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Method for integrating yarrowia lipolytica genome based on non-homologous end-linking mechanism

A Yarrowia lipolytica, non-homologous technology, applied in the biological field, can solve the problems of low homologous recombination efficiency and unrealizable application

Active Publication Date: 2021-01-22
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the homologous recombination efficiency of Yarrowia lipolytica is far lower than that of Saccharomyces cerevisiae, the efficiency of non-homologous recombination is dominant, and these conventional biotechnologies cannot be effectively applied in Yarrowia lipolytica

Method used

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  • Method for integrating yarrowia lipolytica genome based on non-homologous end-linking mechanism
  • Method for integrating yarrowia lipolytica genome based on non-homologous end-linking mechanism
  • Method for integrating yarrowia lipolytica genome based on non-homologous end-linking mechanism

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Embodiment 1

[0038] A method for Yarrowia lipolytica genome integration based on a non-homologous end-joining mechanism (see figure 1 ), including the following steps:

[0039] (1) Obtain the gene DL4 (SEQ ID NO.1) and XRCC4 (SEQ ID NO.3) derived from Yarrowia lipolytica CLIB122 from the NCBI database, the gene PAXX amino acid sequence derived from Homo sapiens, and artificially synthesize the PAXX gene (SEQ ID NO.3) through codon optimization NO.2), the expression cassettes of three genes were assembled simultaneously.

[0040] Through the method of homologous recombination, the DL4 gene expression cassette, the PAXX gene expression cassette and the XRCC4 gene expression cassette were integrated into the genomic rDNA site of Yarrowia lipolytica ATCC201249 strain, and the non-homologous recombination repair mechanism of Yarrowia lipolytica was enhanced The recombinant strain YNH01.

[0041] Use YNH01 as the chassis to transfer the green fluorescent protein GFP gene expression cassette (S...

Embodiment 2

[0049] A method for Yarrowia lipolytica genome integration based on a non-homologous end joining mechanism, comprising the steps of:

[0050] (1) Preparation of the recombinant strain YNH01 with step (1) of Example 1;

[0051] (2) The construction method of the second high-pressure screening label strain, comprises the following steps:

[0052] URA3 nutrition label U11 with promoter truncated to 11bp and lycopene synthesis gene CrtE (SEQ ID NO.5) expression cassette H0-LD01-CrtE-H1, lycopene synthesis gene CrtB (SEQ ID NO.6) expression cassette H1-LD02in-CrtB-H2 and lycopene synthesis gene CrtI (SEQ ID NO.7) expression box H2-LD03-CrtI-H3, 4 fragments are connected to obtain the connection fragment-2; the connection fragment-2 is transformed into a recombinant After the strain YNH01, the second high-pressure screening tag strain was obtained;

[0053] CrtE (SEQ ID NO.5), CrtB (SEQ ID NO.6) and CrtI (SEQ ID NO.7) are artificially synthesized from Erwinia Stawartii Pantoea ste...

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Abstract

The invention discloses a method for integrating a yarrowia lipolytica genome based on a non-homologous end-linking mechanism. The method comprises the following steps of: integrating yarrowia lipolytica endogenous genes DL4 and XRCC4 and an exogenous wisdom gene PAXX into a genome rDNA site of a yarrowia lipolytica ATCC201249 strain in an rDNA site integration manner, so as to obtain a recombinant strain YNH01; linking a to-be-transferred gene expression cassette with a URA3 nutrition tag U11 truncated to 11bp by a promoter, and transforming an obtained linking fragment into the strain YNH01,so as to obtain a high-pressure screening tag strain; transforming the linking fragment into the high-pressure screening tag strain, so as to obtain a recombinant strain subjected to two-round transformation; and transforming the linking fragment into the recombinant strain subjected to two-round transformation again, so as to obtain a transferred gene high-expression strain. According to the method, a large number of diversified strains are obtained within a short time, and the efficient application of an NHEJ integration technology in microbial synthesis of natural products is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method for Yarrowia lipolytica genome integration based on a non-homologous end joining mechanism. Background technique [0002] In organisms, DNA molecules, as the carrier of life information, play a vital role in the storage, replication and transmission of genetic information. The damage of DNA molecules seriously affects the growth and function of cells. Among them, DNA double-strand breaks (DNA double-strand breaks, DSBs) are one of the most harmful DNA damages, which not only lead to the loss of potential sequence information, but also affect the growth state. In order to effectively repair DNA double-strand breaks that occur during growth, cells mainly implement two pathways, namely homologous recombination (Homologous recombination, HR) and non-homologous end-joining (Non-homologous end-joining, NHEJ). The fundamental difference between the two repair path...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/65C12P5/02C12R1/645
CPCC12N15/815C12N15/65C12P5/007
Inventor 曹英秀白秋艳程帅张金来
Owner TIANJIN UNIV
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