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Recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol using xylose and construction method

A technology of Saccharomyces cerevisiae and dammarenediol, applied in the field of recombinant Saccharomyces cerevisiae and construction, and can solve problems such as low efficiency

Active Publication Date: 2018-12-07
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after a lot of research, it is found that the efficiency of ethanol production from xylose is still not as good as that of glucose

Method used

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  • Recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol using xylose and construction method
  • Recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol using xylose and construction method
  • Recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol using xylose and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, amplification and preparation of genetic elements

[0042] The xylulokinase XKS1 promoter is replaced by the promoter P FBA1 (Promoter P FBA1 ; Nucleotide sequence as shown in SEQ ID NO.1; simply marked as SEQ ID NO.1, the same below);

[0043] The expression cassette of the xylose reductase gene XYL1 is composed of a promoter P CCW12 (SEQ ID NO.15), xylose reductase gene XYL1 (SEQ ID NO.2) and terminator T HSP26 (SEQ ID NO.16) composition;

[0044] The xylitol dehydrogenase gene XYL2 expression cassette is composed of promoter P HXT7 (SEQ ID NO.17), xylitol dehydrogenase gene XYL2 (SEQ ID NO.3) and terminator T HXT7 (SEQ ID NO.18) composition;

[0045] The expression cassette of the transaldolase gene TAL1 is composed of a promoter P HXT7 , transaldolase gene TAL1 (SEQ ID NO.4) and terminator THXT7 composition;

[0046] The expression cassette of the transketolase gene TKL1 is controlled by the promoter P CCW12 , transketolase gene TKL1 (SEQ ID N...

Embodiment 2

[0073] Example 2 Transformation and Construction of Recombinant Saccharomyces cerevisiae Using Xylose to Produce Dammarenediol and Protopanaxadiol

[0074] DNA fragment XKS1t2-his3-P FBA1 -XKS1t1,P CCW12 -XYL1-T HSP26 ,P HXT7 -XYL2-T HXT7 ,P CCW12 -TKL1-T HSP26 and P HXT7 -TAL1-T HXT7 Import Saccharomyces cerevisiae to obtain recombinant strain 1;

[0075] DNA fragment P PGK1 -DS-T CYC1 P TEF1 -ERG1-T ADH1 and P TPI1 -ERG9-T ADH2 Introduce recombinant bacterium 1 to obtain recombinant bacterium 2;

[0076] DNA fragment P TPI1 -ERG20,P PGK1 -NADH-HMGr-T CYC1 and P TDH3 -PPDS-ATR1-T ADH1 Introducing the recombinant bacterium 2 to obtain the recombinant bacterium 3;

[0077] DNA fragment P HXT7 -ERG10, P PGK1 -ERG12 and P TEF1 -ERG13 was introduced into recombinant strain 3 to obtain recombinant strain 4.

[0078] The conversion method is as follows:

[0079] After Saccharomyces cerevisiae W303-1A was cultured in YPD medium for 12 hours, 300 μL was added ...

Embodiment 3

[0080] Example 3 Production of Dammarenediol and Protopanaxadiol by Fermentation of Recombinant Saccharomyces cerevisiae Using Xylose

[0081] Single colonies of recombinant bacteria 2, recombinant bacteria 3 and recombinant bacteria 4 were picked for shake flask fermentation. The fermentation conditions were 30° C., 220 rpm, and cultured for 3 days. Fermentation medium is YPX liquid medium, and wherein each component and final concentration thereof are as follows: final concentration is the xylose of 4% (mass percentage), the peptone of 2% (mass percentage), the yeast extract of 1% (mass percentage) powder, make up to volume with water.

[0082] After the fermentation is finished, extract with n-butanol, and the volume ratio of n-butanol to fermentation broth is 1:4. The n-butanol phase was taken for LC-MS detection, and it was found that recombinant bacteria 2 could produce 10.69 mg / L dammarene diol, and recombinant bacteria 3 could produce 3.17 mg / L dammarene diol and 11....

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Abstract

The invention discloses recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol using xylose and a construction method. The construction method comprises the steps of replacing a promoter of a saccharomyces cerevisiae xylulokinase gene XKS1 with a promoter PFBA1 by virtue of a homologous recombination method, introducing xylose reductase XYL1 and a xylitol dehydrogenase XYL2 expression cassette, increasing the activities of transketolase TKL1 and transaldolase TAL1 so as to obtain recombinant bacteria 1, introducing a farnesyl-diphosphate farnesyltransferase gene ERG9, a squalene monooxygenase gene ERG1 and a dammarenediol synthase gene DS into the recombinant bacteria 1 so as to obtain recombinant bacteria 2, and introducing a nicotinamide adenine dinucleotide-hydroxymethylglutaryl coenzyme A reductase gene NADH-HMGr, farnesyl diphosphatesynthase ERG20 and a protopanoxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1 into the recombinant bacteria 2, so as to obtain recombinant bacteria 3. According to the recombinant saccharomyces cerevisiae, dammarenediol and protopanoxadiol can be artificially synthesized by virtue of xylose.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Saccharomyces cerevisiae strain for producing dammarenediol and protopanaxadiol by utilizing xylose, a construction method and application thereof. Background technique [0002] Xylose is a pentose sugar. Natural D-xylose exists in plants in the form of polysaccharides. No free xylose has been found in nature so far. As the main component of plant cell walls, lignocellulose mainly includes three components: cellulose, hemicellulose and lignin. Lignocellulosic biomass produced through photosynthesis on the earth is more than 100 billion tons every year, which is the most abundant renewable energy substance on the earth. By hydrolyzing lignocellulose, rich monosaccharide substances such as glucose and xylose can be obtained. As the monosaccharide in lignocellulose hydrolyzate, which is second only to glucose, the utilization of xylose has attracted extensive attention o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/90C12P33/00C12R1/865
CPCC12N9/0006C12N9/0073C12N9/0081C12N9/1022C12N9/1025C12N9/1029C12N9/1085C12N9/1205C12N9/88C12N15/905C12P33/00C12Y101/01009C12Y101/01034C12Y101/01307C12Y114/13132C12Y114/15006C12Y202/01001C12Y202/01002C12Y203/01009C12Y203/0301C12Y205/0101C12Y205/01021C12Y207/01036C12Y402/01125
Inventor 卢文玉高晓
Owner TIANJIN UNIV
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