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Pseudomonas engineering bacteria for producing epirubicin

A technology of epirubicin and Pseudomonas putida, applied in the field of genetic engineering, can solve problems such as strict requirements, restrictions on effective development and application of epirubicin, environmental pollution and the like

Inactive Publication Date: 2011-08-17
北京赛诺百奥生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, epirubicin is mainly obtained by chemical synthesis, but the chemical route itself has some shortcomings, such as the use of a large amount of solvents, which is easy to pollute the environment; in addition, the chemical synthesis process is complicated, the cost is high, and the conditions are strict.
These factors restrict the effective development and application of epirubicin

Method used

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  • Pseudomonas engineering bacteria for producing epirubicin
  • Pseudomonas engineering bacteria for producing epirubicin
  • Pseudomonas engineering bacteria for producing epirubicin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of doxorubicin biosynthesis gene cluster cloning vector

[0041] In order to construct the expression vector of doxorubicin biosynthetic gene cluster, which was then integrated into the P.putida KT2440 genome for expression, the transformation of low-copy plasmids and the cloning of homologous fragments of the P.putida KT2440 genome were carried out successively. Cloning of homology arms of synthetic gene clusters and cloning of positive and negative selection markers.

[0042] 1. Preparation and DNA transformation of competent Escherichia coli DH10B

[0043] Pick a single colony of Escherichia coli DH10B freshly streaked from the plate, put it into 2ml LB liquid medium and shake overnight at 37°C, transfer it to 50ml LB at 1 / 50 volume, and shake it until the cell OD 600 About 0.6. Pour the bacterial solution into a pre-cooled centrifuge tube, place in an ice bath for 10 minutes, centrifuge at 5000 rpm for 5 minutes at 4°C, and discard the super...

Embodiment 2

[0076] Cloning and modification of embodiment 2 doxorubicin biosynthesis gene cluster

[0077] The invention utilizes the recombination engineering method to clone and modify the biosynthetic gene cluster of doxorubicin.

[0078] 1. Construction of a plasmid expressing the recombinase gene

[0079] Due to the many resistance markers involved in the cloning gene cluster experiment, we first constructed a recombinase expression plasmid with chloramphenicol resistance.

[0080] Design primers RC1: 5'-AAACATGGATATTAATACTGAAACTG-3', RC2: 5'-AAAAAGCTTGTCATCGCCATTGCTCCCCAAATAC-3', using lambda DNA as a template, PCR amplified to obtain a 1.9kb recombinase gene (ie gam, bet and exo gene) fragment, After digestion with NcoI and HindIII, it was cloned into the same site of pBAD322C, and the resulting plasmid was named pBADRed.

[0081] Since pBADRed and pDHA5 contain the same replicon, in order to avoid plasmid incompatibility, pBADRed was digested with NsiI and PstI, and the 3.2kb ar...

Embodiment 3

[0098] Example 3 Gene knockout of dnrH, dnrX and dnrU in the doxorubicin biosynthetic gene cluster

[0099] The genes dnrH, dnrX, and dnrU are negative regulatory genes in the doxorubicin biosynthetic gene cluster, DnrH and DnrX catalyze the conversion of doxorubicin and daunomycin to baumycin-like glycosides, and DnrU catalyzes the conversion of daunomycin Converted into 13(R)-dihydroxydaunomycin, which are all by-products of doxorubicin metabolism, blocking their formation is beneficial to the biosynthetic precursor small molecule metabolic flow to the target product doxorubicin, thereby increasing The yield of doxorubicin while eliminating by-products facilitates the isolation and purification of the target product.

[0100] 1. Knockout of dnrH gene

[0101] The gene knockout of dnrH was carried out by recombination engineering method. Firstly, dnrH gene was replaced by positive selection and negative selection gene cassette rpsL-tet through homologous recombination, and t...

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Abstract

The invention provides a pseudomonas engineering bacteria for producing an epirubicin. The preparation method of the pseudomonas engineering bacteria comprises the following steps: eliminating dnrH gene, dnrX gene and dnrU gene in an adriamycin biosynthesis gene cluster from streptomyces peucetius ATCC 29050; permuting dnmV gene to be aveBIV gene from an abamectin biosynthesis gene cluster; inserting a Pm promoter from pseudomonas before coding doxA gene of oxidation-reduction enzyme; and integrating the modified gene cluster and the screening marker gene into the genome of the pseudomonas putida KT2440 to obtain the pseudomonas engineering bacteria. The pseudomonas engineering bacteria lays a foundation for the heterologous expression and the large-scale industrial production of the epirubicin, thereby being wide in application value.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a pseudomonas engineering bacterium producing epirubicin. Background technique [0002] Epirubicin, doxorubicin and daunorubicin are anthracycline antibiotics, which are commonly used anticancer drugs in clinical practice. Doxorubicin, also known as 14-hydroxydaunorubicin, is the 4′ hydroxylated derivative of daunosamine in the structure of daunorectin. Epirubicin, also known as epirubicin, is an isomer of doxorubicin. In the structure of doxorubicin, the 4′ hydroxyl group of the daunosamine part is cis, while in the structure of epirubicin, the daunuo The 4' hydroxyl of the sugar amine moiety is trans. Strains containing a doxorubicin biosynthetic gene cluster, such as Streptomycespeucetius ATCC 29050, can simultaneously produce doxorubicin and daunomycin. [0003] Epirubicin's anti-tumor spectrum, mechanism of action and metabolic process in the human body are similar to d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/56C12R1/40
Inventor 廖志勇杨小芳
Owner 北京赛诺百奥生物技术有限公司
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