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A multi-inhibitor stress-tolerant Saccharomyces cerevisiae and its preparation method and application

A technology of Saccharomyces cerevisiae and inhibitor, applied in the field of microbial biology, can solve the problem of tolerance relationship to be studied and so on

Active Publication Date: 2021-04-20
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the relationship between overexpression of this gene and tolerance to acetic acid, furfural and vanillin remains to be studied

Method used

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  • A multi-inhibitor stress-tolerant Saccharomyces cerevisiae and its preparation method and application
  • A multi-inhibitor stress-tolerant Saccharomyces cerevisiae and its preparation method and application
  • A multi-inhibitor stress-tolerant Saccharomyces cerevisiae and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022]Example 1: Recombination of phospholipid synthetic protein gene YOL032W, the construction and conversion method of brewing yeast

[0023]The phospholipid synthetic protein gene YOL032W sequence from the present invention comes from the NCBI public database, and the GenBank login number of the YOL032W gene is nm_001183286.1. The promoter of the gene was inserted with the CYC1 as a terminator using CYC1 as a terminator, and the YOL032W gene was inserted between the PGK1 promoter and the CYC1 terminator. The constructed plasmid enzyme was then cooked by limiting endonuclease NOTi, transformed into the Saccharomycessee S288C for integrated expression.

[0024]1.1 Sacchal yeast genome DNA extraction

[0025](1) Picking the yeast single colony from the new YPD plate, in 100 mL of liquid YPD medium, at 30 ° C, 150 rpm, overnight culture.

[0026](2) 4 ml of bacterial liquid, 10000 rpm, centrifuged 1min, and the yeast cells were collected, and 2 times were washed twice with deionized water, and t...

Embodiment 2

[0078]Example 2: Saccharomyces of Yol032W of Phospholipid Synthesis Protein Gene YOL032W

[0079]2.1 Preparation of Saccharomycesmastic Conversion Sensing Cells

[0080](1) Yeast vaccination YPD medium, 30 ° C, 150 rpm culture 12-14h, then transferred into new YPD medium (1% inoculation) culture for 6 h;

[0081](2) Place the culture bottle on ice at least 15 minutes, so that the bacteria stops growing. 50 ml of centrifuge tube, ultra-pure water, 1M sorbitol solution is pre-cooling on ice, and the entire experiment is maintained at a low temperature;

[0082](3) Equivalence of the bacterial collected after centrifugation is gently mixed with the bacteria (up and down, do not use pipe gun to blow), 3000 rpm, 5 min, 4 ° C to collect the bacteria, abandon The supernatant, repeat this step 2;

[0083](4) Pepted 2 times with 20 ml of pre-cooling 1M sorbitol solution, and finally the yeast cells were used to immisteize the yeast cells with 0.5 mL, 1m sorbitol solution.

[0084]2.2 Electricization method to...

Embodiment 3

[0094]Example 3: Recombinant yeast S288C-YOL032W total RNA extraction and purpose gene real-time quantitative analysis of recombinant strain gene expression

[0095](1) Turn the recombinant empty yeast S288C-HO control strain and the recombinant brew yeast S288C-YOL032W to 250ml shake flask containing 100 ml seed medium (30 g / l glucose, 3 g / L protein, 4g / L yeast powder) , 30 ° C, 150 rpm, cultured 24h;

[0096](2) Substituting liquid, measured at 620 nm, and then regulates the bacterial liquid OD to 1.5 with seed medium.

[0097](3) The bacterial liquid that adjusts OD is 10% (V / V) inoculated (100 g / l glucose, 3 g / L protein, 4g / L yeast powder, pH 4.5), Culture was carried out under 30 ° C, 150 rpm;

[0098](4) When the strain is fermented to 12h (for several long-term long-term), the supernatant is separated by 4 ml of bacteria, and the cells are washed twice with sterile water. spare;

[0099](5) Extraction of recombinant bacteria S288C-YOL032W and Control Fungus S288C-HO on the tot...

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Abstract

The invention relates to the field of microbial biotechnology, in particular to a strain of multi-inhibitor stress-tolerant Saccharomyces cerevisiae and its preparation method and application. The main technical scheme is as follows: the name of the strain is S288C-YOL032W, the preservation number of the strain is CGMCC No.13926, and it is preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, and the preservation date is March 24, 2017; the strain carries There is YOL032W gene, the gene GenBank accession number is NM_001183286.1. The Saccharomyces cerevisiae strain is a single-celled fungus, generally oval and round in shape. The shape of the colony is similar to that of bacteria, but it is larger and thicker, milky white, and the surface is moist and sticky. The nutritional mode is heterotrophic facultative anaerobic. It is free and milky white when grown in medium. The recombinant Saccharomyces cerevisiae S288C-YOL032W of the present invention can have good growth and high ethanol fermentation performance under environmental stress conditions containing high concentrations of acetic acid, furfural and vanillin respectively.

Description

Technical field[0001]The invention belongs to the field of microbial biological technology, and in particular, the present invention relates to a multi-suppression of physical stress tolerance, and has a method, and the preparation method of preparation.Background technique[0002]Fuel ethanol is a clean renewable energy source is an important oil alternative, with good development prospects. Source-rich and cheap cellulose biomass farm-forestry waste is a raw material to produce fuel ethanol, which is an important direction of domestic and foreign biological energy development. However, wood cellulose feedstocks can produce by-products that have toxic to cell growth and metabolism during pre-treatment, including weak acids, aldehydes and phenols, etc. (OMICS: A Journal of Integrative Biology, 2011, 15: 647-653 ). These by-products are inhibited the cytotycosis, increasing cell membrane permeability, and disrupted the growth and ethanol fermentation of microbials, 2004, 31: 345-352, e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/06C12R1/865
CPCC07K14/395C12N15/81C12P7/06Y02E50/10
Inventor 何艳艳孜力汗刘源涛许建韧白凤武
Owner DALIAN UNIV OF TECH
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