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Saccharomyces cerevisiae recombinant strain for regulating expression activity of GRE3 genes and application of recombinant strain

A technology of Saccharomyces cerevisiae and genetically engineered strains, applied in the field of genetically engineered strains, can solve the problems of reducing the efficiency of ethanol fermentation of xylose, difficulty in achieving the optimal effect of ethanol fermentation, and the failure of recombinant strains of Saccharomyces cerevisiae to meet the requirements of industrial production, so as to improve growth speed effect

Inactive Publication Date: 2016-10-05
INST OF CROP SCI CHINESE ACAD OF AGRI SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the currently obtained Saccharomyces cerevisiae recombinant strain 1308-P can not meet the requirements of industrial production, and there is still a considerable gap
The bacterium has two main disadvantages. One is that more by-product xylitol is produced during the fermentation process, which reduces the efficiency of ethanol fermentation from xylose; The obvious inhibitory effect of furfural and hydroxymethylfurfural contained in it makes it difficult to achieve the optimal effect of ethanol fermentation

Method used

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  • Saccharomyces cerevisiae recombinant strain for regulating expression activity of GRE3 genes and application of recombinant strain
  • Saccharomyces cerevisiae recombinant strain for regulating expression activity of GRE3 genes and application of recombinant strain
  • Saccharomyces cerevisiae recombinant strain for regulating expression activity of GRE3 genes and application of recombinant strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Knockout of the GRE3 gene in Example 1 Saccharomyces cerevisiae 1308-P

[0054]According to the known GRE3 gene and the nucleotide sequence of the plasmid pUG6, design primers: the design direction is the direction to be amplified, the primer length is 63-64bp, and the annealing temperature is 60-65°C. And they were named knockout 5'GRE3::hphMX, 3'GRE3::hphMX (downstream specific primers) see Table 1.

[0055] Table 1 GRE3 gene knockout and verification primers

[0056]

[0057] The knockout component (1815bp) was obtained by PCR, and the amplified product was gel-recovered and then sent to BGI for sequencing. The sequencing sequence was compared and analyzed, and it was confirmed that the obtained sequence was consistent with the theoretically designed primer sequence.

Embodiment 2

[0058] Example 2 Knockout and modular transformation of Saccharomyces cerevisiae 1308-P

[0059] (1) Activate Saccharomyces cerevisiae 1308-P, wash it twice with sterilized deionized water, add 1ml sterilized deionized water and take 100μl for later use;

[0060] ⑵Put 50μl of salmon essence in boiling water and cook for 10 minutes for later use;

[0061] (3) Take 240 μl of PEG3350 and LiAC36 μl, add the above Saccharomyces cerevisiae, salmon essence and knockout components, vortex and mix well, put them in a water bath at 30°C for 30 minutes, and then put them in a water bath at 42°C for 45 minutes;

[0062] (4) Add 1ml of YPD to culture for 1 hour at 30°C and 120rpm;

[0063] (5) Take 20μl and spread it on the YPD plate containing G418 antibiotic for about 48h to observe.

Embodiment 3

[0064] The above engineering strain of embodiment 3 transforms the excision of pSH65 and Kanr gene (removal of the resistance selection marker in the knockout module)

[0065] (1) Activate the Saccharomyces cerevisiae 1308-P transformed into the knockout component, wash it twice with sterilized deionized water, add 1ml sterilized deionized water and take 100μl for later use;

[0066] ⑵Put 50μl of salmon essence in boiling water and cook for 10 minutes for later use;

[0067] (3) Take 240 μl of PEG3350 and LiAC36 μl, add the above Saccharomyces cerevisiae, salmon essence and knockout components, vortex and mix well, put them in a water bath at 30°C for 30 minutes, and then put them in a water bath at 42°C for 45 minutes;

[0068] (4) Add 1ml of YPD to culture for 1 hour at 30°C and 120rpm;

[0069] (5) Take 20 μl and apply it on the YPD plate containing the antibiotic phleomycin for about 48 hours to observe.

[0070] (6) Culture the strain transformed into pSH65 with YPG med...

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Abstract

The invention relates to a genetic engineering strain for regulating expression activity of GRE3 genes in saccharomyces cerevisiae 1308-P. According to the invention, it discovers that the GRE3 genes in the saccharomyces cerevisiae can induce the generation of a by-product, namely xylitol, from xylose metabolism, and the generation of the xylitol is related to tolerance in a xylose fermenting process. It also discovers that the GRE3 genes, which are although negative regulator genes, cannot be completely knocked out. Experiments prove that the engineering strain, which is obtained by knocking out one of the GRE3 genes in the 1308-P, can improve the efficiency of fermenting ethanol by virtue of the xylose and reduce the generation of the by-product, namely the xylitol. Moreover, the modified bacterium also has the advantages that the original glucose utilization rate of an original strain, an inhibitor is high in tolerance and the like.

Description

Technical field: [0001] The invention relates to a new genetic engineering bacterial strain for metabolizing xylose, in particular to a genetic engineering bacterial strain obtained by adjusting the expression activity of the GRE3 gene in Saccharomyces cerevisiae 1308-P. Background technique: [0002] Most industrial S. cerevisiae strains are unable to metabolize xylose due to their own lack of a xylose-to-xylulose metabolic pathway. [0003] Saccharomyces cerevisiae recombinant strain 1308-P is an engineered strain that has been transformed into the XI gene of Piromyces sp. The ability of xylose to produce ethanol. [0004] However, the currently obtained Saccharomyces cerevisiae recombinant strain 1308-P cannot meet the requirements of industrial production, and there is still a considerable gap. The bacterium has two main disadvantages. One is that more by-product xylitol is produced during the fermentation process, which reduces the efficiency of ethanol fermentation f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/87C12P7/06C12N15/53C12R1/865
CPCY02E50/10
Inventor 王智顿宝庆王林风杨付伟路明
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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