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A stress-tolerant recombinant Saccharomyces cerevisiae strain

A Saccharomyces cerevisiae strain and tolerance technology, applied in the field of microorganisms, can solve problems such as low cellulase hydrolysis efficiency, and achieve the effect of high ethanol fermentation efficiency

Inactive Publication Date: 2015-08-19
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at lower temperatures, the hydrolysis efficiency of cellulase will be very low. Considering the hydrolysis and fermentation process (Biotechnology Advances, 2012, 30: 1207–1218), high temperature resistant yeast is an inevitable requirement

Method used

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  • A stress-tolerant recombinant Saccharomyces cerevisiae strain
  • A stress-tolerant recombinant Saccharomyces cerevisiae strain
  • A stress-tolerant recombinant Saccharomyces cerevisiae strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Construction and transformation of recombinant Saccharomyces cerevisiae containing histone methylase encoding gene SET5

[0018] The sequence of the histone methylase encoding gene SET5 involved in the present invention comes from the NCBI public database, and the GenBank accession number of the gene is NC_001140.6. The PGK1 promoter was used as the promoter of the gene, and the CYC1 terminator was used as the terminator, and the SET5 gene was inserted between the PGK1 promoter and the CYC1 terminator in the manner of enzyme cleavage. Then, the constructed plasmid enzyme was cut linearly through the restriction site NotI, and transformed into Saccharomyces cerevisiae 4126 for expression.

[0019] 1.1 Saccharomyces cerevisiae genomic DNA extraction

[0020] (1) Centrifuge the overnight cultured yeast solution at 12,000 rpm for 2 min, and remove the supernatant;

[0021] (2) Add 480 μL of TE solution (pH 8.0) and 20 μL of lysozyme solution (2 mg / mL) to the pr...

Embodiment 2

[0087] Example 2: Transformation of industrial saccharomyces cerevisiae containing histone methylase encoding gene SET5

[0088] Saccharomyces cerevisiae cell transformation method is as follows:

[0089] 2.1 Preparation of Saccharomyces cerevisiae competent cells for electrotransformation

[0090] (1) Yeast inoculate YPD medium, culture at 30℃, 150rpm for 12-14 hours, then transfer to new YPD medium (1% inoculation) for overnight culture;

[0091] (2) The next day, put the culture bottle on ice for at least 15min to stop the growth of bacteria. Put the 50mL centrifuge tube, ultrapure water, and 1M sorbitol solution on ice to pre-cool at low temperature;

[0092] (3) Collect the cells by centrifugation, gently mix the cells with an equal volume of ultrapure water (shake upside down, do not use a pipette), centrifuge at 3000 g, 5 min, 4 °C to collect the cells, discard the supernatant, Repeat this step twice; (4) Wash the cell pellet twice with 20 mL of pre-cooled 1M sorbito...

Embodiment 3

[0102] Example 3: Comparison of plate growth of recombinant empty yeast 4126-HO control strain and recombinant Saccharomyces cerevisiae 4126-SET5 under different stress factors

[0103] 3.1 Growth comparison of recombinant empty yeast 4126-HO control strain and recombinant Saccharomyces cerevisiae 4126-SET5 on high temperature plates

[0104] (1) Inoculate the recombinant empty yeast 4126-HO control strain and recombinant Saccharomyces cerevisiae 4126-SET5 into a 250 mL shake flask containing 50 mL of seed medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) , 30°C, 150 rpm, overnight culture;

[0105] (2) Repeat step (1);

[0106] (3) Take the bacterial liquid separately, measure its absorbance value OD at 620nm, then adjust the OD to 0.3 with the seed medium, inoculate the seed medium with 10% of the inoculum respectively (same as step 1), and cultivate for 5h;

[0107] (4) Take the bacterial liquid separately, and measure its absorbance value OD at 620nm. At this...

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Abstract

The invention discloses recombined saccharomyces cerevisiae (4126-SET5) with stress tolerance and belongs to the technical field of microorganisms. The strain is classified and named as Saccharomyces cerevisiae, the accession number of the strain is CGMCC No.8724, and the strain is preserved in the common microorganism center of China microorganism strain preservation administration committee, wherein the address of the preservation enterprise is 3#, Yard No.1, West Beichen Road, Chaoyang District, Beijing City, and the preservation data is Jan 15th, 2014. The invention discloses a gene engineering construction method of the recombined strain 4126-SET5, and the method comprises acquisition of a gene, construction of a chromosome integration vector and the growth condition of the strain under various environmental stress conditions, including on a flat plate containing high-concentration acetic acid, hydrogen peroxide and ethanol and under a high temperature condition. Compared with a contrast strain of an empty integration vector, the recombined saccharomyces cerevisiae 4126-SET5 can be rapidly fermented in the presence of 5g / L of acetic acid, thereby not only providing theoretical support for further research on tolerance mechanism of the saccharomyces cerevisiae but also being used as a good strain for fermentation of cellulosic ethanol.

Description

technical field [0001] The invention relates to a strain of recombinant Saccharomyces cerevisiae (4126-SET5) with stress tolerance, belonging to the technical field of microorganisms. Background technique [0002] Saccharomyces cerevisiae is widely used in different fields such as food, brewing and bioenergy production. Good cell activity is conducive to increasing the accumulation of biomass, promoting cell recycling, and improving fermentation efficiency. However, during the growth and fermentation process of Saccharomyces cerevisiae, especially in Under industrial production conditions, it is often affected by environmental stress factors such as high concentration of ethanol, extreme temperature (freezing or high temperature, etc.), low pH and high osmotic pressure. These environmental stress conditions inhibit cell growth and metabolism, thus affecting production efficiency (Journal of Biotechnology, 2009, 144: 23-30). Therefore, the response and tolerance mechanism of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/63C12R1/865
Inventor 赵心清张明明白凤武
Owner DALIAN UNIV OF TECH
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