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Recombinant Yarrowia lipolytica for producing Valencene and (+)-Nootkatone, and construction method thereof

A technology of valencia citrurene and Yarrowia lipolytica, which is applied to the field of recombinant Yarrowia lipolytica producing valencia citruene and nokadone and its construction, which can solve the problems of low yield

Inactive Publication Date: 2018-02-23
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The extraction of nokatone from natural plants such as citrus is limited by various factors such as climate, environment and harvest season, and the extraction yield is extremely low

Method used

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  • Recombinant Yarrowia lipolytica for producing Valencene and (+)-Nootkatone, and construction method thereof
  • Recombinant Yarrowia lipolytica for producing Valencene and (+)-Nootkatone, and construction method thereof
  • Recombinant Yarrowia lipolytica for producing Valencene and (+)-Nootkatone, and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, amplification and preparation of genetic elements

[0044] According to the DNA sequences of valencia citruene synthase coding gene CVS and nokatone synthase coding gene CYP706M1 provided on NCBI, valencia citruene synthase was synthesized by chemical synthesis (Wuhan Jinkairui Biological Engineering Co., Ltd.) Enzyme gene coding CVS (SEQ ID NO.1), nokatone synthase coding gene CYP706M1 (SEQ ID NO.2), cytochrome P450 reductase coding gene AtCPR (SEQ ID NO.3).

[0045] The valencia tangerene synthase encoding gene expression cassette is composed of promoter P TEF1 , Valencia citruene synthase coding gene CVS and terminator T xpr2 composition;

[0046] Described noka ketone synthase coding gene expression cassette is by promoter P EXP1 , nokatone synthase coding gene CYP706M1 and terminator T mig1 composition;

[0047] The cytochrome P450 reductase encoding gene expression cassette is composed of a promoter P GPD1 , cytochrome P450 reductase coding gen...

Embodiment 2

[0083] Embodiment 2, transformation and construction of recombinant Yarrowia lipolytica producing valencia citrurene and nokadone

[0084] The DNA fragment rDNAup, P TEF1 -CVS-T xpr2 ,P EXP1 -CYP706M1-T mig1 ,P GPD1 -AtCPR-T lip2 , and ura-rDNAdown were introduced into Yarrowia lipolytica ATCC 201249 to obtain recombinant bacterium 1;

[0085] The DNA fragment rDNAup, P TEF1 -CVS-T xpr2 ,P EXP1 -CYP706M1-tAtCPR-T lip2 And ura-rDNAdown was introduced into Yarrowia lipolytica ATCC201249 to obtain recombinant bacterium 2. The conversion method is as follows:

[0086] Yarrowia lipolytica ATCC 201249 was cultured in YPD medium (1% yeast extract, 2% peptone, 2% glucose) for 12 hours, then 300 μL was added to 3 mL of fresh YPD medium, and cultured for 5 hours. Centrifuge at 6000rpm at room temperature for 5min to collect the bacterial cells, discard the supernatant, wash the bacterial cells with sterilized ddH2O, collect the bacterial cells by centrifuging at 6000rpm at ro...

Embodiment 3

[0087] Example 3, Preparation and linearization of expression plasmid ptHMG1

[0088]Using Saccharomyces cerevisiae genome as a template, tHMG1-BamHI-F (SEQ ID NO.38) and tHMG1-BamHI-R as primers (SEQ ID NO.39) (see primer list 2 for the sequence) to amplify tHMG1. Plasmid PINA1269 was extracted with a small plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.). The plasmids PINA1269 and tHMG1 were digested with restriction endonuclease BamHI, and the digested plasmids PINA1269 and tHMG1 were recovered by agarose gel electrophoresis.

[0089] Primer List 2

[0090]

[0091] Use T4 ligase to ligate the digested plasmid PINA1269 and tHMG1, the system is as follows:

[0092]

[0093] React at 22°C for 30 minutes.

[0094] 5 μL of the connected system was used to transform Escherichia coli Trans T1 competent. The conversion method is as follows:

[0095] (1) Take out Escherichia coli Trans T1 competent cells from the -80°C refrigerator, place them in an ice ...

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Abstract

The invention discloses a construction method of a recombinant Yarrowia lipolytica for producing Valencene and (+)-Nootkatone. The method is characterized in that a Valencene synthase coding gene CVSexpression cassette, a (+)-Nootkatone synthase coding gene CYP706M1 expression cassette and a cytochrome P450 reductase coding gene AtCPR expression cassette are introduced into the rDNA site of Yarrowia lipolytica through a homologous recombination technology to obtain the recombinant Yarrowia lipolytica 1 for producing Valencene and (+)-Nootkatone. Experiments prove that the recombinant Yarrowialipolytica for producing Valencene and (+)-Nootkatone, obtained by the homologous recombination technology, can increase the yield of the Valencene and the (+)-Nootkatone, and allows the yield of theValencene to be 6.5 to 18 mg / L and the yield of the (+)-Nootkatone to be 30 [mu]g / L to 0.5 mg / L. The method provides a basis for the artificial synthesis of the Valencene and (+)-Nootkatone.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Yarrowia lipolytica producing valenciarne and nokadone and a construction method thereof. Background technique [0002] Nootkatone ((+)-nootkatone) belongs to sesquiterpenes, which was isolated from Nootka cypress for the first time, and also exists in small amounts in grapes, pomelo and other fruits. Nokatone has a unique aroma and a low odor threshold, which makes nokatone a popular fragrance and widely used as an additive in food and cosmetics. Compositional analysis and research on Nootka cypress wood has shown that nokatone also has insecticidal, acaricidal and antimicrobial properties, which can keep Nootka cypress against decay for decades. In recent years, the application of nokadone in medicine has received more and more attention. Studies have shown that nokadone has anti-platelet aggregation effects, anti-cancer cell proliferation, and improves energy metab...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P7/26C12P5/00C12R1/645
CPCC12N9/0042C12N9/1085C12N9/88C12P5/002C12P7/26C12Y106/02004C12Y402/03073
Inventor 卢文玉郭校燕
Owner TIANJIN UNIV
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