A method for constructing a Saccharomyces cerevisiae strain with high tolerance to isobutanol
A Saccharomyces cerevisiae strain and technology of Saccharomyces cerevisiae are applied in the field of constructing Saccharomyces cerevisiae strains with high tolerance to isobutanol, and can solve problems such as inability to adapt, decline in isobutanol yield and the like
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Embodiment 1
[0049] Using molecular biology and gene recombination technology to transform Saccharomyces cerevisiae cells, transform the plasmid YCplac22-spt15-30 overexpressing the Saccharomyces cerevisiae mutant gene spt15-30 into the host bacteria into Saccharomyces cerevisiae wild-type strain W303-1A into MATaleu2-3,112ura3-1 trp1-92 his3-11, 15ade2-1 can1-100 (Thomas and Rothstein, 1989), the specific steps are as follows:
[0050] The first step is to construct a plasmid for overexpressing the Saccharomyces cerevisiae mutant gene spt15-30:
[0051] (1.1) Synthesis of mutant gene spt15-30 fragment:
[0052] Design the mutation site according to the SPT15 gene sequence on the Saccharomyces cerevisiae W303-1A chromosome, and entrust a gene synthesis company to synthesize the fragment of the mutant gene spt15-30. The DNA sequence of the mutant gene spt15-30 is as follows image 3 shown;
[0053] (1.2) Construction of the plasmid YCplac22-spt15-30 overexpressing the Saccharomyces cerevi...
Embodiment 2
[0070] Divide with T 4 Ligase to connect the plasmid YCplac22 and the mutant gene spt15-30 fragment, the connection operation is as follows: in a 1.5mL centrifuge tube, add 10×T 4 Ligase buffer 2 μL, T 4 Add 0.2 μL of ligase, plasmid YCplac22 and the mutant gene spt15-30 fragment obtained in the above step (1.1) to 0.6 μL respectively, then fill the total volume to 20 μL with sterile water, and ligate at 16°C for 1.5 h. The ligation product was obtained, and 10 μL of the ligation product was transferred into Escherichia coli competent cells for cultivation, and then the plasmid was extracted, verified by restriction enzyme digestion, and the plasmid YCplac22-spt15-30 overexpressing the Saccharomyces cerevisiae mutant gene spt15-30 was constructed; before use The obtained Saccharomyces cerevisiae strain HBGDAL-104 with high tolerance to isobutanol was connected to the CM-Tryptophan plate for 3 times of activation treatment, cultivated in CM-Tryptophan liquid medium containing ...
Embodiment 3
[0072] Divide with T 4 Ligase to connect the plasmid YCplac22 and the mutant gene spt15-30 fragment, the connection operation is as follows: in a 1.5mL centrifuge tube, add 10×T 4 Ligase buffer 2 μL, T 4 Add 0.2 μL of ligase, plasmid YCplac22, and 8 μL of the mutant gene spt15-30 fragment obtained in the above step (1.1), and then make up the total volume to 20 μL with sterile water, and connect at 16°C for 2 hours to obtain the connection For the product, take 10 μL of the ligated product and transfer it into Escherichia coli competent cells for culture, then extract the plasmid, and verify it by restriction enzyme digestion to construct a plasmid YCplac22-spt15-30 that overexpresses the Saccharomyces cerevisiae mutant gene spt15-30; The obtained Saccharomyces cerevisiae strain HBGDAL-104 with high tolerance to isobutanol was connected to the CM-Tryptophan plate and carried out 3 times of activation treatment, and was cultivated in the CM-Tryptophan liquid medium containing ...
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