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A kind of construction method of Saccharomyces cerevisiae isobutanol high-yielding strain

A technology of Saccharomyces cerevisiae and Saccharomyces cerevisiae, which is applied in the field of DNA recombination technology to prepare microorganisms, and can solve problems such as imbalance and restricting the yield of isobutanol.

Active Publication Date: 2016-05-18
HEBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the prior art, due to overexpression of ILV2, ILV3 and ILV5 genes in isobutanol-producing Saccharomyces cerevisiae cells, the coenzymes NADPH / NADP from pyruvate to 2-ketoisovalerate + The imbalance restricts the further improvement of isobutanol production, these defects are in Image 6 displayed in

Method used

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  • A kind of construction method of Saccharomyces cerevisiae isobutanol high-yielding strain
  • A kind of construction method of Saccharomyces cerevisiae isobutanol high-yielding strain
  • A kind of construction method of Saccharomyces cerevisiae isobutanol high-yielding strain

Examples

Experimental program
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Effect test

Embodiment 1

[0061] The host strain was Saccharomyces cerevisiae strain W303-1A (MATaleu2-3, 112ura3-1trp1-92his3-11, 15ade2-1can1-100 (Thomas and Rothstein, 1989)), and the plasmid YEplac195- PGK1p-ILV2, YEplac181-PGK1p-ILV3 and YEplac112-PGK1p-ZWF1-PGK1p-ILV5,

[0062] The first step, construction of plasmids for overexpressing Saccharomyces cerevisiae ILV2, ILV3, ILV5 and ZWF1 genes

[0063] (1.1) Preparation of Saccharomyces cerevisiae W303-1A chromosome: inoculate Saccharomyces cerevisiae W303-1A in 5 mL of YPD medium, vibrate at 30°C overnight to obtain a culture solution, and divide the culture solution into two 1.5 mL In the centrifuge tube, centrifuge at 12000rpm for 30s, take the supernatant, add 0.5mL water to resuspend, repeatedly suck the tip of the pipette to disperse the sedimentation of bacteria, then centrifuge at 12000rpm for 30s, collect the bacteria, and put them in Add 200 μL of bacteriostasis buffer to resuspend the cells in each centrifuge tube, then add 200 μL of g...

Embodiment 2

[0084] Except that the host bacterium is Saccharomyces cerevisiae strain and is selected as an industrial Saccharomyces cerevisiae strain, the others are the same as in Example 1.

[0085] The raw materials used in the above examples are all known and commercially available, and the operating techniques used are within the grasp of those skilled in the art.

Embodiment 3

[0087] Divide with T 4 Ligase to connect YEplac195-PGK1p and ILV2ORF genes: In a 1.5mL centrifuge tube, add 10×T 4 Ligase buffer 2 μL, T 4 The amount of ligase 0.2 μL, YEplac195-PGK1p and ILV2ORF gene fragments added was 4 μL, and then the total volume was made up to 20 μL with sterile water, and the ligation product was obtained at 16°C for 1.5 h; 4 Ligase to connect YEplac181-PGK1p and ILV3ORF genes: In a 1.5mL centrifuge tube, add 10×T 4 Ligase buffer 2 μL, T 4 Add 0.2 μL of ligase, 4 μL of YEplac181-PGK1p and ILV3ORF gene fragments, add sterile water to a total volume of 20 μL, and connect at 16°C for 1.5 h to obtain the ligation product; 4 Ligase to connect YEplac112-PGK1p and ILV5ORF genes: In a 1.5mL centrifuge tube, add 10×T 4 Ligase buffer 2 μL, T 4 Add 0.2 μL of ligase, 4 μL of YEplac112-PGK1p and ILV5ORF gene fragments, add sterile water to a total volume of 20 μL, and connect at 16°C for 1.5 h to obtain the ligation product; 4 Ligase the YIplac211-PGK1p and Z...

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Abstract

The invention discloses a construction method of a saccharomyces cerevisiae strain with a high yield of isobutanol and relates to preparation of microorganism by a recombinant DNA (Deoxyribonucleic Acid) technology. According to the method, the saccharomyces cerevisiae strain with the high yield of isobutanol is constructed by reforming saccharomyces cerevisiae cells by using molecular biology and the gene recombination technology. The method comprises the following steps: S1, constructing plasmids of over-expressing ILV2 (Induced Leukemia Virus), ILV3, ILV5 and ZWF1 genes of saccharomyces cerevisiae; and S2, constructing a saccharomyces cerevisiae strain HBGDAL-103 with the high yield of isobutanol. The method disclosed by the invention overcomes the deficiency that further improvement of the yield of isobutanol is restricted by factors such as imbalance of a coenzyme NADPH / NADP<+> (Reduced Form of Nicotinamide-Adenine Dinucleotide Phosphate) / (Nicotinamide-Adenine Dinucleotide Phosphate<+>) from pyruvic acid to 2-one isovaleric acid due to over-expressed ILV2, ILV3 and ILV5 in the saccharomyces cerevisiae cells of yielding isobutanol in the prior art.

Description

technical field [0001] The technical scheme of the invention relates to the preparation of microorganisms by DNA recombination technology, in particular to a method for constructing a high-yield isobutanol strain of Saccharomyces cerevisiae. Background technique [0002] Isobutanol has low volatility, corrosiveness, high octane number and energy density, and is easy to store and transport. It is a new type of biofuel with huge market potential. Saccharomyces cerevisiae can be used as the dominant host strain for isobutanol production due to its good tolerance to isobutanol, the advantage of utilizing cellulose hydrolyzate and its own advantage of synthesizing isobutanol gene. [0003] In the prior art, due to overexpression of ILV2, ILV3 and ILV5 genes in isobutanol-producing Saccharomyces cerevisiae cells, the coenzymes NADPH / NADP from pyruvate to 2-ketoisovalerate + The imbalance restricts the further improvement of isobutanol production, these defects are in Image 6 dis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12R1/865
Inventor 张爱利李杨高玉菡
Owner HEBEI UNIV OF TECH
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