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Multi-inhibitor stress tolerant saccharomyces cerevisiae as well as preparation method and application thereof

A technology of Saccharomyces cerevisiae and inhibitors, applied in the field of microbial biology, can solve the problems of tolerance relationship to be studied, and achieve the effect of good growth and high ethanol fermentation efficiency

Active Publication Date: 2018-04-20
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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  • Multi-inhibitor stress tolerant saccharomyces cerevisiae as well as preparation method and application thereof
  • Multi-inhibitor stress tolerant saccharomyces cerevisiae as well as preparation method and application thereof
  • Multi-inhibitor stress tolerant saccharomyces cerevisiae as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: Construction and transformation method of recombinant Saccharomyces cerevisiae of phospholipid synthesis protein gene YOL032W

[0023] The YOL032W sequence of the phospholipid synthesis protein gene in the present invention comes from the NCBI public database, and the GenBank accession number of the YOL032W gene is NM_001183286.1. PGK1 is used as the promoter of the gene, and CYC1 is used as the terminator, and the YOL032W gene is respectively inserted between the PGK1 promoter and the CYC1 terminator in the manner of enzyme-cut ligation. Then, the constructed plasmid was linearized by the restriction endonuclease Not I, and transformed into Saccharomyces cerevisiae S288C for integrated expression.

[0024] 1.1 Saccharomyces cerevisiae genomic DNA extraction

[0025] (1) Pick a single yeast colony from a newly cultivated YPD plate, insert it into 100 mL liquid YPD medium, and culture overnight at 30° C. and 150 rpm.

[0026] (2) Take 4 mL of bacterial li...

Embodiment 2

[0078] Embodiment 2: Saccharomyces cerevisiae electrotransformation method of phospholipid synthesis protein gene YOL032W

[0079] 2.1 Preparation of Saccharomyces cerevisiae electroporation competent cells

[0080] (1) Yeast was inoculated with YPD medium, cultured at 30°C, 150rpm for 12-14h, and then transferred to new YPD medium (1% inoculated) for 6h;

[0081] (2) 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 at a low temperature throughout the experiment;

[0082] (3) Gently mix the bacterial cells collected after centrifugation with an equal volume of ultrapure water (shake upside down, do not blow with a pipette), centrifuge at 3000rpm, 5min, 4°C to collect the bacterial cells, discard Supernatant, repeat this step 2 times;

[0083] (4) Wash the bacterial pellet twice with 20 mL of pre-cooled 1M sorbitol solution, and finally resu...

Embodiment 3

[0094] Example 3: Extraction of total RNA from recombinant yeast S288C-YOL032W and real-time quantitative analysis of gene expression in recombinant strains

[0095] (1) Inoculate the recombinant empty yeast S288C-HO control strain and recombinant Saccharomyces cerevisiae S288C-YOL032W into a 250mL shake flask containing 100mL seed medium (30g / L glucose, 3 g / L peptone, 4g / L yeast extract powder) medium, 30°C, 150rpm, cultured for 24h;

[0096] (2) Take the bacterial liquid respectively, measure its absorbance at 620nm, and then use the seed medium to adjust the OD of the bacterial liquid to 1.5;

[0097] (3) Inject the OD-adjusted bacterial solution into the fermentation medium (100 g / L glucose, 3 g / L peptone, 4 g / L yeast extract powder, pH 4.5) with an inoculum size of 10% (v / v), Cultivate at 30°C, 150 rpm;

[0098] (4) When the strain was fermented for 12 hours (logarithmic growth phase), 4 mL of the bacterial liquid was centrifuged to discard the supernatant, the cells we...

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Abstract

The invention relates to the field of microbial biotechnology, in particular to a multi-inhibitor stress tolerant saccharomyces cerevisiae as well as a preparation method and application thereof. Themain technical scheme is as follows: the name of the strain is S288C-YOL032W, the preservation number of the strain is CGMCC No.13926, the strain is preserved in the China General Microbiological Culture Collection Center on March 24, 2017; and the strain carries a YOL032W gene with a GenBank accession number of NM_001183286.1. The saccharomyces cerevisiae strain is a unicellular fungus which is generally oval and round. The colony morphology is similar to that of bacteria, but is larger and thicker, and the strain is milky white, and has a wet and sticky surface. The nutrient mode is a heterotrophic facultative anaerobic mode. The strain is free and milky white when growing in a culture medium. The recombinant saccharomyces cerevisiae S288C-YOL032W provided by the invention can have goodgrowth and higher ethanol fermentation performance under the environmental stress condition of containing high-concentration acetic acid, furfural and vanillin respectively.

Description

technical field [0001] The invention belongs to the field of microbial biotechnology, and in particular relates to a multi-inhibitor stress-tolerant Saccharomyces cerevisiae and its preparation method and application. Background technique [0002] As a clean and renewable energy, fuel ethanol is an important substitute for petroleum and has a good development prospect. The production of fuel ethanol from abundant and cheap cellulosic biomass and agricultural and forestry wastes is an important direction for the development of bioenergy at home and abroad. However, during the pretreatment of lignocellulosic raw materials, by-products that are toxic to cell growth and metabolism can be produced, including weak acids, aldehydes and phenols (Omics: a Journal of Integrative Biology, 2011, 15: 647-653 ). These by-products inhibit microbial cell growth and ethanol fermentation by inhibiting yeast aerobic respiration, increasing cell membrane permeability, and destroying intracell...

Claims

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Application Information

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Patent Type & Authority Applications(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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