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Gene cluster for biological synthesis of Nosiheptide

A biosynthesis and gene technology, applied in the field of microbial genetic resources and genetic engineering, can solve the problems of low yield, high production cost, cumbersome operation, etc.

Active Publication Date: 2011-06-22
国科新研国际技术转移有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the extremely complex molecular structure of Northeptide, even in today's highly developed organic synthesis chemistry, it is a difficult challenge to obtain products through chemical total synthesis
It was not until 2008 that the partial chemical synthesis of nosiheptide was reported for the first time [Marc C. Kimber and Christopher J. Moody. 593.], the whole synthetic process is cumbersome to operate and the yield is low
Structural analogues obtained by chemical synthesis, even with improved properties in terms of activity and water solubility, may be too expensive for practical production due to the complexity of the synthesis

Method used

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  • Gene cluster for biological synthesis of Nosiheptide
  • Gene cluster for biological synthesis of Nosiheptide
  • Gene cluster for biological synthesis of Nosiheptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Extraction of the total DNA of Streptomyces actuosus ATCC25421, a bacterium producing heptapeptide:

[0077] 100 μL 1 x 10 8S.actuosus spore suspension was inoculated into 3mL TSB liquid medium, cultivated at 30°C, 230rpm for about 24hrs and reached the late logarithmic growth phase, then inoculated 2mL into 50mL TSB (containing 10mM magnesium chloride, 0.1% glycine), 30°C, 250rpm After cultivating for about 23 hours, it reached the early stage of the stable growth period, and it was milky yellow and turbid. The mycelium was collected by centrifuging the bacterial solution at 4°C and 3500 rpm for 15 minutes, washed with lysate, and 0.5 mL of pale milky yellow mycelium was collected. Add 10 mL of lysate (containing 5 mg / mL of lysozyme) to 1 mL of mycelium, a total of four tubes, vortex until uniform, and bathe in 37 ° C for 15 min. Add 0.1mL proteinase K (10mg / mL, freshly prepared with lysate), 1mL 10% SDS, mix well and quickly put in 70°C water bath for 15mim, it becom...

Embodiment 2

[0079] The establishment of the genetic transfer system of Streptomyces actuosus ATCC25421, which produces Nossy heptapeptide:

[0080] Culture E.coli ET12567 containing appropriate plasmids to OD 600 0.3-0.4, the bacterial cells in 30mL LB culture medium were collected by centrifugation, washed twice with an equal volume of LB, resuspended in 2mL LB, and used as E. coli donor cells. Take 500 μL of 20% glycerol spore suspension of Streptomyces actuosus ATCC25421 frozen at -80°C, wash twice with an equal volume of TES buffer (50 mM TES Na, pH 8.0), resuspend in an equal volume of TES buffer, Heat shock at 50°C for 10 min to germinate the spores. Add an equal volume of TSB medium and incubate at 37°C for 2-5hr. Centrifuge and resuspend in 0.5-1 mL LB as Streptomyces recipient cells. Mix 100 μL of different concentrations of recipient cells with an equal volume of donor cells and directly spread it on a medium containing 10 mM MgCl 2 After incubating at 30°C for 20 hours, gen...

Embodiment 3

[0083] Construction of Genome Library of Streptomyces actuosus ATCC25421 Genome Library

[0084] Firstly, the total DNA of Streptomyces actuosus ATCC25421 was sucked several times with a 1ml syringe, so that the total DNA was randomly broken, and the DNA was subjected to gradient centrifugation using sucrose gradient centrifugation, and the DNA fragments slightly larger than 40kb were collected, dephosphorized, and set aside. pJTU2554 was first cut from the middle of the two cos sequences with EcoRV, and ligated with the prepared 40kb DNA fragment overnight. Thaw the PromegaPackagene extract stored at -80°C on ice, immediately add 10ul of the ligation product, mix well by flicking, and place it at room temperature (about 22°C) for 3hr. Add 445ul Phage buffer, invert to mix; add 25ul chloroform to terminate the reaction, centrifuge to make the chloroform sink to the bottom, and store at 4°C. The strain E.coli LE392 frozen at -80°C was spread on LB medium for recovery. Take a ...

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Abstract

The invention relates to gene cluster for biological synthesis of Nosiheptide, particularly relates to cloning, sequencing, analyzing, researching function and use thereof of gene cluster for biological synthesis of streptomycete produced Nosiheptide with anti-Gram positive antibiotics. The entire gene cluster contain 16 genes: six genes related to macrocyclic biosynthesis of Nosiheptide, five genes related to biological synthesis of lateral chain, four genes related to modification after translation, and one regulatory gene. Synthesis of Nosiheptide can be blocked by genetic operations of thegenes for biological synthesis. The gene and protein thereof provided by the invention can be used for looking for and discovering compound or gene, protein for medicine, industry or agricultural.

Description

Technical field: [0001] The invention belongs to the field of microbial genetic resources and genetic engineering, and in particular relates to the cloning, analysis, functional research and application of biosynthetic gene clusters of anti-gram-positive bacteria antibiotic Nosiheptide. technical background: [0002] Nosiheptide is a well-known member of the thiopeptide antibiotic family, which can be produced by Streptomyces actuosus ATCC25421, Streptomyces azureus ATCC14921, and Streptomyceshawaiiensis ATCC12236. Its molecular structure formula was first analyzed and identified by Depaire.H et al. [Depaire, H.; Thomas, J.-P.; Brun, A.; Olesker, A.; Lukacs, G. Tetrahedron Lett.1977, 1397.]. Structurally, Nosiheptide consists of five thiazole rings and one pyridine ring to form a macrocyclic skeleton. The side chain is a 3-methyl-5-methoxy-2-indole acid connected to the main macrocyclic structure through a thioester bond. [0003] Nosepeptide has good biological activity. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N15/55C12N15/54C12N15/53C12N15/52C12N15/31C12P21/02C12P17/12C12P17/14C12P17/10C12R1/465
Inventor 刘文虞沂段炼雷春丁莹廖日晶
Owner 国科新研国际技术转移有限公司
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