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Gene cluster related to biosynthesis of cyclo-lipopeptide antibiotics locillomycin

A technology of roximycin and cyclolipopeptide, which is applied in the field of microbial genetic resources and genetic engineering, and can solve problems such as strain restrictions

Inactive Publication Date: 2015-05-27
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional production strains of antibacterial compounds with improved fermentation conditions or chemical mutagenesis are increasingly limited and can easily reach saturation.

Method used

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  • Gene cluster related to biosynthesis of cyclo-lipopeptide antibiotics locillomycin
  • Gene cluster related to biosynthesis of cyclo-lipopeptide antibiotics locillomycin
  • Gene cluster related to biosynthesis of cyclo-lipopeptide antibiotics locillomycin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Extraction of Roximycin producing bacteria Bacillus subitilis916 total DNA

[0096] Inoculate Bacillus subtilis916 into 50ml LB liquid medium, and shake overnight at 37°C and 200r / min; cultivate the culture solution at 5000r / min, centrifuge for 10 minutes, and air-dry the medium; the cells float in 40ml TE buffer, Add lysozyme to a concentration of 100ug / ml, incubate at 37°C for 20min; then add RNAase (1mg / ml) 100ul, 10% SDS 5ml, at 37°C, incubate for 30min; add proteinase K to a final concentration of 50ug / ml, at 37°C Keep warm for 75 minutes, if it is viscous, continue; then use phenol, phenol chloroform (1:1), and chloroform to extract twice; finally add 1 / 5 sodium acetate (3M), 2.5 times hexyl alcohol, precipitate for 60 minutes, and shake the tube gently , pick out the DNA filaments with a thin glass rod, wash with 70% ethanol, and dry in vacuum, which is the total DNA extracted.

Embodiment 2

[0097] Construction of Roximycin-producing Bacillus subitilis916 Genomic Library

[0098] The extracted Bacillus subtilis 916 total DNA was partially digested with Sau3A I, used 1% low-melting point agarose gel, and contained 0.5 g of TBE electrophoresis buffer in pulse field electrophoresis (Pulse Field Gel Electrophoresis, PEGE, Bio-Rad) separate. Recover a DNA fragment of about 40kb in size, and then treat it with alkaline phosphatase to dephosphorylate the end, which is used for vector ligation and packaging transfection. The processed genomic DNA and the cosmid vector were ligated with T4 DNA ligase (NEB) according to the molecular ratio of 1:1. Add the phage packaging protein dissolved on ice (MaxPlaxLambda Packing Extracts, EPICENTER Biotechnologies company) to the ligation product, mix well to avoid steam pockets, centrifuge briefly, incubate at 30°C for 90min, add another packaging protein, and continue to incubate. After incubation for 90min, add phage dilution bu...

Embodiment 3

[0099] Acquisition of Gene Disruption Mutants

[0100] The method of homologous double crossover is used to destroy the reading frame related to the development of the roximycin synthetic gene cluster, and the corresponding gene disruption mutants are obtained. Homologous double crossover plasmid vector construction. According to the nucleic acid sequences of different open reading frames of Roximycin, primers were designed to amplify the upstream homologous fragment and the downstream homologous fragment (about 750bp) respectively from the Bacillus subtilis916 genomic DNA, and the amplified upstream and downstream homologous fragments were inserted in pairs Construct a series of spare recombinant plasmid vectors to pUC19. The spare recombinant plasmid vector was then inserted into the neomycin sulfate resistance gene derived from the pBEST501 plasmid to construct a series of homologous double exchange plasmid vectors. The constructed homologous recombination plasmid vector...

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Abstract

The invention relates to a gene cluster related to biosynthesis of cyclo-lipopeptide antibiotics locillomycin and particularly relates to clone, sequencing, analysis, function research and application of the gene cluster relates to the biosynthesis of the cyclo-lipopeptide antibiotics locillomycin having functions of bacteria resistance and virus resistance and being generated from bacillus subtilis. The whole gene cluster includes ten genes, wherein four genes are related to the biosynthesis of the locillomycin macro ring, two genes are related to regulation and the rest four genes are related to transfer. By means of genetic operations to the genes hereinbefore, synthesis of the locillomycin can be blocked or influenced. The genes and protein thereof also can be used for searching and finding compounds, genes and proteins capable of being used in medicines, industry or agriculture.

Description

Technical field: [0001] The invention belongs to the field of microbial genetic resources and genetic engineering, and specifically relates to the biosynthesis, analysis, functional research and application of cyclolipopeptide antibiotic locillomycin with broad-spectrum antibacterial activity, especially antiviral activity. technical background: [0002] Lipopeptide antibiotics are a class of secondary metabolites composed of a hydrophilic cyclic short peptide head and a long-chain hydrophobic fatty acid tail synthesized by Bacillus subtilis and its related species through a non-ribosomal synthesis pathway. They have antibacterial activity and It has hemolytic properties and has wide application value in medicine, pesticide and industry. Lipopeptide antibiotics can be divided into three major families according to their synthetic gene clusters and chemical structure differences: Surfactin, Iturin and Fengycin. [0003] The common feature of the surfactant family cluster str...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12R1/125
Inventor 罗楚平郭俊尧王晓宇陈志谊
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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