Recombinant yarrowia lipolytica with high homologous recombination efficiency, and construction method and application thereof

A technology of lipolysis and homologous recombination, applied in the field of bioengineering, can solve the problems of non-homologous recombination mechanism damage, unfavorable industrial scale cultivation, high cost, etc., to improve the efficiency of homologous recombination, improve genetic stability, Simple operation effect

Active Publication Date: 2020-02-28
NANJING UNIV OF TECH
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  • Claims
  • Application Information

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Problems solved by technology

The physicochemical method mainly uses DNA inhibitors to synchronize the cell cycle in the cell cycle prone to homologous recombination, but this method is short-term and expensive; while the biological method mainly focuses on knocking out or destroying the non-homologous cells of the strain itself. Recombination of key genes, such as Ku protein, LigD gene, etc. Although this method ca

Method used

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  • Recombinant yarrowia lipolytica with high homologous recombination efficiency, and construction method and application thereof
  • Recombinant yarrowia lipolytica with high homologous recombination efficiency, and construction method and application thereof
  • Recombinant yarrowia lipolytica with high homologous recombination efficiency, and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, the amplification of gene element and the preparation of target plasmid

[0032] (1) Preparation of target gene

[0033]The nucleotide sequence of the scRad52 gene of the Saccharomyces cerevisiae Rad52 epistatic gene cluster, after codon optimization, the sequence is shown in SEQ ID NO.1, entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the optimized scRad52, and insert it into pUC57, The plasmid pUC57-scRad52 was obtained.

[0034] According to the nucleotide sequence (M37309.1) of the 3(β)-isopropylmalate dehydrogenase coding gene leu in Yarrowia lipolytica provided by NCBI, Suzhou Jinweizhi Biotechnology Co., Ltd. was entrusted to synthesize leu, and 3( β)-Isopropylmalate dehydrogenase encoding gene expression cassette was inserted into plasmid pUC57 to obtain plasmid pUC57-leu. The promoter in the expression cassette of 3(β)-isopropylmalate dehydrogenase gene is P of Yarrowia lipolytica leu Promoter (SEQ ID NO.4), terminator is the T o...

Embodiment 2

[0097] Embodiment 2, the construction of recombinant bacteria

[0098] (1) Construction of Recombinant Bacteria 1

[0099] The plasmid pUC-leu-A08-scRad52 containing the scRad52 gene expression cassette was introduced into Yarrowialipolytica Po1f, and the scRad52 expression cassette and the 3(β)-isopropylmalate dehydrogenase gene expression cassette were integrated into the A08 site of the genome to obtain recombinant Bacteria 1. The specific method is as follows: (1) Yarrowia lipolytica Po1f was cultured overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose) to prepare competent cells. (2) Using the Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation, pUC-leu-A08-scRad52 was transformed into Yarrowia lipolytica Po1f for homologous recombination. (3) The screening medium SD-Leu was used to screen, and the positive clone identified by PCR was named recombinant bacteria 1. The screening medium SD-Leu contains: glucose 20...

Embodiment 3

[0102]Example 3. The application of recombinant bacteria 1-2 in mediating homologous recombination of homologous arms of different lengths for genetic modification of genomic DNA and the comparison of homologous recombination efficiency.

[0103] (1) Construction of homologous recombination strains

[0104] The recombinant plasmids pUC-HUH-ADE2-1000, pUC-HUH-ADE2-500, pUC-HUH-ADE2-250 and pUC-HUH-ADE2-100 were introduced into recombinant bacteria 1 respectively. The specific method is as follows: (1) After culturing the recombinant bacteria 1 overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract, and 2% glucose), competent cells were prepared. (2) Transform the recombinant plasmids pUC-HUH-ADE2-1000, pUC-HUH-ADE2-500, pUC-HUH-ADE2-250, pUC-HUH-ADE2-100 using the Zymogen Frozen EZ Yeast Transformation Kit II of ZymoResearch Corporation Into recombinant bacteria 1, homologous recombination was performed, and the knockout cassettes of ADE2 homology arms of diff...

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Abstract

The invention provides recombinant yarrowia lipolytica with high homologous recombination efficiency and a construction method thereof, and relates to the field of biological engineering. The recombinant yarrowia lipolytica with the high homologous recombination efficiency is obtained by inserting an Rad52 gene expression cassette derived from a saccharomyces cerevisiae Rad52 epistatic gene cluster into a yarrowia lipolytica genome. The recombinant yarrowia lipolytica can have high homologous recombination efficiency, and the construction method is efficient, and simple to operate.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a recombinant Yarrowia lipolytica with high homologous recombination efficiency and its construction method and application. Background technique [0002] Yarrowia lipolytica is an organism recognized as safe by the FDA. It is an excellent strain for studying unconventional yeast. It has great application potential as a microbial platform for the synthesis of various biochemical products. With the rapid development of metabolic engineering and synthetic biology, the genetic tools of Yarrowia lipolytica have been gradually perfected. However, in wild-type Yarrowia lipolytica cells, non-homologous recombination occupies Most foreign genes are randomly inserted into the genome, and the probability of targeted knockout or knockin through homologous recombination is extremely low. [0003] Rad52 is an important homologous recombination protein, both yeast cells and human cells contain h...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/31C12R1/645
CPCC07K14/395C12N2800/22
Inventor 纪晓俊丁颖王凯峰马一榕王维鉴
Owner NANJING UNIV OF TECH
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