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Genetically engineered strain of streptomyces coeruleorubidus producing epi-daunorubicin and preparing method thereof

A technology of genetically engineered bacteria and epidaunorubicin, applied in the field of genetic engineering, can solve the problems of low epidaunorubicin yield and low genetic stability, and achieve the effects of accelerated screening, good repeatability and increased yield

Inactive Publication Date: 2013-07-31
SHANGHAI INST OF PHARMA IND CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Therefore, the technical problem to be solved in the present invention is exactly the low yield of the genetically engineered bacterium epidaunorubicin aimed at the existing production of epidaunorubicin Streptomyces coelicolor, low genetic stability, to provide an improved The genetic engineering strain of Streptomyces coelicolor producing epidaunorubicin, the production of epidaunorubicin of this strain is much higher than that of the existing epidaunorubicin producing strains, and the passage is very stable

Method used

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  • Genetically engineered strain of streptomyces coeruleorubidus producing epi-daunorubicin and preparing method thereof
  • Genetically engineered strain of streptomyces coeruleorubidus producing epi-daunorubicin and preparing method thereof
  • Genetically engineered strain of streptomyces coeruleorubidus producing epi-daunorubicin and preparing method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Double exchange of left and right arms and the cloning of aveBIV sequence

[0034] In Streptomyces coelicolor SIPI-1482, dnmZ and dnmU are sequentially upstream of dnmV, and dnmJ and dnmI are sequentially downstream of dnmV. The present invention takes dnmZ, dmnU plus a part of dmnV as the left arm of the double exchange, and dnmI and dnmJ plus a part dmnV was used as the right arm of the double crossover to construct the double crossover plasmid. Firstly, primers were designed according to the published corresponding sequence of Streptomyces coelicolor SIPI-1482 (GenBank accession number: AF006633):

[0035] The left arm upstream primer is 5'-AAAAAGCTTCTGGGACGGAATGGGC-3',

[0036] The downstream primer of the left arm is 5'-AAATTCGAATCGCACCAGTCGCAGATG-3';

[0037] The upstream primer on the right arm is 5'-AAATCTAGAGGGCTGGTCGTCAACATC-3',

[0038] The downstream primer of the right arm is 5'-AAAGGATCCCATCAAACTCCGCAAGACAT-3'.

[0039] The above primers were...

Embodiment 2

[0042] Example 2 Construction and conjugative transfer of double exchange site-directed insertion plasmids

[0043] The right arm in Example 1 was linked into plasmid pKC1139 (purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.), and the access sites XbaI and EcoRV were inserted to obtain a plasmid, which was named pSL-02-7-1. The left arm was positively connected to the plasmid pSL-02-7-1, and the access sites HindIII and XbaI were used to obtain the plasmid, which was named pSL-02-12-1. Use the restriction endonuclease XbaI to cut out the ErmEP+aveBIV gene from the pSL-02-26-1 plasmid in Example 1 and insert it forward into pSL-02-12-1. This plasmid is the double exchange knockout dmnV , and at the same time site-specific insertion of the epidaunorubicin expression integration plasmid of aveBIV. This plasmid was used to transform large intestine DH5α, and the transformants were cultured in LB. The plasmid was extracted for enzyme digestion and PCR ver...

Embodiment 3

[0045] The screening of embodiment 3 double exchange engineering bacteria

[0046] The zygotes obtained in Example 2 were picked and placed in a small test tube containing 4 ml of TSB medium (containing Am50 μg / ml, 2 to 3 glass beads with a diameter of 2 to 5 mm were added to the test tube), and cultured with shaking at 30°C. After the bacterial solution was shaken, take 1 μl of the bacterial solution and dilute it in 1 ml of sterile water, mix well, take 100 μl of the Gao No. 1 plate coated with Am 50 μg / ml, and incubate at 37°C. Because the pKC1139 plasmid is a temperature-sensitive plasmid, it can survive only when it is integrated into the chromosome, and all integrants that come out have undergone homologous recombination exchange. After 4 to 5 days, the integrants were picked and cultured in antibiotic-free TSB medium for continuous passage to promote double crossover. Take 1 μl of the third-generation bacterial solution, dilute it in 10ml of sterile water, mix well, ta...

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Abstract

This invention discloses a genetically engineered strain of streptomyces coeruleorubidus highly producing epi-daunorubicin, which is a genetically engineered strain to block dnmV gene and express aveBIV (avermectins keto-reductase) by inserting a first foreign gene containing aveBIV gene in the dnmV gene of a wild strain of the streptomyces coeruleorubidus or changing the dnmV gene of the wild strain of the streptomyces coeruleorubidus into the first foreign gene containing aveBIV gene. A second foreign gene is further integrated in the genome of the genetically engineered strain, and the first and second foreign genes are chain segments of a promoter and the aveBIV gene. The constructed genetically engineered strain does not contain any resistance marker and is convenient for further genetic engineering construction. The yield of epi-daunorubicin is preferably 70mg / L and is far greater than that of the epi-daunorubicin made by existing methods; through continuous passage, the yield of epi-daunorubicin is very stable with good repeatability.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered strain of Streptomyces coelicolor with high yield of epidaunorubicin, a preparation method thereof, and a used recombinant vector and transformant. Background technique [0002] Daunorubicin and doxorubin and epirubicin synthesized from it are very important anti-tumor anthracycline antibiotics in clinical practice. Epirubicin has lower cardiotoxicity and bone marrow toxicity than doxorubicin, while the antitumor effect is equivalent or higher. Epi-daunorubicin (epi-daunorubicin) is an important precursor for industrial production of epirubicin. The difference between it and daunorubicin lies in the configuration of the hydroxyl group at the C4 position of the deoxysugar in the molecule. The chemical synthesis from daunorubicin to epidaunorubicin needs to go through a multi-step reaction process with harsh conditions and will caus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/63C12N15/03C12P19/56C12R1/19C12R1/465
Inventor 邵雷黄佳李继安陈代杰景兰
Owner SHANGHAI INST OF PHARMA IND CO LTD
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