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Genetic screening method for negative regulatory factor of streptomyces biosynthesis gene cluster

A negative regulatory factor and biosynthetic technology, applied in the fields of biochemistry and molecular biology, can solve the problems affecting the transcription and translation process and the loss of target gene function, and achieve high screening throughput, convenient operation, and good genetic stability

Pending Publication Date: 2018-10-02
ZHEJIANG UNIV
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Problems solved by technology

Transposons can be randomly or specifically inserted into a certain position in the genome, thereby affecting the subsequent transcription and translation process of the gene at this position, resulting in the loss of the function of the target gene in the organism

Method used

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  • Genetic screening method for negative regulatory factor of streptomyces biosynthesis gene cluster
  • Genetic screening method for negative regulatory factor of streptomyces biosynthesis gene cluster
  • Genetic screening method for negative regulatory factor of streptomyces biosynthesis gene cluster

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Embodiment 1

[0041] Take the method for screening the negative regulatory protein of the dptE gene in the daptomycin-producing strain Streptomyces roseospora L30 as an example. Streptomyces roseospora L30 is a streptomyces strain that produces daptomycin industrially. Its genome sequence was also determined and the daptomycin synthesis gene cluster was mapped. In the whole daptomycin synthesis gene cluster, the direct synthesis protein of daptomycin is encoded by 5 genes including dptE, dptF, dptA, dptBC and dptD. These five genes form a cistron, and the promoter dptEp of the dptE gene regulates the transcription and translation steps of the whole cistron. Existing studies have shown that the high expression of five genes including dptE, dptF, dptA, dptBC, and dptD can effectively increase the industrial fermentation yield of daptomycin. Therefore, we screened the negative regulator of promoter dptEp in the genome of Streptomyces roseospora L30 by this method and knocked it out, so as to...

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Abstract

The invention provides a screening method for a negative regulatory factor of a novel biosynthesis gene cluster of streptomyces. The screening method comprises the following steps: constructing a promoter-mediated reporting system of genes of self-interest in a streptomyces cell, then using a random mutation system based on a transposon Himar1 to mutate the streptomyces with the reporting system,intensively screening a mutant streptomyces strain to obtain a streptomyces strain with high expression of target genes, performing bacteriophage packaging on genomes of the streptomyces strain with high expression of target genes and screening out cosmids with randomly inserted fragments, sequencing DNA of the cosmids to determine the location of the randomly inserted fragments in the genomes ofthe streptomyces strain with high expression of target genes. The screening method, provided by the invention, is stable in screening environment, high in screening flux, low in false positive, high in efficiency, accurate and convenient to operate, and can be widely used in the field of high-yield screening and transformation of industrial streptomyces metabolites, and the screened and transformed streptomyces metabolites are high in yield, good in genetic stability and suitable for industrial production.

Description

technical field [0001] The invention relates to the fields of biochemistry and molecular biology, in particular to a new method for genetic screening of Streptomyces biosynthetic gene cluster negative regulators. Background technique [0002] Over the past few centuries, scientists have isolated and screened tens of thousands of natural products from nature. In recent decades, however, it has become increasingly difficult for scientists to discover and isolate new natural products. The vigorous development of genomics since the beginning of this century has pointed out a new direction for us to discover new natural products. Many proteins have regulatory functions in organisms, and a class of regulatory proteins can participate in the regulation of gene expression, and they can activate or inhibit the transcription level of specific genes. Researchers are trying to discover and isolate more interesting compounds by finding specific regulatory factors to activate and highly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/76C12N15/65
CPCC12N15/65C12N15/76C12N2800/90C12N15/1086C12Q1/6897
Inventor 李永泉毛旭明罗帅
Owner ZHEJIANG UNIV
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