Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Engineering bacterial strain for heterogenous expression of histone deacetylases inhibitor and application thereof

A deacetylase, engineering strain technology, applied in the field of biosynthesis, to achieve the effect of increasing yield

Active Publication Date: 2018-01-19
SHANDONG UNIV
View PDF1 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After searching, there is no report on the literature of using the biosynthetic gene cluster (tdp) of Thailandepsins to achieve expression in the heterologous host strain Burkholderia sp.DSM7029 to obtain an engineering strain capable of expressing the histone deacetylase inhibitor Thailandepsin A

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Engineering bacterial strain for heterogenous expression of histone deacetylases inhibitor and application thereof
  • Engineering bacterial strain for heterogenous expression of histone deacetylases inhibitor and application thereof
  • Engineering bacterial strain for heterogenous expression of histone deacetylases inhibitor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Construction of Thailandepsins biosynthetic gene cluster (tdp) expression plasmid

[0029] (1) Direct cloning of Thailandepsins biosynthetic gene cluster (tdp)

[0030] The process of direct cloning of Thailandepsins biosynthetic gene cluster (tdp) see figure 1 .

[0031] The specific steps are: restriction endonuclease AvaI digests the plasmid p15A-cm-tetR-tetO-hyg-ccdB to obtain the fragment p15A-cm-tetR-tetO (enzyme digestion and recovery of large fragments, the glue runs to the bottom and then cuts the glue, and the glue is recovered For specific methods, refer to the instructions of the Tiangen kit). Then use p15A-cm-tetR-tetO as a PCR template, use primers p15A-cm-tet-5 and p15A-cm-tet-3 PCR to amplify the fragment p15A-cm-tetR-tetO, and obtain the PCR product p15A-cm- The two ends of the tetR-tetO vector fortdp have homology arms of the sequences at both ends of the Thailandepsins biosynthetic gene cluster (tdp). Then, the genomic DNA of the PCR...

Embodiment 2

[0049] Example 2: Construction of engineering strains expressing histone deacetylase inhibitor Thailandepsin A heterologously

[0050] The plasmid p15A-tnpA-apra-tetR-tetO-tdp was electrotransformed into Burkholderia sp.DSM7029, and the electrotransformation step was: Burkholderia sp.DSM7029 was placed in CYMG (Casitone 8g / L, MgCl 2 4g / L, Yeastextract 4g / L, 50% glycerol 10ml / L) culture medium at 30°C for 12h. Transfer 40 μl of culture to CYMG medium (OD 600 ≈0.1), placed on an Eppendorf thermomixer at 30°C, 950rpm for 14h (OD 600 ≈1.8). Cells were collected by centrifugation at 9,400g for 30sec. Discard the supernatant and pellet with 1 ml H 2 O suspension. Repeat centrifugation, resuspension, and centrifugation again, with 20 μl H 2O Suspension cells. Add 1 μg of plasmid p15A-tnpA-apra-tetR-tetO-tdp, transfer the mixture of cells and DNA into a 1mm electric shock cup, and use Eppendorf electroporator 2510 for electric shock with a voltage of 1250V, a capacitance of 10...

Embodiment 3

[0066] Example 3: Application of the engineering strain 7029-tdp-A of the present invention in the preparation of histone deacetylase inhibitor Thailandepsin A

[0067] The engineering strain 7029-tdp-A was inoculated in the CYMG medium added with apramycin (20 μg / ml), and cultured at 30° C. for 24 hours. The overnight culture was inoculated into 50 ml of fresh CYMG medium (apramycin 20 μg / ml) at an inoculum volume of 1%. After culturing for 2 days at 30° C. and 200 rpm, XAD-16 macroporous adsorption resin with a final concentration of 2% was added to continue culturing for 2 days. Cells and macroporous resin were collected by centrifugation at 8000rpm for 10min, and then extracted with methanol. Methanol was removed by rotary evaporation and the remaining extract was dissolved in 1 ml methanol. After filtration with a 0.22 μm filter membrane, 3 μl was taken for HPLC-MS analysis. The high performance liquid chromatograph model is UltiMate TM 3000 RSLC. The chromatographic...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an engineering bacteria strain for heterogenous expression of a histone deacetylases inhibitor Thailandepsin A. The bacterial strain is named as the engineering bacterial strain 7029-tdp-A, which uses Burkholderia sp.DSM7029 as an original strain, and a biosynthetic gene cluster (tdp) of Thailandepsins is integrated on a genome through a transposition mode. The invention also discloses an application of the engineering bacterial strain in preparation of the histone deacetylases inhibitor Thailandepsin A. The experiment proves that the engineering bacterial strain 7029-tdp-A and a wild bacterial strain Burkholderia thailandepsis E264 are compared, the output of Thailandepsin A is increased by one time, and the engineering bacteria strain establishes a base for large-scale preparation and exploitation of the histone deacetylases inhibitor drugs.

Description

technical field [0001] The invention relates to an engineering strain and its construction and application, in particular to an engineering strain expressing histone deacetylase inhibitor Thailandepsin A heterologously and its construction and application, belonging to the technical field of biosynthesis. Background technique [0002] Histone deacetylases (HDACs) can remove acetyl groups from the N-terminal lysine residues of histones, maintaining a more open state of transcriptional activity in chromatin, thereby regulating the expression of silent tumor suppressor genes . Romidepsin, the first HDAC inhibitor exhibiting antitumor activity, was originally isolated from the rod-shaped Gram-negative bacterium Chromobacterium violaceum in Japanese soil samples. The anticancer drug preparation (injection) of Romidepsin is called Istodax, which is developed by Gloucester Pharmaceuticals of the U.S. and was approved by the U.S. FDA on November 9, 2009. Romidepsin, also known as ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N1/21C12N15/74C12P17/18C12R1/01
Inventor 张友明李瑞娟宋超逸涂强
Owner SHANDONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com