Recombinant yarrowia lipolytica strain with high oleic acid yield as well as construction method and application thereof

A technology of Yarrowia lipolytica and Yarrowia lipolytica is applied in the field of bioengineering to achieve the effects of efficient synthesis, improved genetic stability and simple operation

Active Publication Date: 2021-03-23
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the prior art, there is a lack of microorgan

Method used

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  • Recombinant yarrowia lipolytica strain with high oleic acid yield as well as construction method and application thereof
  • Recombinant yarrowia lipolytica strain with high oleic acid yield as well as construction method and application thereof
  • Recombinant yarrowia lipolytica strain with high oleic acid yield as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the amplification of gene element and the preparation of target plasmid (one) the preparation of target gene

[0036] According to the nucleotide sequence of the gene encoding stearoyl-CoA desaturase from Puccinia graminis (GenBank accession number: NW_003526568.1) provided by NCBI, after codon optimization, entrusted Suzhou Jinweizhi Biology Co., Ltd. Technology Co., Ltd. synthesized the optimized stearoyl-CoA desaturase coding gene PgSCD (SEQ ID No: 1), and inserted it into the plasmid pUC57 to obtain the plasmid pUC57-PgSCD.

[0037] According to the nucleotide sequence of the gene encoding fatty acid elongase 2 from Mortierella alpina (GenBank accession number: AB468587.1) provided by NCBI, after codon optimization, entrust Suzhou Jinweizhi Biotechnology Co., Ltd. The optimized fatty acid elongase 2 encoding gene MaELO2 (SEQ ID No: 2) was synthesized and inserted into plasmid pUC57 to obtain plasmid pUC57-MaELO2.

[0038] According to the nucleotide s...

Embodiment 2

[0102] Example 2, Construction of Recombinant Yarrowia lipolytica (1) Construction of Recombinant Bacteria 1

[0103] The plasmid pUC-HUH-IntC-ACC1-DGA1 containing the ACC1 and DGA1 gene expression cassettes was introduced into Yarrowialipolytica Po1fΔku70, and the ACC1 and DGA1 expression cassettes were integrated at the IntC site of the genome, and then one was lost under the selection pressure of 5-fluoroorotic acid After hisG tag and Ura selection marker, the recombinant strain 1 was obtained.

[0104] The specific method is as follows:

[0105] ①Yarrowia lipolytica Po1fΔku70 was cultured overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose) to prepare competent cells.

[0106] ② Transform Yarrowia lipolytica Po1fΔku70 competent cells with pUC-HUH-IntC-ACC1-DGA1 using the Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation for homologous recombination.

[0107] ③ Positive clones were screened by screening medium SD...

Embodiment 3

[0130] Example 3, Application of Recombinant Yarrowia lipolytica in the Production of Oleic Acid (1) Cultivation of Engineering Bacteria and Product Extraction

[0131] The original bacteria Yarrowia lipolytica (Yarrowia lipolytica) Po1fΔku70, and the recombinant bacteria 2 and 5 in Example 2 were used to produce oleic acid, respectively. The specific method is as follows: activate the bacterial strain, culture in YPD liquid medium (containing 2% peptone, 1% yeast extract, 2% glucose, and water as a solvent) at 30° C. and 220 rpm for 16 hours to obtain seed liquid. The seed solution was inoculated in 50 ml of fermentation medium with an inoculum amount of 1%, and cultured with shaking at 30° C. and 220 rpm for 5 days. After the fermentation, the fermentation broth was transferred to a 50ml centrifuge tube, centrifuged at 5000rpm for 15min, the supernatant was removed, and dried in an oven (75°C) to constant weight.

[0132] The fermentation medium formula is: glucose 60g / L, a...

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Abstract

The invention provides recombinant yarrowia lipolytica with high oleic acid yield as well as a construction method and application thereof, and belongs to the field of bioengineering. The recombinantyarrowia lipolytica is a yarrowia lipolytica XJ-9 strain, is deposited in the China Center for Type Culture Collection (CCTCC), and has a deposit number of CCTCC NO: M 2020707. According to the yarrowia lipolytica XJ-9 strain, oleate desaturase is knocked out, endogenous acetyl coenzyme A carboxylase, diacylglycerol acyltransferase and fatty acid elongase 1 are overexpressed, heterologous fatty acid elongase 2 and stearoyl coenzyme A desaturase are overexpressed, and experiments prove that the recombinant yarrowia lipolytica can be efficiently fermented to produce oleic acid, and efficient synthesis of plant-derived natural product oleic acid in yarrowia lipolytica is realized.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a recombinant Yarrowia lipolytica with high oleic acid production and its construction method and application. Background technique [0002] Oleic acid (C 18 h 34 o 2 ), scientific name: cis-9-octadecenoic acid, is an Omega-9 type monounsaturated fatty acid, mainly present in Brassica napus, Camellia japonica and other oil crops. In the field of medicine, oleic acid has the function of regulating physiological functions, can selectively regulate high-density and low-density cholesterol in human blood, and reduce the risk of cardiovascular disease. In addition, oleic acid has a good adjuvant therapeutic effect on cancer, autoimmune diseases and inflammatory diseases. In the energy sector, oleic acid is an excellent biodiesel component due to its low temperature fluidity and oxidation stability. In the field of materials, oleic acid can be converted into fatty acid de...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/90C12N15/52C12N15/53C12N15/54C12P7/64C12R1/645
CPCC12N15/52C12N9/93C12N9/1029C12N9/0071C12N15/815C12N15/905C12P7/6409C12Y114/19C12Y604/01002C12Y203/0102C12Y203/01199C12Y114/19001Y02E50/10
Inventor 王金鹏纪晓俊王凯峰陆然支咏妍朱芮莹胡佳颖
Owner NANJING UNIV OF TECH
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