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Daunorubicin C-14 hydroxylase mutant and production method of genetically engineered bacteria thereof

A hydroxylase mutant, a technology of genetically engineered bacteria, applied in genetic engineering, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as low productivity and pollution of the environment

Inactive Publication Date: 2014-09-24
JIANGSU HECHANG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Aiming at the above-mentioned defects and problems of low productivity and environmental pollution caused by the chemical semi-synthesis method mainly used in the existing production of doxorubicin, the purpose of the embodiments of the present invention is to provide a soft drug with better safety, higher productivity and environmental protection. The production method of the erythromycin C-14 hydroxylase mutant and its genetically engineered bacteria, through the directed evolution technique, the target gene is randomly mutated, and the high-throughput screening method is used to obtain the C-14 hydroxylation with improved enzyme activity Enzyme Mutant

Method used

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  • Daunorubicin C-14 hydroxylase mutant and production method of genetically engineered bacteria thereof
  • Daunorubicin C-14 hydroxylase mutant and production method of genetically engineered bacteria thereof
  • Daunorubicin C-14 hydroxylase mutant and production method of genetically engineered bacteria thereof

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Embodiment 1

[0078] Step Q1: Cloning of the C-14 hydroxylase gene of Streptomyces C5

[0079] Q1: The cloning method of the C-14 hydroxylase gene of Streptomyces C5, comprising the following sub-steps:

[0080] Q11: PCR amplification:

[0081] Q111: Design a pair of primers, where the underlined part is the introduced BamHI site and HindIII site:

[0082] 5'-CT GGATCC ATGGGCGGTGGGCGGTCC-3' and

[0083] 5'-AT AAGCTT CACGGGGCCGGCTTCTCG-3';

[0084] Q112: Using the Streptomyces C5 cell as a template, design PCR amplification primers for PCR amplification. The PCR system is: 2μl PCR buffer (that is, the buffer of the PCR reaction, the purpose is to provide an optimal enzymatic reaction condition); 25mMol / L MgCl 2 1μl, 2.5mMol / L dNTP1.5μl, take 1μl of the two primers in the above Q111 step, pick a small amount of Streptomyces C5 cells as a template with a toothpick, 2 units of Taq enzyme (purchased by Shanghai Sangon Company), two Methyl sulfoxide 1 μl, and finally make up to 20 μl wi...

Embodiment 2

[0092] Step Q2: Random mutations to the doxA gene

[0093] Q21: Take the primer pair:

[0094] 5'-AGCCTGAATCACTTCCGAATTCA-3' and

[0095] 5'-TTCATTGGACCTAATACCGATCA-3';

[0096] Q22: Take 50 μl of the error-prone PCR reaction system, including: 20 ng of the E. coli plasmid containing the doxA gene after the completion of the Q1 step, 30 pmol each of a pair of primers in the Q21 step, 7 mMol / LMgCl 2 , 50mMol / L KCl, 10mMol / L Tris-HCl, (pH 8.3), 0.2mMol / L dGTP, 0.2mMol / L dATP, 1mMol / L dCTP, 1mMol / L dTTP, 0.05mMol / L MnC1 2 , and 5 enzyme activity units of Taq enzyme (Shanghai Sangon Company);

[0097] Q23: Set the PCR reaction conditions as follows: denature at 94°C for 10 minutes, then maintain denaturation at 94°C for 30 seconds, then anneal at 72°C for 30 seconds, and extend at 72°C for 2 minutes;

[0098] Q24: Repeat step Q23 for 30 cycles, and then fully extend for 10 minutes at 72°C;

[0099] Q25: Use 1% agarose gel to perform nucleotide electrophoresis at a voltage of ...

Embodiment 3

[0106] Q3: Screening of mutant library:

[0107] Q31: Transformation of mutants: transform the mutants constructed after step 2 into competent cells E.coli DH10B by electric shock method (Invitrogen);

[0108] Q32: Spread the competent E.coli DH10B cells transformed in step Q31 on LB plates containing ampicillin antibiotics, and culture them at 37°C; the LB plates also contain 1% peptone, yeast extract 0.5% sodium chloride, 1% sodium chloride, 2% agar;

[0109] Q33: Screening and identification of mutants:

[0110] Q331: Pick the bacteria grown on the LB plate and transfer them to liquid LB test tubes, culture them overnight in a shaker at 220 rpm in a culture environment at 37°C, and transfer them to 50ml LB the next day In the 250ml shake flask of the culture medium, after 3 hours, the bacterium was induced with 1mMIPTG, after continuing to cultivate for 18h, after centrifugation at 4000 rpm for 15 minutes, the bacterium was collected;

[0111] Q332: Carry out daunorubici...

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Abstract

The invention provides a daunorubicin C-14 hydroxylase mutant and a production method of genetically engineered bacteria thereof. In a C-14 hydroxylase sequence coded by genes of the daunorubicin C-14 hydroxylase mutant, the glutamic acid at the 121 position is mutated into valine; through directed evolution technology, a target gene is subjected to random mutation, and a C-14 hydroxylase mutant with improved enzyme activity is obtained through a high-throughput screening method; the safety is better, the production rate is higher, and the method is environment-friendly.

Description

technical field [0001] The invention relates to the technical field of bioengineering pharmacy, in particular to a production method of a daunorubicin C-14 hydroxylase mutant and a genetically engineered bacterium. Background technique [0002] Anthracyclines (Anthracyclines) are an important class of anti-tumor drugs, they are all composed of anthracyclone chromophores linked with one or several different sugars through glycosidic bonds. Anthracycline antibiotics are mainly distinguished by the following structural changes: different ligands on the anthracycline; different number, type and sequence of sugars; different positions and stereochemical structures of glycosidic bonds. Most anthracycline antibiotics have strong bone marrow suppression and cardiotoxicity and cannot be used clinically. Only daunorubicin-doxorubicin-type anthracycline antibiotics have become the most clinically applicable antitumor drugs, including daunorubicin-doxorubicin. Erythromycin, Adriamycin,...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N1/21C12N15/70C12P19/56C12R1/19
CPCC12N9/0004C12N15/70
Inventor 余宏徐超波王波陈欢斌张道生
Owner JIANGSU HECHANG BIOLOGICAL TECH
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