Biosynthesis gene cluster of polyenoid and polyol macrolide compound

A technology for biosynthesis and compound application in the field of biomedicine, which can solve the problems of unclear mechanism of action and no antifungal activity.

Inactive Publication Date: 2014-05-28
SICHUAN INDAL INST OF ANTIBIOTICS CHINA NAT PHARMA GROUP CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unlike other polyene antibiotics, WSS2277 has almost no antifungal activity (IC against Candida albicans, Aspergillus fumigatus 90

Method used

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  • Biosynthesis gene cluster of polyenoid and polyol macrolide compound
  • Biosynthesis gene cluster of polyenoid and polyol macrolide compound
  • Biosynthesis gene cluster of polyenoid and polyol macrolide compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Extraction of WSS2277-producing strain Streptomyces sp. SIIA-H8968 genomic DNA:

[0063]Inoculate 20 μl of 20% glycerol-preserved Streptomyces sp. SIIA-H8968 spores into 10 ml of seed medium (TSB medium, Oxoid Company), culture on a shaker at 30°C for 20 hours, and centrifuge to collect the bacteria for later use. Resuspend 50 mg mycelia in 500 μl lysozyme solution and incubate at 37°C for about 30 min, or until the cells become translucent. Add 100 μl 10% SDS, mix and shake for about 1 min until the viscosity of the solution drops significantly, incubate at 37°C for 20 min, then add 250 μl neutral phenol / chloroform, mix and shake evenly, centrifuge at 12000 r / min for 5 min, pipette Supernatant, discard the white intermediate layer. Repeat the second extraction with neutral phenol / chloroform until the middle layer is invisible (or very little), and finally add 0.1 volume of 3 M sodium acetate (pH natural), mix and add 1 volume of isopropanol (or 2.2 times th...

Embodiment 2

[0064] Example 2 Construction and screening of the genome library of Streptomyces sp. SIIA-H8968, which produces WSS2277:

[0065] The construction method of the genomic library refers to the experimental method provided by the kit CopyControl? Fosmid Library Production Kit.

[0066] A pair of polymerase chain reaction (PCR) primers (5'-GAGGACCCGGTCWCSTWCGCSGTSAAC-3 ') and (5'-AGCAGCCSGASCCGTASGAGAA -3') successfully amplified a specific product of the expected size from WSS2277-producing strain Streptomyces sp. SIIA-H8968, and sequence analysis showed that the amino acid sequence homology between the two was 70%. Based on this primer, the polymerase chain reaction (PCR) is used to screen the genomic library. First, the clones of each 96-well plate are mixed and cultured, and the mixed cosmid is used as a template, and then the PCR reaction is carried out. The positive signal is located in each For 96-well plates, the same method is followed by positioning in rows and finally...

Embodiment 3

[0067] Example 3 Establishment of WSS2277-producing bacteria Streptomyces sp. SIIA-H8968 conjugative transfer system:

[0068] Fresh spores of Streptomyces sp. SIIA-H8968 grown in SFM medium were collected for use. The oriT-containing target plasmid must be assisted by the helper plasmid pUZ8002 to be introduced into recipient Streptomyces cells by conjugative transfer. First, transform the plasmid to be transferred into Streptomyces sp. SIIA-H8968 into Escherichia coli ET12567 / pUZ8002, then culture the Escherichia coli ET12567 / pUZ8002 containing the target plasmid, collect the cells after 4 hours, and wash the cells twice with fresh LB medium spare. Streptomyces spores used as recipients need to be heat-shocked and pre-germinated. Streptomyces spores were resuspended in 5 ml of 0.05 M TES buffer at pH 8.0, heat-shocked in a water bath at 50°C for 10 min, cooled to room temperature, and then added with an equal volume of 2× spore pre-germination medium (Oxoid yeast extract 1...

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Abstract

The invention discloses cloning, sequencing, analysis, function study and application of a biosynthesis gene cluster of a novel anti-microbial and anti-tumor compound WSS2277 in the field of biotechnologies. The gene cluster comprises nucleotide sequences of twenty-one genes or complementary sequences which are formed by the sequences 1, wherein the fourteen genes including siaA, siaB1, siaB2, siaC, siaD, siaE, siaF, siaG, siaH, siaI, siaJ, siaK, siaL and siaM are responsible for the synthesis of a carbon skeleton, and the seven genes incluidng siaN, siaO, siaP, siaQ, siaR, siaS and siaT are responsible for regulating biosynthesis. The information of the genes which are related to the biosynthesis of the WSS2277 is provided by the invention, and a foundation is provided for the study and the genetic modification of the biosynthesis of the WSS2277. A novel structural analogue can be generated through the modification of carbon skeleton synthesis genes in the WSS2277, and the yield of the WSS2277 or a derivative thereof can be provided through the interruption or the high replication of regulator genes in the WSS2277. The genes and proteins, which are provided by the invention, can also be used for searching and discovering compounds or genes and proteins, which can be applied to the fields of medicine, industry or agriculture.

Description

technical field [0001] The invention relates to a gene cluster in the technical field of biomedicine and its application, in particular to the cloning, sequencing, analysis and application of the biosynthetic gene cluster of antibiotic WSS2277 with anti-gram-positive bacteria and anti-tumor activities. Background technique [0002] Chinese patent CN201010151873.4 (application date April 14, 2010) discloses a new polyene polyol macrolide antibiotic WSS2277 produced by the fermentation of Streptomyces sp. SIIA-H8968. Polyene polyol antibiotics are a class of microbial natural products with a specific structure, and have a wide range of antibacterial, antifungal, and antitumor activities. Their mechanism of action is mainly to bind to ergosterol in the cell membrane, interfere with metabolism, and increase cell membrane permeability. , leading to cell death. [0003] Studies have confirmed that WSS2277 has strong anti-Gram-positive bacteria and anti-tumor cell activity, and it...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/53C12N15/60C12N15/61C12N15/31C12R1/465
Inventor 褚以文张春然翟龙飞孙敏
Owner SICHUAN INDAL INST OF ANTIBIOTICS CHINA NAT PHARMA GROUP CORP
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