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

A biosynthetic gene cluster of pactamide and its application

A technology of pactamide and biosynthesis, applied in the direction of plant genetic improvement, application, microorganism, etc.

Active Publication Date: 2019-06-21
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far, there have been no reports at home and abroad about the biosynthetic gene cluster of pactamide, cyclase and other biocatalyzed preparation of pactamide and promoter engineering to obtain anti-tumor pactamide and its analogues.

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
  • A biosynthetic gene cluster of pactamide and its application
  • A biosynthetic gene cluster of pactamide and its application
  • A biosynthetic gene cluster of pactamide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0164] Extraction of total DNA from Streptomyces pactum SCSIO 02999:

[0165] Fresh Streptomyces pactum (Streptomyces pactum) SCSIO 02999 mycelium was inoculated in 50 mL of TSB medium (Tryptone 17g, Plant Peptone 3g, Sodium Chloride 5g, Dipotassium Hydrogen Phosphate 2.5g, Glucose 2.5%) g, add water to 1L, pH 7.2-7.4), 28-30°C, shake culture for about 3-4 days, centrifuge at 4000rpm for 10 minutes to collect mycelium. The mycelium was washed twice with STE solution (NaCl 75mM, EDTA 25mM, Tris-Cl 20mM), 30mL STE solution and lysozyme at a final concentration of 3mg / mL were added to the washed mycelium, vortexed evenly, 37℃ Warm for 3 hours, add proteinase K to a final concentration of 0.1-0.2mg / mL, mix well, incubate at 37°C for 10 minutes, add SDS to a final concentration of 1-2%, mix well, and put it in a 55°C water bath for about 1 hour. The period was reversed several times. Add an equal volume of phenol-chloroform-isoamyl alcohol (V / V / V=25:24:1), mix well, and cool on ice ...

Embodiment 2

[0167] The establishment of the whole genome library of Streptomyces pactum SCSIO 02999:

[0168] First, determine the amount of restriction endonuclease Sau3A I through a series of dilution experiments. In a 20μL system, 17μL of Streptomyces pactum SCSIO 02999 total DNA, 2μL of 10× reaction buffer and 1μL of different dilutions For high-degree Sau3A I, the termination reaction is 4μL 0.5mol / L EDTA and a suitable loading buffer. Through exploration, it was determined that 0.025-0.05U enzyme activity unit is more appropriate. On this basis, a genomic DNA fragment slightly larger than 40kb was obtained by a large number of partial enzyme digestion, and dephosphorylation treatment was performed with a dephosphorylase.

[0169] The vector SuperCos l plasmid used to construct the library was first cut from the middle of the two cos sequences with restriction endonuclease Xba I, then dephosphorylated, and then restriction endonuclease Bam was used from the multiple cloning site HI inci...

Embodiment 3

[0171] Example 3: Determination of the cytotoxic activity of pactamide A-F

[0172] The activity of pactamide AF was tested against four tumor cell lines: human breast cancer MCF-7 cells, human liver cancer cells HepG2, human lung cancer H460 cells, and human glioma cells SF-268. The experimental methods are referenced in the literature [Wu,ZC; Li,DL; Chen,YC; Zhang,WM,A new isofuranonaphthalenone and benzopyrans from the endophytic fungus Nodulisporium sp.A4from Aquilariasinensis.Helv.Chim.Acta 2010,93,(5),920-924.], with Cisplatin (Cisplatin ) As a control, its IC against human cancer cells including breast cancer MCF-7, human lung cancer H460 cells, human liver cancer HepG2 and human glioma cells SF-268 50 They are 9.2μM, 1.5μM, 1.4μM and 3.9μM respectively. The results showed that the IC of pactamide A on human cancer cells including breast cancer MCF-7, human lung cancer H460 cells, human liver cancer HepG2 and human glioma cells SF-268 50 They are 0.26μM, 0.24μM, 0.37μM and...

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 a biosynthetic gene cluster of paquete amide and an application thereof. The biosynthetic gene cluster of paquete amide comes from (Streptomyces pactum)SCSIO 02999, and the cluster comprises five genes including heterozygous polyketone / nonribosomal peptide synthetases genes ptmA, FAD dependent redox enzyme genes ptmB1, phytoene dehydrogenase genes ptmB2, cyclase genes ptmC and hydroxylase genes ptmD. According to the biosynthetic gene cluster of paquete amide and the application thereof, information in genes and proteins related to biosynthesis of the paquete amide provides theoretical foundations and materials for conducting genetic modification on the biosynthesis of multi-ring tetramate macrocylic lactam family. By conducting genetic modifications on the biological synthetic genes, 6 new structures antineoplastic paquete amide compounds pactamide A-F are obtained, and thus effective compound entities are provided for research and development of antineoplastic drugs.

Description

Technical field [0001] The invention belongs to the field of microbial genetic engineering, and specifically relates to a biosynthetic gene cluster of Pacotamide and its application. Background technique [0002] Synthetic biology technology, which has developed vigorously in recent years, has brought new opportunities for activating the silent biosynthetic gene clusters contained in deep-sea microorganisms and discovering new anti-tumor lead compounds. On the basis of clarifying natural biosynthetic pathways and understanding the mechanism of combinatorial biosynthesis, using combinatorial biosynthesis technology to knock out and recombine the biosynthetic genes of antibiotics in vivo can not only produce structural analogs of "non-natural" natural products. It can also increase the yield of natural products, or directional accumulation of required natural products, and provide diversification of compound structure and biological activity for natural product discovery and drug d...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N9/00C12N1/21C12N15/53C12P17/18C07D487/08A61K31/407A61P35/00C12R1/465
CPCC07D487/08C12N9/00C12N9/0004C12N9/001C12P17/18C12Y103/05005
Inventor 张长生张光涛张文军朱义广袁呈山张庆波张海波张丽萍
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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