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Recombinant yarrowia lipolytica as well as construction method and application thereof

A technology of Yarrowia lipolytica and Yarrowia lipolytica, applied in the field of bioengineering, can solve problems such as poor heterologous high-efficiency expression effect, and achieve the effects of enhancing homologous recombination ability, improving genetic stability, and efficient construction method

Pending Publication Date: 2021-11-30
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using Yarrowia lipolytica to produce OA through genetic engineering is more green and efficient than traditional methods, but the heterologous high-level expression of cyclooxygenase OAC gene and polyketide synthase OLS gene in Yarrowia lipolytica is not effective

Method used

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  • Recombinant yarrowia lipolytica as well as construction method and application thereof
  • Recombinant yarrowia lipolytica as well as construction method and application thereof
  • Recombinant yarrowia lipolytica as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 A kind of construction method of recombinant Yarrowia lipolytica

[0049] The construction method of recombinant Yarrowia lipolytica in the present embodiment comprises the following steps carried out in sequence: 1) preparation of target gene

[0050] i) Target gene sequence optimization

[0051] According to the nucleotide sequence of the cyclooxygenase OAC gene from cannabis provided by NCBI, after codon optimization, Qingke Biological Co., Ltd. was commissioned to synthesize the optimized cyclooxygenase OAC gene and insert it into the plasmid pUC57. The plasmid pUC57-OAC was obtained, and the nucleotide sequence of the optimized artificially synthesized OAC is shown in SEQ ID NO.1.

[0052] According to the nucleotide sequence of the polyketide synthase OLS gene from alfalfa provided by NCBI, after codon optimization, we entrusted Qingke Biological Co., Ltd. to synthesize the optimized polyketide synthase OLS gene and insert it into the plasmid In pUC...

Embodiment 2～5

[0092] Embodiment 2-5 Construction method of recombinant Yarrowia lipolytica

[0093] Embodiments 2 to 5 are respectively a method for constructing a recombinant Yarrowia lipolytica, and their steps are basically the same as in Example 1, the only difference being that II) the selection of promoters and terminators in the construction of recombinant plasmids is different , see Table 5 for details:

[0094] The selection list of promoter and terminator in the construction of table 5 recombinant plasmid

[0095]

[0096] The content of other parts of embodiment 2-5 is the same as embodiment 1.

Embodiment 6

[0097] A kind of application of embodiment 6 recombinant Yarrowia lipolytica

[0098] The recombinant Yarrowia lipolytica J1 constructed in Example 1 was used to ferment and produce olive oil, and the obtained olive acid was detected and quantitatively analyzed by LC-MS.

[0099] Fermentation production: Cultivate recombinant Yarrowia lipolytica J1 under the condition of 30°C and 220rpm in YPD liquid medium, and obtain seed liquid after 16h; inoculate the seed liquid into 50ml fermentation medium (containing 60g / L glucose, 10g / L yeast powder and 20g / L tryptone were fermented at a temperature of 30°C and a rotation speed of 220rpm for 4 days with shaking culture; The four-day fermentation broth was extracted with ethyl acetate, spin-dried, and the olive oil was dissolved in chromatographic grade methanol and stored for later use.

[0100] Quantitative analysis: the organic phase in the fermentation broth is passed through an organic nylon filter membrane (0.22um), and detected...

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Abstract

The invention discloses recombinant yarrowia lipolytica as well as a construction method and application thereof. The recombinant yarrowia lipolytica is obtained by inserting a codon-optimized cyclooxygenase OAC gene expression cassette and a polyketide synthase OLS gene expression cassette into a yarrowia lipolytica genome. The recombinant yarrowia lipolytica disclosed by the invention is used for efficiently synthesizing olivetolic acid, the construction method is efficient, the operation is simple, and the olivetolic acid can be efficiently produced in a green manner.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and relates to a recombinant Yarrowia lipolytica, in particular to a recombinant Yarrowia lipolytica and its construction method and application. Background technique [0002] In addition to its antimicrobial, cytotoxic and photoprotective activities, the monoaromatic compound olivolic acid (OA) can also act as an alkyl resorcinol during the biosynthesis of cannabinoids. It has broad application prospects in the synthesis of pharmacological compounds. [0003] At present, the acquisition of OA and its derivatives still relies on direct extraction from plants. This plant extraction method is severely limited by plant growth cycles and conditions. Geographical and climatic conditions significantly affect the quality of the synthesized OA precursor compounds. The extraction cycle extremely long. However, the existing chemical synthesis methods of OA often have complicated steps and low yield...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/53C12N15/81C12P7/42C12R1/645
CPCC12N9/0083C12N9/1029C12N15/815C12P7/42C12Y114/99001C12N2800/22C12N2800/102
Inventor 施天穹沈琦杨彩玲彭倩倩黄和
Owner NANJING NORMAL UNIVERSITY
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