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Recombinant yarrowia lipolytica as well as construction method and application thereof

A technology for Yarrowia lipolytica and yeast, applied in the field of bioengineering, can solve problems such as low yield and industrial application, and achieve the effects of simple operation, enhanced homologous recombination ability, and improved genetic stability

Active Publication Date: 2020-05-15
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been relevant studies using this point to produce β-elemene in microorganisms, but due to the low yield, industrial applications have not been realized.

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

[0038] Embodiment 1, the amplification of gene element and the preparation of target plasmid

[0039] (1) Preparation of target gene

[0040] According to the nucleotide sequence of the gene gas (genbank accession number KU589283.1) encoding germane A synthase from Roman chamomile provided by NCBI, after codon optimization, it was synthesized and optimized by entrusting Suzhou Jinweizhi Biotechnology Co., Ltd. The gene Optgas encoding germane A synthase was inserted into the plasmid pUC57 to obtain the plasmid pUC57-Optgas. The nucleotide sequence of Optgas is shown in SEQ ID No:1.

[0041] According to the nucleotide sequence of the gene leu encoding 3(β)-isopropylmalate dehydrogenase in Yarrowia lipolytica provided by NCBI (genbank accession number M37309.1), Suzhou Jinweizhi Biotechnology Co., Ltd. was entrusted to synthesize the gene leu, The 3(β)-isopropylmalate dehydrogenase encoding gene expression cassette (including the endogenous promoter P of Yarrowia lipolytica ...

Embodiment 2

[0092] The construction of embodiment 2 recombinant bacteria

[0093] (1) Construction of Recombinant Bacteria 1

[0094](1) The plasmid pUC-leu-A08-Optgas containing the Optgas gene expression cassette was introduced into Yarrowialipolytica Po1fΔku70, and the Optgas expression cassette was integrated into the A08 site of the genome to obtain recombinant bacterium 1. The specific method is as follows: (1) Yarrowia lipolytica Po1fΔku70 was cultured overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose) to prepare competent cells. (2) Use Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation to transform pUC-leu-A08-Optgas into Yarrowia lipolytica Po1fΔku70 competent cells for homologous recombination.

[0095] (2) 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 20g / L, Yeast Nitrogen Base (YNB, yeast nitr...

Embodiment 3

[0131] Embodiment 3, the application of recombinant bacteria 1-8 in the production of β-elemene

[0132] 1. Cultivation of engineering bacteria and product extraction

[0133] The recombinant bacteria 1-8 in Example 2 were used to produce β-elemene respectively. The specific method is as follows: activate the recombinant bacteria, culture in YPD liquid medium 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 size of 1%, and cultured with shaking at 30° C. and 220 rpm for 1 day, then added n-dodecane with 10% of the volume of the fermentation liquid, and continued shaking for 3 days. After the fermentation, the fermentation broth was transferred to a 50ml centrifuge tube, centrifuged at 5000rpm for 15min, and the organic phase was collected for later use.

[0134] Wherein the fermentation medium contains 55g / L glucose, 10g / L yeast extract and 20g / L tryptone.

[0135] 2. Qualitative and ...

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Abstract

The invention provides recombinant yarrowia lipolytica as well as a construction method and application thereof, and relates to the field of bioengineering. The recombinant yarrowia lipolytica is a yarrowia lipolytica XJ-8 strain collected in CGMCC, with the collection number of CGMCC No.19064. The construction method of the yarrowia lipolytica is simple and efficient to operate, and can efficiently synthesize beta-elemene and / or germacrene A.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a recombinant Yarrowia lipolytica for producing β-elemene / or rhizomerene A, its construction method and application. Background technique [0002] β-elemene (β-elemene), scientific name: 1-methyl-1-vinyl-2,4-diisopropylcyclohexane, is a volatile sesquiterpene, mainly present in Curcuma zedoaria), citronella (Cymbopogon citratus) and other essential oils. β-Elemene can pass through the blood-brain barrier, not only directly anti-tumor, but also has immune protection; it also has the characteristics of relieving cancer pain, increasing white blood cells and inhibiting platelet aggregation (Edris AE, Current Clinical Pharmacology, 2009, 4 (1):43-46.; Chen M et al., Evidence-Based Complementary and Alternative Medicine, 2012, 2012.). Moreover, compared with most chemotherapy drugs, the drug has less toxic and side effects, no obvious liver and kidney damage, no bone marrow suppression...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P5/02C12P5/00C12R1/645
CPCC12N9/88C12N9/1085C12N9/0006C12P5/026C12P5/002C12Y402/03023C12Y205/0101C12Y101/01088
Inventor 纪晓俊王凯峰马一榕李文娟买洁
Owner NANJING UNIV OF TECH
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