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Method for improving cell viability and multiple stress resistance of saccharomyces cerevisiae and application of method

A technology of Saccharomyces cerevisiae and stress resistance, applied in the field of high-viability yeast strains and gene expression cassettes, to achieve the effect of achieving high efficiency, improving stress resistance, and strong multiple stress resistance

Active Publication Date: 2018-05-01
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the single and technical deficiencies of the prior art, to provide a gene expression cassette that can make yeast engineering strains possess multiple stress resistance and enhance their cell viability, an expression vector inserted with the gene expression cassette, Method for preparing engineering strains using the gene expression cassette and its application in industrial production of ethanol

Method used

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  • Method for improving cell viability and multiple stress resistance of saccharomyces cerevisiae and application of method
  • Method for improving cell viability and multiple stress resistance of saccharomyces cerevisiae and application of method
  • Method for improving cell viability and multiple stress resistance of saccharomyces cerevisiae and application of method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Gene Expression Cassette and High Activity Multiple Stress Resistance Construction

[0039] The starting strain Saccharomyces cerevisiae (INVSC1) used in this paper is an auxotrophic yeast strain. The shuttle plasmid pRS42K used was purchased from EUROSCARF in Frankfurt, Germany. The YPD medium used is a universal complete medium, and the solid medium contains 2% agar powder. Antibiotic G418 was added to the medium of plasmid-containing strains such as LIP-P11 and the control strain WT / V.

[0040] Design primers to clone the promoter gene, target gene sequence SEQ ID NO.1 sequence, terminator gene, and then perform gene combination by OE-PCR to obtain the gene expression cassette. The gene expression cassette was transformed into the yeast cell INVSC1 by chemical transformation together with the plasmid pRS42K after homozygous double enzyme digestion (BamHI enzyme, Slal enzyme), (see schematic diagram figure 1 ) utilize the homologous recombination mechanis...

Embodiment 2

[0042] Example 2. Detection of the transcription level of the target gene sequence in the expression cassette

[0043] After cultivating Saccharomyces cerevisiae engineered bacteria in liquid YPD medium at 30°C and 200rpm for 36h, the initial OD 660 =0.1 was transferred to 40 mL fresh YPD liquid medium, and after culturing at 30°C for 12 hours, the temperature was raised to 40°C to continue culturing for 24 hours. The bacteria were collected using a kit for RNA extraction. Then use TAKARA's "PrimeScript TM The RT Master Mix (Perfect Real Time)” kit reverse-transcribed the extracted RNA using the relative quantitative method of qRT-PCR, that is, selecting an internal reference gene ATC1, using the internal reference characteristic gene in the sample as the calibration gene, and the target gene The content is reflected by comparison with the internal reference gene. Numerical value is not a deterministic sample concentration, but a relative value compared with the internal ref...

Embodiment 3

[0044] Embodiment 3. high temperature fermentation experiment

[0045] Activation of strains

[0046] Pick the strains stored on the plates stored in the refrigerator at 4°C, inoculate them in liquid YPD medium, culture them in a constant temperature shaker at 30°C with a rotation speed of 200rpm for 36 hours, and then transfer them to fresh liquid YPD with an inoculum of 1%. Continue culturing in the culture medium for 12 h as the seed solution.

[0047] Gradient heating (37°C-45°C) fermentation experiment

[0048] Inoculate a certain amount of activated seed solution into a 250mL Erlenmeyer flask containing 40mL of fresh YPD liquid medium, so that the initial OD of the cell culture solution 660 0.1 and cultured in a constant temperature shaker at 30°C for 12 hours, then raise the culture temperature to 37°C. Then, based on 37°C, the culture temperature was gradually increased to 39°C, 40°C, 41°C, 42°C, and 43°C every 12 hours, and co-cultivated for 84 hours. Sampling eve...

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Abstract

The invention discloses a gene expression cassette, which is constituted by sequentially connecting a promoter, a nucleotide sequence shown as SEQ ID NO.1 and a terminator sequentially, an expressionvector which is interpolated into the gene expression cassette and two multiple-stress-resistance yeast strains which are prepared via the gene expression cassette and are high in cell viability. According to the technical scheme, saccharomyces cerevisiae is transformed by virtue of the gene expression cassette of the invention, so that the fine transformation of ubiquitin proteasome is achieved;the invention provides a method which is capable of simultaneously improving cell viability and multiple stress resistance of the saccharomyces cerevisiae and an application of the method; and the obtained saccharomyces cerevisiae engineering strain is stable in genetic performance, and has a broad application range and a broad application prospect in industrial fermentation.

Description

[0001] The invention relates to a method for simultaneously improving cell viability and multiple stress resistance of Saccharomyces cerevisiae and its application. Specifically, it relates to a gene expression cassette, an expression vector inserted into the gene expression cassette, a method for transforming the expression vector into yeast cells or inserting the expression cassette into the yeast genome to prepare a highly active yeast strain with multiple stress resistance The invention and its application belong to the field of biochemical industry. Background technique [0002] At present, the annual output value of industrial biotechnology products is increasing year by year, occupying a very high proportion in the national economy, among which microbial fermentation is the main component of industrial biotechnology. One of the decisive factors for the benefit of industrial microbial fermentation is the microbial strain used. In the actual industrial high-density microb...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/81C12N1/19C12R1/865
CPCC12N9/60C12N15/81
Inventor 李珺俞利平刘护李春
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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