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Daunorubicin and adriamycin producing engineered pseudomonas

A technology of Pseudomonas putida and doxorubicin, which is applied in the field of genetic engineering, can solve the problems of slow growth of bacteria, restrictions on the effective development and application of daunorubicin and doxorubicin, and the difficulty of mutagens to exert their efficacy.

Inactive Publication Date: 2011-11-02
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

And adopt S.peucetius ATCC 29050 to carry out some deficiencies in the production of daunorubicin and doxorubicin, such as slow cell growth (the doubling time of the cell is nearly 48 hours), difficult to cultivate (need strict oxygen-rich environment , and needs to be fully stirred to make the bacteria evenly mixed) and genetic breeding is difficult to carry out (the strain is Gram-positive bacteria with a thick cell wall, so it is difficult for mutagen to exert its effect), etc.
These factors restrict the effective development and application of daunorubicin and doxorubicin

Method used

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  • Daunorubicin and adriamycin producing engineered pseudomonas
  • Daunorubicin and adriamycin producing engineered pseudomonas
  • Daunorubicin and adriamycin producing engineered pseudomonas

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Construction of doxorubicin biosynthesis gene cluster cloning vector

[0037] 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.

[0038] 1. Preparation and DNA transformation of competent Escherichia coli DH10B

[0039] 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

[0072] Cloning and modification of embodiment 2 doxorubicin biosynthesis gene cluster

[0073] The invention utilizes the recombination engineering method to clone and modify the biosynthetic gene cluster of doxorubicin.

[0074] 1. Construction of a plasmid expressing the recombinase gene

[0075] Due to the many resistance markers involved in the cloning gene cluster experiment, we first constructed a recombinase expression plasmid with chloramphenicol resistance.

[0076] Design primers RC1: 5'-AAACATGGATATTAATACTGAAACTG-3', RC2: 5'-AAAAAGCTTGTCATCGCCATTGCTCCCCAAATAC-3', using lambda DNA as a template, and 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.

[0077] Since pBADRed and pDHA5 contain the same replicon, in order to avoid plasmid incompatibility, pBADRed was digested with NsiI and PstI, and the 3.2k...

Embodiment 3

[0094] Example 3 Heterologous Expression of Doxorubicin Biosynthesis Gene Cluster

[0095] 1. Transformation of Pseudomonas putida KT2440 by three-parent combination transfer method

[0096] Use P.putida KT2440 as the host bacterium, E.coli HB101 / pRK2073 as the assisted transfer strain, and E.coli DH10B / pSino-202 as the strain containing the DNA to be transformed.

[0097] Take out the cryopreservation tube, streak the host strain P.putida KT2440 on the LB plate, culture overnight at 30°C; streak the assisted transfer strain E.coliHB101 / pRK2073 on the LB plate containing 50 μg / ml streptomycin, culture overnight at 37°C ; Streak E.coli DH10B / pSino-202 on an LB plate containing 30 μg / ml kanamycin and culture overnight at 37°C.

[0098] Pick a single colony of P.putida KT2440 and put it into 2ml LB liquid medium, 30°C 220rpm, and shake it overnight; pick a single colony of E.coli HB101 / pRK2073 and put it into 2ml of LB liquid medium containing 50μg / ml streptomycin, Shake at 220...

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Abstract

The invention provides daunorubicin and adriamycin producing engineered pseudomonas, which is constructed by substituting doxE for dnrM in a TDP-D-glucose 4,6-dehydratase gene lacking frame shift from adriamycin biological synthesis gene cluster of Streptomyces peucetius ATCC 29050, and integrating the TDP-D-glucose 4,6-dehydratase gene and a screened marker gene into genome of Pseudomonasputida KT2440. The daunorubicin and adriamycin producing engineered pseudomonas provides a basis for the heterologous expression and large-scale industrial production of daunorubicin and adriamycin and has high wide application value.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a pseudomonas engineering bacterium producing daunomycin and doxorubicin. Background technique [0002] Daunorubicin and doxorubicin are anthracycline antibiotics, which are the basic drugs for tumor chemotherapy recommended by the United Nations World Health Organization (WHO). Daunorubicin, also known as daunorubicin, was first discovered by Dimarco in 1963. It has anti-Gram-positive bacteria, anti-Gram-negative bacteria, tumors and viruses, and it also has immunosuppressive properties. Doxorubicin, also known as 14-hydroxydaunorubicin, is the 4′ hydroxylated derivative of daunosamine in the structure of daunorectin. [0003] Daunomycin and doxorubicin are mainly used clinically for the treatment of acute lymphocytic or myeloid leukemia, neuroblastoma, rhabdomyosarcoma and other diseases, and also have different degrees of effects on various solid tumors, brain tumors, heman...

Claims

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Application Information

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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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