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Biosynthetic gene cluster of paquete amide and application thereof

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

Active Publication Date: 2017-02-22
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • 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

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  • Biosynthetic gene cluster of paquete amide and application thereof
  • Biosynthetic gene cluster of paquete amide and application thereof
  • Biosynthetic gene cluster of paquete amide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0165] The mycelium of fresh Streptomyces pactum (Streptomyces pactum) SCSIO 02999 was inoculated into 50 mL of TSB medium (17 g of tryptone, 3 g of plant peptone, 5 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose) according to an inoculum size of 5%. g, add water to 1 L, pH 7.2-7.4), 28-30 ° C, shaking culture for about 3-4 days, 4000 rpm for 10 minutes to collect mycelia. Wash the mycelia twice with STE solution (NaCl 75mM, EDTA 25mM, Tris-Cl 20mM), add 30mL of STE solution and lysozyme with a final concentration of 3mg / mL to the washed mycelium, vortex evenly, and store at 37°C Incubate 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, put in a 55°C water bath for about 1 hour, Invert several times during this period. Add an equal volume of phenol-chloroform-is...

Embodiment 2

[0167] Streptomyces pactum (Streptomyces pactum) SCSIO 02999 whole genome library establishment:

[0168] First, through a series of dilution experiments to determine the amount of restriction endonuclease Sau3A I, in a 20 μL system, containing 17 μL of Streptomyces pactum (Streptomyces pactum) SCSIO 02999 total DNA, 2 μL of 10 × reaction buffer and 1 μL of different dilutions Sau3A I, the stop reaction is 4 μL 0.5mol / L EDTA and the appropriate loading buffer. Through exploration, it is determined that the enzyme activity unit of 0.025-0.05U is more appropriate. On this basis, a genomic DNA fragment slightly larger than 40kb was obtained by a large number of partial enzyme digestions, and dephosphorylated 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 used restriction endonuclease Bam from the multiple clonin...

Embodiment 3

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

[0172] The activity of pactamide A-F was measured 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. For experimental methods, refer to [Wu, Z.C.; Li,D.L.; Chen,Y.C.; Zhang,W.M.,A new isofuranonaphthalenone and benzopyransfrom the endophytic fungus Nodulisporium sp.A4from Aquilariasinensis.Helv.Chim.Acta 2010,93,(5),920-924.] ) as a control, its IC to 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 9.2 μM, 1.5 μM, 1.4 μM and 3.9 μM, 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 0.26μM, 0.24μM, 0.37μM and 0.51μM respectively; the IC of pactamide B on h...

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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 in particular relates to a biosynthetic gene cluster of pactamide and its application. Background technique [0002] The vigorous development of synthetic biology technology in recent years has brought new opportunities for the activation of silent biosynthetic gene clusters in deep-sea microorganisms and the discovery of new anti-tumor lead compounds. On the basis of elucidating the biosynthetic pathway in nature and understanding the mechanism of combinatorial biosynthesis, using combinatorial biosynthesis technology to knock out and recombine antibiotic biosynthetic genes in vivo can not only produce "unnatural" natural product structural analogs , and can also increase the yield of natural products, or directional accumulation of the required natural products, providing diversification of compound structure and biological activity for the discovery of natural products and drug...

Claims

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Application Information

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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
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