High efficient homologous recombination engineering strain of Amycobacter, its construction method and application

A mycobacteria pseudomycoides and engineering strain technology, which is applied in the field of highly efficient homologous recombination and efficient Amycobacter pseudomycoides engineering strains, and can solve the problems of low homologous recombination efficiency, restricting the application of gene targeting technology and the like

Active Publication Date: 2022-08-02
SHAANXI HEALTHFUL BIOLOGICAL ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The repair of DNA double-strand breaks in Amycolatopsis mainly relies on non-homologous end joining (NHEJ), which leads to the integration of exogenous DNA into the genome chromosome mainly by random insertion, and the efficiency of homologous recombination is low. , which seriously restricts the application of gene targeting technology in Amycolatopsis

Method used

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  • High efficient homologous recombination engineering strain of Amycobacter, its construction method and application
  • High efficient homologous recombination engineering strain of Amycobacter, its construction method and application
  • High efficient homologous recombination engineering strain of Amycobacter, its construction method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Example 1: Construction of ku1 gene deletion strain

[0058] The sequence of ku1 of Amycolatopsis is compared on the NCBI website according to the gene sequence of Amycolatopsis sp.ATCC 39116 strain, the gene sequence is as shown in SEQ ID NO: 1, and the encoded protein accession number is WP_027936589.1.

[0059] Construction of ku1 gene deletion strains: In order to knock out the ku1 gene in Amycobacterium pseudomycoides, ku1-U-FOR / ku1-U-REV and ku1-D-FOR / ku1-D-REV were used as primers, respectively. Amycobacter HM-141 genome was used as template, and the upstream and downstream homology arms of ku1 gene were amplified by PCR. At the same time, the above two ku1-up and ku1-down fragments were connected to the HindIII / EcoRI site of the pKC1139 plasmid by the Assembly kit to complete the construction of the plasmid pKC1139-ku1. The plasmid map of pKC1139-ku1 is as follows figure 1 shown.

[0060] The pKC1139-ku1 plasmid was transformed into Amycobacterium pseudomycoi...

Embodiment 2

[0061] Example 2: Construction of ku2 gene deletion strain

[0062] Similarly, the sequence of ku2 in the Amycobacterium pseudomycetes strain was aligned on the NCBI website according to the gene sequence of the Amycobacterium pseudomycoides ATCC 39116 strain, and the encoded protein accession number was WP_020416491.1.

[0063] Construction of ku2 gene deletion strains: In order to knock out the ku2 gene in Amycobacterium pseudomycoides, ku2-U-FOR / ku2-U-REV and ku2-D-FOR / ku2-D-REV were used as primers, respectively. The genome of Mycobacterium HM-141 was used as the template, and the upstream and downstream homology arms of the ku2 gene each about 2 kb were amplified by PCR.

[0064] The above two ku2-up and ku2-down fragments were connected to the HindIII / EcoRI site of the pKC1139 plasmid by the Assembly kit to complete the construction of the plasmid pKC1139-ku2. The plasmid map of pKC1139-ku2 is shown in figure 2 shown. The pKC1139-ku2 plasmid was transformed into Amyco...

Embodiment 3

[0065] Example 3: Construction of strains with both ku1 and ku2 genes deleted

[0066] On the basis of the Δku1 strain constructed in Example 1, the ku2 gene was further knocked out:

[0067] The pKC1139-ku2 plasmid of Example 2 was transformed into the Δku1 strain using the conjugation transfer experiment method, and positive mutants were screened according to abramycin resistance to obtain a genetically engineered strain Δku1ku2 with both ku1 and ku2 genes deleted.

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Abstract

High-efficiency homologous recombination engineering strain of Amycolatopsis, its construction method and application. The invention provides the application of ku1 and / or ku2 gene deletion in improving the efficiency of homologous recombination of Amycolatopsis sp. The engineering strain of Amycobacter lacks the ku1 gene and / or the ku2 gene of Amycolatopsis, and the invention also provides a construction method and application of the strain. In the present invention, it is verified through experiments that the deletion of the ku1 gene, the ku2 gene or the simultaneous deletion of the ku1 gene and the ku2 gene can significantly improve the homologous recombination efficiency of Amycolatopsis. Among them, the homologous recombination efficiency of the engineered strain with deletion of ku1 gene reaches 95%, which has the most prominent effect.

Description

【Technical field】 [0001] The invention belongs to the technical field of genetic engineering. More specifically, the present invention relates to a high-efficiency homologous recombination and high-efficiency Amycobacterium pseudomycoides engineered strain, and also relates to a construction method and application of the engineered strain. 【Background technique】 [0002] Vanillin, also known as vanillin (3-methoxy-4-hydroxybenzaldehyde), is one of the most widely used seasonings in the world and is widely used in food, medicine, cosmetics, agriculture and other fields. Vanillin can be biotransformed from the natural substrate ferulic acid, and Amycobacterium pseudomycoides has been reported to be able to use ferulic acid to produce vanillin, which has very good industrial application value. However, due to the low efficiency of homologous homology, genetic manipulation such as gene targeting is difficult, which limits the metabolic engineering in vanillin synthesis. high i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/31C12N13/00C12N15/01C12P7/24C12R1/01
CPCC07K14/195C12N15/74C12N13/00C12N15/01C12P7/24
Inventor 孟永宏强珊郭建琦牛永洁杨璐
Owner SHAANXI HEALTHFUL BIOLOGICAL ENG
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