Corynebacterium gene continuous knockout system, as well as construction method and application thereof

A coryneform bacterium and a construction method technology, applied in the field of microbial genetic engineering, can solve problems such as the trouble of the second round of exchange screening of sacB gene
CN103409446AInactive Publication Date: 2013-11-27JIANGNAN UNIV
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
JIANGNAN UNIV
Publication Date
2013-11-27
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention discloses a construction method and an application of a corynebacterium gene continuous knockout system, belonging to the technical field of genetic engineering. The corynebacterium gene continuous knockout system comprises a template plasmid and a temperature-sensitive expression plasmid, wherein the template plasmid provides a kan fragment (also known as a kan box) with loxp or variant loxL / R sites at two ends; and the temperature-sensitive expression plasmid carries a Cm resistance gene cat and a temperature-sensitive C. glutamicum replicon, expresses a Cre recombinant enzyme and is used for removing a Km resistance gene kan. The corynebacterium continuous knockout system disclosed by the invention can be generally used for continuous gene transformation of corynebacterium and has the advantages of simplicity and convenience in operation and high efficiency. By utilizing the knockout system, gene (continuous) knockout of three major typical subspecies genomes of the corynebacterium can be successfully completed, and the system is proved to be applicable to research and production of corynebacterium metabolites.
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Description

technical field

[0001] The invention relates to a coryneform bacterium gene continuous knockout system and its construction method and application, in particular to a knockout system based on the combination of homologous recombination and specific site recombination, which belongs to the field of microbial genetic engineering. Background technique

[0002] Corynebacteria are a class of Gram-positive bacteria belonging to the actinomycetes with moderate to high GC content. Since Corynebacterium glutamicum was first isolated to produce L-glutamate (Kinoshita et al., 1985), three major representatives of Corynebacterium: Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum, have been It is widely used in the production of amino acids.

[0003] Since a large amount of knowledge on the physiology, biochemistry and genetics of C. glutamicum has accumulated (Eggeling and Bott, 2005; Burkovski, 2008), metabolic engineering has replaced classical ran...

Claims

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