Sorangium cellulosum highly producing epothilone D as well as construction method and application of sorangium cellulosum

A technology of S. cellulosus and epothilone, which is applied in the fields of biochemistry and molecular biology, can solve the problems of unseen production and less application of S. cellulosus epothilones, and achieve broad-spectrum and high-efficiency anti-tumor Activity, the effect of expanding the application

Active Publication Date: 2019-12-31
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TALE elements mainly include TALEN elements and TALE-TF elements. Due to the advantages of high gene knockout efficiency and almost no off-target effects, TALEN elements have been widely used in genome editing of eukaryotic cells such as mammali...

Method used

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  • Sorangium cellulosum highly producing epothilone D as well as construction method and application of sorangium cellulosum
  • Sorangium cellulosum highly producing epothilone D as well as construction method and application of sorangium cellulosum
  • Sorangium cellulosum highly producing epothilone D as well as construction method and application of sorangium cellulosum

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Amplification of the epoK gene in S. cellulosus So ce M4 and construction of TALEN vector:

[0022] The primers were designed according to the known epoK gene of S. cellulosus, and the epoK gene of S. cellulosus So ce M4 was amplified. The left arm target sequence (5'-GAGACGAAGCCTGCTTT-3') and right arm target sequence (5'-CCTCCGCGTACCCAGGCG-3') of TALEN were selected from the amplified epoK gene as the target sequence. Using the Fast TALENassembly kit (Shanghai Stance Biological Engineering Co., Ltd., article number: 2801-005), TAL repeat units were designed according to the target base sequences of the left and right arms of the TALEN, and the TALE repeat units targeting the left arm and right arm of the epoK gene were respectively obtained. The TALE repeat unit targeting the left arm of the epoK gene was linked to the left arm vector PtalenL48 to obtain a recombinant TALEN-Ptalen L48 vector, and the TALE repeat unit targeting the right arm of the epoK gene was linked...

Embodiment 2

[0025] Introduction of recombinant TALEN vector and knockout of epoK gene in S. cellulosus So ce M4 strain:

[0026]The recombinant TALEN backbone vectors (epoK-TALEN-L48-P43 and epoK-TALEN-R36-P43) containing the target sequence of the left arm and the target sequence of the right arm were simultaneously introduced into the competent cells of S. cellulosus SoceM4 by electroporation In , the G52 plate containing kanamycin was used for screening. The successful introduction of the recombinant TALEN vector into S. cellulosus Soce M4 was verified by amplifying the ColE1 replicon. Using the expanded cultured bacterial solution as a template, the nucleotide sequence (F: 5'-TGAGATCCTTTTTTTCTGCG-3', R: 5'-TTTCCATAGGCTCCGCCCCC-3') as shown in SEQ ID NO.1 and SEQ ID NO.2 is used as The primers were used for PCR amplification, and the PCR amplification of the bacteria solution successfully obtained the ColE1 replicon, which confirmed the introduction of the recombinant TALEN backbone v...

Embodiment 3

[0029] Analysis of epothilone production in wild So ce M4 and recombinant So ce M4 strains (△epoK So ce M4):

[0030] Ferment the wild So ce M4 and △epoK So ce M4 strains, add macroporous adsorption resin, and use methanol to elute the product on the macroporous adsorption resin. The obtained products were loaded on HPLC-MS respectively, and the yields of epothilones B and epothilones D in the products were compared and analyzed. The results showed that the yield of epothilone D in the wild So ce M4 strain was 4.35±0.16mg / L, and the yield of epothilone D in the △epoK So ce M4 strain was 5.82±0.25mg / L. And the output of epothilone B in wild So ce M4 bacterial strain is 7.64 ± 1.18mg / L, the output of epothilone B in △ epoK So ce M4 bacterial strain is 5.06 ± 0.16mg / L ( Figure 5 ). The above results show that knocking out the epoK gene in the Socemus cellulosum So ce M4 strain using TALEN technology can significantly increase the production of epothilone D, while reducing the ...

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Abstract

The invention discloses sorangium cellulosum highly producing epothilone D as well as a construction method and application of the sorangium cellulosum. The invention discloses construction of a recombinant sorangium cellulosum So ce M4 strain with an epoK gene knocked out by a TALEN technology for the first time. Tests prove that by knocking out the epoK gene in the sorangium cellulosum So ce M4strain by the TALEN technology, the yield of the epothilone D can be significantly increased, and meanwhile, the yield of epothilone B can be reduced, so that directed biosynthesis of epothilone in the sorangium cellulosum can be realized. The invention discloses the increase of the yield of the epothilone produced by the sorangium cellulosum by the TALEN for the first time, the TALEN technology is applied to increase of the yield of the epothilone D produced by the sorangium cellulosum, biosynthesis of the epothilone is promoted while the development of a sorangium cellulosum genetic engineering modification technology is promoted, and the application of the epothilone D in antitumor aspects is enlarged, which has quite important significance in development of biomedicine in China.

Description

technical field [0001] The invention relates to the fields of biochemistry and molecular biology, and in particular to a Sopramyces cellulosus high-yielding epothilone D and its construction method and application. Background technique [0002] Epothilone is a sixteen-membered macrolide compound with spectrally efficient antitumor activity. Due to its good water solubility, more efficient anti-tumor activity and lower side effects, it is an anti-tumor drug that is expected to replace paclitaxel. At present, ixabepilone, a derivative of epothilone B, has been used in the clinical treatment of advanced breast cancer, and epothilone D has also been used in clinical phase III trials for the treatment of breast cancer. However, the low yield of epothilone limits its wide application. The cytotoxicity of epothilone leads to low yield of its heterologous expression, and the lack of genetic manipulation system of S. cellulosus limits its metabolic engineering. TALE is a class of ...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12P17/16C12R1/01
CPCC12N9/0004C12N15/74C12P17/16
Inventor 叶伟章卫民刘桃妹孔亚丽朱牧孜刘珊
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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