1 (see figure 2 ) Construction of HS023 genome library. First, establish a suitable partial digestion system. Use Sau3A1 to partially digest 50-100μg of high molecular weight chromosomal DNA, and then use Tris saturated phenol: chloroform: isoamyl alcohol (25:24:1) to extract twice. Chloroform: isoamyl alcohol (24:1) was extracted once. Transfer the upper aqueous phase to a new tube, add 0.1 volume of 3mol / L sodium acetate and twice the volume of ethanol, mix well, and place at -20°C for 30 minutes. The DNA was precipitated by centrifugation (12000r / min, 10min), the DNA pellet was washed with 70% ethanol, the DNA pellet was dried, and the DNA pellet was suspended in an appropriate volume of TE buffer and stored at 4°C for later use. The double-cos site vector SuperCos 1 first fully digested the plasmid DNA with NheI, then fully treated with CIAP, phenol-chlo...

Embodiment 3

[0091] Example 3: Gene library screening

[0092] Design the universal primer PcaF / PcaR (SEQ ID NO:1 / SEQ ID NO:2), screen the genomic library constructed in Example 2 by colony PCR, screen a total of 1000 single colonies, and obtain two Cosmids that may contain the desired sequence, respectively Numbering pSCM-7C11 and pSCM-4C12, using universal primers T7 and T3 both ends of sequencing, the results showed that the four genes in the pSCM-7C11 vector are located in the center of the sequence (see image 3 ), suitable for subsequent gene manipulation, and pSCM-7C11 was sequenced at the same time to obtain the full sequence of the four genes (see SEQ ID NO: 7).

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Abstract

The invention relates to milbemycins producing recombinant streptomyces. In comparison with corresponding wild streptomyces or start streptomyces generating the recombinant streptomyces, the milbemycins producing recombinant streptomyces is characterized in that at least one of the following genes of the recombinant streptomyces is inactivated: gene mpca5 with code SEQ ID NO:8 or amino acid sequence with at least 80% of sequence identity, gene mpca3 with code SEQ ID NO:9 or amino acid sequence with at least 80% of sequence identity, gene mpca4 with code SEQ ID NO:10 or amino acid sequence with at least 80% of sequence identity and gene mpca5 with code SEQ ID NO:11 or amino acid sequence with at least 80% of sequence identity. The invention also relates to a method for generating the recombinant streptomyces and a method for producing milbemycins by use of the recombinant streptomyces.

Description

Technical field [0001] The present invention relates to the technical field of molecular biology, in particular to a method for preparing recombinant engineered streptomyces for producing antibiotics, especially milbemycin, the obtained recombinant streptomyces and applications thereof. [0002] Background of the invention [0003] Milbemycin is a macrolide anthelmintic. It was discovered by Sankyo Co., Ltd. in 1967. After years of improvement, it was officially marketed under the trade name milbemycin oxime in 1986. Milbemycin A3 and Milbemycin A4 are oxime derivatives of Milbemycin A3 and Milbemycin A4. Milbemycin has a good effect on 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 canine whipworm disease. As Milbex oxime has high insecticidal activity and low toxicity, the LD50 is more than 2000 times the recommended clinical dosage. At the same time, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C07K14/36C12P17/18C12R1/465C12R1/55
CPCC07K14/36C12P17/181
Inventor 黄隽党福军周军徐晴雨李娜
Owner ZHEJIANG HISUN PHARMA CO LTD
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