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Cyclothiazomycin analogue generated by mutation of precursor peptide groups, and uses

A technology of cyclic thiazomycin and precursor peptide, which is applied in the field of microbial resources and genetic engineering, can solve the problems of low solubility and slow transformation speed, and achieve the effect of enriching methods and means

Inactive Publication Date: 2012-07-04
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is precisely because cyclic thiazomycin has a tricyclic structure and contains multiple thiazole rings or thiazolines that its solubility is low, which poses a huge challenge for scientists to analyze its spatial structure
In 2006, Japanese scientists successfully described the three-dimensional structure of cyclic thiazomycin by NMR method, and separated two derivative isomers B1 and B2 of cyclic thiazomycin in different conformations from the fermentation product. They found that these two The derivative isomers are very stable in the solid state, but can be converted into each other in solution, but the conversion rate is very slow [Bioorg Med Chem (2006) 14, 8259-70]

Method used

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  • Cyclothiazomycin analogue generated by mutation of precursor peptide groups, and uses
  • Cyclothiazomycin analogue generated by mutation of precursor peptide groups, and uses
  • Cyclothiazomycin analogue generated by mutation of precursor peptide groups, and uses

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Embodiment 1, the extraction of escherichia coli plasmid DNA

[0057] Take 1-1.5ml of Escherichia coli overnight culture into EP tube, centrifuge at 12000rpm for 30 seconds and remove the supernatant thoroughly. Add 150 μl of alkaline lysis solution I to the EP tube with a pipette gun, break up the precipitated bacteria on the shaker, and suspend it; then add 300 μl of alkaline lysis solution II, and mix gently to fully lyse the bacteria; then add 230 μl alkaline lysis solution III; add 100 μl chloroform (chloroform), shake evenly; then put the EP tube into the centrifuge and centrifuge at 12000 rpm for 5 minutes. After centrifugation, carefully remove the supernatant to a new EP tube; add an equal volume of iso Propanol, invert the centrifuge tube up and down, mix well, and place at -20°C for 5 minutes; then centrifuge at 12,000 rpm for 1 minute, discard the supernatant; wash the precipitate twice with 75% ethanol, and finally dry the precipitate and dissolve it in a...

Embodiment 2

[0058] The extraction of embodiment 2, streptomyces total DNA

[0059] Take 50-100 μl of mycelium and put it in an EP centrifuge tube, centrifuge to remove the supernatant to collect the streptomyces mycelium, add 500 μl of lysozyme solution, and bathe in water at 37°C until the mycelium becomes viscous, usually 30-60 minutes; Add 500 μl of 2% SDS, mix and oscillate until the solution is not viscous and more transparent; add 100 μl of mesophenol, oscillate, centrifuge at 12,000 rpm for 5 minutes, take the supernatant, then add an appropriate amount of mesophenol, oscillate, centrifuge, and take the supernatant; add 1 / 10 times the volume of 3mol / L NaAc (natural pH) and equal volume of isopropanol, mix them upside down, place at -20°C for 5 minutes, then pick out the flocculent precipitate with a pipette tip and wash it twice in 75% ethanol, and discard it at last All supernatants were dried and dissolved in an appropriate amount of TE buffer or sterilized ultrapure water.

Embodiment 3

[0060] Example 3, Preparation of Escherichia coli Calcium Transduction Competent Cells and Transformation with Exogenous DNA

[0061] The Escherichia coli DH10B strain was activated on a new LA plate, and a single colony of Escherichia coli on a fresh LA plate was picked, inserted into 5ml of LB liquid medium, and cultured on a shaker at 37°C for 12 hours. According to the inoculum size of 1 / 100, the overnight culture of Escherichia coli was transferred to 100ml of LB liquid medium (20mmol / L MgCl could be added in LB 2 ), cultured on a shaker at 37°C for 2.5 hours to 3 hours so that the OD600 value of the bacteria is about 0.6, and quickly put the bacteria in an ice bath, and the cells should be kept in an environment of 0°C to 4°C since then. Centrifuge at 3500rpm at 4°C for 6min to collect the bacteria, discard the supernatant, and use ice-cold 0.1M CaCl 2 Resuspend the cells and place them on an ice bath; centrifuge at 4°C, remove the supernatant, collect the cells, and ...

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Abstract

The invention relates to a cyclothiazomycin analogue generated by mutation of precursor peptide groups, and uses. Amino acid residues at seven sites of a cyclothiazomycin precursor peptide are subjected to point mutation, so that 14 mutant strains are obtained, and 23 novel cyclothiazomycin analogue antibiotics are generated by the heterologous expression of type strain streptomyces lividans 1326 of streptomyces, wherein the products of six mutant strains can inhibit the growth of bipolaris maydis. Compared with the prior art, the cyclothiazomycin analogue can allow the mutant strain building process to be quick and efficient, and random mutant strains can be obtained, the research of biosynthetic mechanism can be conducted by the products generated by the cyclothiazomycin mutant strains so as to provide richer methods and means for the research of the biosynthetic mechanism of sulfur peptide antibiotics. Most importantly, the obtained novel sulfur peptide antibiotics open a way for the artificial design and synthesis of novel sulfur peptide antibiotics.

Description

technical field [0001] The invention belongs to the field of microbial resources and genetic engineering, and in particular relates to the generation of cyclic thiazomycin analogs by mutation of precursor peptide groups and its method and application. Background technique [0002] Antibiotics are a class of secondary metabolites with anti-pathogenic or other activities produced by microorganisms (including bacteria, fungi, and actinomycetes) or higher animals and plants during their life, which can interfere with the developmental functions of other living cells chemicals. In the 1940s, the clinical application of penicillin marked the birth of antibiotics. Since Alexander Fleming discovered the first antibiotic—penicillin in 1928, scientists have discovered nearly ten thousand kinds of antibiotics. Most of them are too toxic to be used as medicines for treating infectious diseases of humans and livestock. There are nearly a hundred kinds of antibiotics used, some can inhi...

Claims

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Application Information

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IPC IPC(8): C07K7/56C12N15/11C12N15/76A01N43/90A01P21/00
Inventor 邓子新邬益鸣黄曦殷俊
Owner SHANGHAI JIAO TONG UNIV