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A recombinant plasmid py16tef1-△spt15 capable of reducing the ethanol yield of Saccharomyces cerevisiae and its application

A py16tef1-, recombinant plasmid technology, applied in the field of genetic engineering, can solve the problems of inability to grow, affect the growth of microbial cells, slow growth, etc., and achieve the effect of reducing the yield of ethanol

Inactive Publication Date: 2019-07-09
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is very regrettable that the traditional method of simply knocking out or overexpressing single (multiple) genes cannot reduce or is difficult to significantly reduce the ethanol synthesis ability of Saccharomyces cerevisiae. These metabolic by-products will seriously affect the growth of microbial cells and damage the sensory quality of wine, as well as lead to changes in the fermentation characteristics of engineered strains, such as slow growth or inability to grow when glucose is used as the only carbon source.
These problems show that the modification of single (multiple) genes of the ethanol synthesis pathway or / and glycerol synthesis pathway only changes the local elements of the cell, and realizes the local balance including the redox balance system and the carbon metabolism network, and does not touch and Addressing the complex and fundamental issues involved in weakening ethanol metabolic pathways

Method used

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  • A recombinant plasmid py16tef1-△spt15 capable of reducing the ethanol yield of Saccharomyces cerevisiae and its application
  • A recombinant plasmid py16tef1-△spt15 capable of reducing the ethanol yield of Saccharomyces cerevisiae and its application
  • A recombinant plasmid py16tef1-△spt15 capable of reducing the ethanol yield of Saccharomyces cerevisiae and its application

Examples

Experimental program
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Effect test

Embodiment example 2

[0100] Implementation Case 2 Screening of Low Ethanol Yielding Saccharomyces cerevisiae Engineering Strains

[0101] 1) The SPT15 random mutation plasmid library pY16TEF1-△SPT15 was transformed into Saccharomyces cerevisiae YS59. Spread the transformation solution evenly on the SD-Ura solid medium, and culture it at 30°C for about 96 hours to obtain transformants. After culturing all the transformants with liquid SD-Ura medium in shake flasks, carry out strain preservation to obtain S.cerevisia YS59-pY16TEF1-△SPT15 mutation library;

[0102] 2) The obtained S. cerevisia YS59pY16TEF1-ΔSPT15 mutant library strains were respectively activated for 24 hours, inoculated in SD-Ura liquid medium, and fermented statically at 25°C. After the fermentation, use the SBA biosensor analyzer to measure the residual sugar and ethanol content and other indicators, and obtain the strain S. cerevisiae pY16TEF1-△SPT15-409 with a 30% lower ethanol yield compared with S. cerevisiae YS59 and the eth...

Embodiment example 3S

[0106] Implementation case 3 Application of S.cerevisiae pY16TEF1-△SPT15-409 and S.cerevisiae pY16TEF1-△SPT15-615 in simulated grape juice

[0107]After activating the obtained S.cerevisia pY16TEF1-△SPT15-409 and S.cerevisiae pY16TEF1-△SPT15-615 strains respectively for 24 hours, they were inoculated in 800mL simulated grape juice (fermentation vessel The volume is 1L), and it is left to ferment under the condition of 25°C until the residual sugar≦2g / L. After the fermentation, the SBA biosensor analyzer was used to measure the residual sugar and ethanol content and other indicators, and compared with the control strain S.cerevisiae YS59, the ethanol yield was reduced by 16% and 18%, respectively, as shown in Table 1.

[0108] The simulated grape juice in this implementation case is: ergo stock: 12.5mL Tween80, 37.5mL 95% ethanol, 0.125g ergosterol; solution A: add 100g glucose, 100g fructose, 4mL ergo stock to 375mL deionized water, dissolve and remove Make up to 500mL with d...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a recombinant plasmid pY16TEF1-deltaSPT15 capable of lowering ethanol yield of saccharomyces cerevisiae and application thereof. The recombinant plasmid pY16TEF1-deltaSPT15 comprises a recombinant plasmid pY16TEF1-deltaSPT15-409 and a recombinant plasmid py16TEF1-deltaSPT15-619, wherein deltaSPT15-409 in the recombinant plasmid pY16TEF1-deltaSPT15-409 is obtained by mutating a gene SPT15, a base sequence of the deltaSPT15-409 is shown in SEQ ID No.2, and an amino acid sequence of a transcription factor spt15p translated by the deltaSPT15-409 is shown in SEQ ID No.3; and deltaSPT15-615 in the recombinant plasmid py16TEF1-deltaSPT15-615 is obtained by mutating the gene SPT15, the base sequence of the deltaSPT15-615 is shown in SEQ ID No.4, and an amino acid sequence of a transcription factor spt15p translated by the deltaSPT15-615 is shown in SEQ ID No.5. The recombinant plasmid provided by the invention can lower the ethanol yield of the saccharomyces cerevisiae.

Description

[0001] 1. Technical field: [0002] The invention relates to the technical field of genetic engineering, in particular to a recombinant plasmid pY16TEF1-ΔSPT15 capable of reducing the ethanol yield of Saccharomyces cerevisiae and its application. [0003] 2. Background technology: [0004] With the warming of the global climate, the sugar content of grapes is increasing year by year, resulting in an increase in the ethanol content of wine. In the past 20 years, the alcohol content has increased by an average of 2% (V / V). Higher alcohol content brings various adverse sensory effects such as pungentness to wine, and also imposes drinking restrictions on drivers, children, pregnant women and other groups. Low-alcohol wine not only contains the nutrients of ordinary wine, but also avoids the damage to the sensory quality of wine caused by high alcohol content, and can satisfy consumers' pursuit of health and sensory pleasure at the same time. Therefore, appropriately reducing the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12P7/06C12R1/865
CPCC12N15/81C12N2800/102C12P7/06Y02E50/10
Inventor 秦义杜青宋育阳刘延琳
Owner NORTHWEST A & F UNIV