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Method for constructing bacillus licheniformis by knocking out lysE gene, strain and application of strain

A technology of Bacillus licheniformis and Bacillus licheniformis, applied in the direction of microorganism-based methods, other methods of inserting foreign genetic materials, applications, etc., can solve problems such as unclear content and unclear specific functions

Inactive Publication Date: 2018-08-24
LIFECOME BIOCHEM
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Problems solved by technology

At present, the content of these 12 amino acids constituting bacitracin in Bacillus subtilis and Bacillus licheniformis is not clear, and which amino acid or amino acids are the key amino acids that limit the high yield of bacitracin is also unclear, that is to say, Lai Whether bacitracin is the limiting amino acid for bacitracin synthesis and the relationship between lysine supply and bacitracin production is still inconclusive; and, the lysine transporter LysE (its synthetic gene is: lys E licheniformis gene) in Bacillus licheniformis is not yet clear; in addition, the amino acid in the cell has a strict and complex regulatory mechanism, therefore, whether the lysine transporter LysE in Bacillus licheniformis can improve The production of antimicrobial peptides is unpredictable

Method used

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  • Method for constructing bacillus licheniformis by knocking out lysE gene, strain and application of strain
  • Method for constructing bacillus licheniformis by knocking out lysE gene, strain and application of strain
  • Method for constructing bacillus licheniformis by knocking out lysE gene, strain and application of strain

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Embodiment Construction

[0025] by knockout lys E The method for genetically constructing bacillus licheniformis comprises the following steps:

[0026] (1) Using the genomic DNA of Bacillus licheniformis DW2 as a template, PCR amplified lys E The upstream homology arm of the gene and lys E The downstream homology arm of the gene; and then use the overlap extension PCR method to lys E The upstream homology arm of the gene and lys E The downstream homology arms of the gene are spliced ​​together to obtain the fusion gene sequence A;

[0027] (2) Using restriction endonucleases Xba I and Sac I to perform double enzyme digestion on the fusion gene sequence A obtained in step (1) to obtain the enzyme cut gene sequence A;

[0028] (3) Prepare the plasmid T2(2)-ori, and use restriction endonucleases XbaI and SacI to double-enzyme-digest the plasmid T2(2)-ori to obtain the restriction endonuclease T2(2)-ori;

[0029] (4) Link the enzyme-cut gene sequence A obtained in step (2) to the enzyme-cut plasmid ...

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Abstract

The invention aims to provides a method for constructing bacillus licheniformis by knocking out an lysE gene, a strain and an application of the strain. The bacillus licheniformis DW2 delta lysE is constructed by knocking out the lysE gene in a bacillus licheniformis DW2 genome with a genetic engineering method, and the yield of bacitracin of the strain in a fermentation liquid in the bacitracin fermentation process is increased by 10% or above.

Description

technical field [0001] The invention relates to the field of Bacillus licheniformis strain transformation, especially a lys E The method and strain of gene constructing Bacillus licheniformis and its application. Background technique [0002] Bacitracin is a cyclic peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis. As a broad-spectrum antibiotic, bacitracin can effectively inhibit Gram-positive bacteria (such as Clostridium, Staphylococcus, Streptococcus, Corynebacterium and Neisseria, etc.) and some Gram-positive bacteria. Negative bacteria (such as meningococcus, influenza bacillus, actinomycetes, etc.). Moreover, bacitracin is hardly absorbed in the intestinal tract of animals, and is excreted quickly without residue, so it is widely used in feed addition. [0003] Bacitracin is a class of cyclic peptide antibiotics composed of 12 amino acid residues. The constituent amino acids of bacitracin include ornithine (Orn), D-phenylalanine (D-Phe), i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/90C12N15/66C12N15/31C12N1/21C12P21/04C12R1/10
CPCC07K7/58C07K14/32C12N15/66C12N15/75C12N15/902C12P21/02
Inventor 陈守文蔡冬波李阳吴非李俊辉楼丽君陈晓斌
Owner LIFECOME BIOCHEM
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