Recombined saccharomyces cerevisiae strain with stress tolerance

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

Inactive Publication Date: 2014-06-11
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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  • Recombined saccharomyces cerevisiae strain with stress tolerance
  • Recombined saccharomyces cerevisiae strain with stress tolerance
  • Recombined saccharomyces cerevisiae strain with stress tolerance

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. A PGK1 promoter is used as a gene promoter, a CYC1 terminator is used as a terminator, and the SET5 gene is inserted between the PGK1 promoter and the CYC1 terminator in an enzyme-cut ligated manner. Then, the constructed plasmid was linearized through the restriction site NotI, and transformed into industrial Saccharomyces cerevisiae 4126 for expression.

[0019] 1.1 Saccharomyces cerevisiae genomic DNA extraction

[0020] (1) Centrifuge the overnight cultured yeast liquid at 12000rpm for 2min, remove the supernatant;

[0021] (2) Add 480 μL TE solution (pH 8.0) and 20 μL lysozyme solution (2 mg / mL) to the precipitate, vortex and mix well, and ...

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 electroporation competent cells

[0090] (1) Yeast was inoculated with YPD medium, cultured at 30°C, 150rpm for 12-14 hours, and then transferred to new YPD medium (1% inoculation) for overnight culture;

[0091] (2) The next day, place the culture bottle on ice for at least 15 minutes to stop the growth of bacteria. Pre-cool the 50mL centrifuge tube, ultrapure water, and 1M sorbitol solution on ice, and keep it in a low temperature state;

[0092] (3) Collect the bacteria by centrifugation, mix the bacteria gently with an equal volume of ultrapure water (shake upside down, do not blow with a pipette gun), centrifuge at 3000g, 5min, 4°C to collect the bacteria, discard the supernatant, Repeat this step twice; (4) Wash the bacteri...

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 Comparison of growth of recombinant empty yeast 4126-HO control strain and recombinant Saccharomyces cerevisiae 4126-SET5 on high temperature plate

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

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

[0106] (3) Take the bacterial solution separately, measure its absorbance value OD at 620nm, then use the seed medium to adjust the OD to 0.3, inoculate the seed medium with 10% inoculation amount (same as step 1), and incubate for 5 hours;

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

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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 stress-tolerant recombinant Saccharomyces cerevisiae strain (4126-SET5), which belongs 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 biomass accumulation, promoting cell recycling, and improving fermentation efficiency. However, during the growth and fermentation process of Saccharomyces cerevisiae, especially 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, etc. These environmental stress conditions inhibit cell growth and metabolism, thereby affecting production efficiency (Journal of Biotechnology, 2009, 144:23-30). Therefore, the response and tolerance mechanism of Saccha...

Claims

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Application Information

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Patent Type & Authority Applications(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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