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Gene blocking mutant for streptomyces coeruleorubidus and preparation method thereof

A technology of streptomyces and mutant bacteria, applied in the biological field, to achieve the effect of increasing production and increasing fermentation units

Inactive Publication Date: 2011-04-13
SHANGHAI INST OF PHARMA IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But how to use the dauW gene to increase the production of daunorubicin is still unknown

Method used

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  • Gene blocking mutant for streptomyces coeruleorubidus and preparation method thereof
  • Gene blocking mutant for streptomyces coeruleorubidus and preparation method thereof
  • Gene blocking mutant for streptomyces coeruleorubidus and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 dauW partial gene fragment amplification

[0025] Strain: Streptomyces coeruleorubidus SIPI-1482, which produces daunorubicin, can be obtained from Shanghai Pharmaceutical Industry Research Institute.

[0026] Medium: YMB liquid medium, YEME liquid medium (Experimental Manual for Genetic Manipulation of Streptomyces hopwood [M], first edition, Changsha: Hunan Science and Technology Press, 1988).

[0027] Main solutions and buffers: TSE, TEL (Experimental Manual of Genetic Manipulation of Streptomyces Hopwood [M], first edition, Changsha: Hunan Science and Technology Press, 1988), saturated phenol / chloroform.

[0028] method:

[0029] Genomic DNA Extraction of Streptomyces coelicolor

[0030] Inoculate fresh slant spores into a 250ml Erlenmeyer flask filled with 20ml of YMB liquid medium, and culture on a shaking table at 30°C and 230r / min for about 48h. The above-mentioned liquid seeds were inserted into a 250ml Erlenmeyer flask filled with 20ml of YEME ...

Embodiment 2da

[0036] The construction of embodiment 2dauW blocking plasmid

[0037] Strain: Escherichia coli DH5α, purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0038]Plasmid: pKC1139, Escherichia coli-Streptomyces shuttle plasmid, apramycin resistance has a selective effect on both Escherichia coli and Streptomyces, Streptomyces replicons are temperature-sensitive, and cannot replicate autonomously when the temperature is higher than 34 °C (references Bierman M, Logan R, O'Brien K, Seno ET, Nagaraja Rao R, Schoner BE, Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene, 1992, 116:43-49).

[0039] Main solutions and buffers: Solution I, Solution II, Solution III (Sambrook J, Fritsch EF, Maniartis T. Molecular Cloning Experiment Guide [M], Second Edition, Beijing: Science Press, 1992.).

[0040] method:

[0041] The PCR fragment amplified above was recovered using the GeneClean II kit from BIO101 Company. Place the DNA elect...

Embodiment 3

[0050] Example 3 dauW blocking plasmid conjugative transfer to Streptomyces coelicolor SIPI-1482

[0051] Strains: Escherichia coli ET12567 (available from ATCC, Cat. No. BAA-525); Streptomyces coelicolor SIPI-1482.

[0052] Plasmid: pYG770.

[0053] Medium: MS (Kieser T, Bibb M. Practical Streptomyces Genetics [J]. Norwich: The John Innes Foundation, 2000.).

[0054] method:

[0055] The correct pYG770 plasmid verified above was transformed into Escherichia coli ET12567, and the transformant ET12567 / pYG770 was obtained by screening on the LB+Apr plate. The transformant ET12567 / pYG770 was inoculated in 2 ml of LB liquid medium (containing chloramphenicol 34 μg / ml and kanamycin 25 μg / ml), and cultured overnight at 37° C. with shaking. Inoculate 20ml of fresh LB (containing 34 μg / ml chloramphenicol and 25 μg / ml kanamycin) at a ratio of 1:100, and culture until the OD value is preferably 0.4-0.6. Centrifuge with a 50ml centrifuge tube to remove the supernatant (Hitachi CR21G,...

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Abstract

The invention discloses a gene blocking mutant for streptomyces coeruleorubidus and a preparation method thereof. In the gene blocking mutant for streptomyces coeruleorubidus, a dauW gene is blocked. In the technical scheme, the dauW gene in streptomyces coeruleorubidus is blocked through homologous recombination exchange, the yield of daunorubicin of streptomyces coeruleorubidus is greatly improved after the dauW gene is blocked and has increased by five times, and the effect is much better than that of blocking other genes, e.g. dnrX, dnrH and the like, the yield of which only can be increased by three times. Therefore, the blocking of the dauW gene substantially improves the fermentation unit of daunorubicin.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular to a gene-blocking mutant of Streptomyces coelicolor and a preparation method thereof. Background technique [0002] Daunorubicin (DNR) and doxorubicin (Doxorubicin) and epirubicin (Epirubicin) synthesized from it are clinically important anthracycline antitumor antibiotics. For the treatment of various solid tumors and acute leukemia. In industry, daunorubicin is directly produced by microbial fermentation. At present, the biosynthetic pathway of daunorubicin has been studied clearly, and the structure and function distribution map of daunorubicin biosynthetic gene cluster has been drawn accordingly. People have extensively analyzed and modified the structure and function of these genes in order to further improve the yield of daunorubicin-producing bacteria. Hu He, Shang Ke, Hu Youjia, etc. (Cloning of the dauW gene of Streptomyces coelicolor SIPI-1482 and its expression in...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/76C12P19/56C12R1/465
Inventor 胡又佳殷承慧朱春宝朱宝泉
Owner SHANGHAI INST OF PHARMA IND
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