Strain construction method for improving robustness of saccharomyces cerevisiae
A technology of Saccharomyces cerevisiae and a construction method, applied in the field of bioengineering, can solve problems such as the difficulty in regulating the robustness of Saccharomyces cerevisiae in a rational system, achieve low energy and metabolic losses, achieve robustness, reduce cell metabolism and inhibit growth Effect
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Embodiment 1
[0016] The industrial Saccharomyces cerevisiae strains were cultured in liquid media at 28°C, glucose concentration 20g / L, pH=5.0 (no stress condition) and 45°C, glucose concentration 100g / L, pH=5.0 (stress condition). The cultured cells were subjected to transcriptomic sequencing, and two sets of transcriptome data were obtained. The two sets of data were compared, and the gene HSP104 whose transcription level was higher under stress than under no stress was selected. It was found in the reference genome GCF_000146045.2_R64 The sequence 500 bases upstream of the start codon ATG of the corresponding gene was used as the stress-driven promoter HSP104p.
[0017] HSP104p was cloned from the genome, connected with the red fluorescent protein gene to form an expression cassette, and integrated into the chromosome of industrial Saccharomyces cerevisiae. The strains were cultured under the above-mentioned non-stress conditions and stress conditions respectively, and then the intensit...
Embodiment 2
[0020] The laboratory Saccharomyces cerevisiae strains were respectively incubated at 30°C, glucose concentration 30g / L, pH = 6.0 (no stress condition) and 33°C, glucose concentration 40g / L, pH = 3.0, acetic acid concentration 15g / L (stress condition) liquid cultured in culture medium. The cultured cells were subjected to transcriptomic sequencing, and two sets of transcriptome data were obtained. The two sets of data were compared, and the genes TPO2 and TPO3 whose transcription level was higher under stress than under no stress were selected. In the reference genome GCF_000146045.2_R64 The sequence of 700 bases upstream of the start codon ATG of the corresponding gene was found in the corresponding gene as the stress-driven promoter TPO2p, TPO3p.
[0021] TPO2p and TPO3p were cloned from the genome, respectively connected with the yellow fluorescent protein gene and the green fluorescent protein gene to form expression cassettes, and integrated into the chromosome of industr...
Embodiment 3
[0024] The industrial Saccharomyces cerevisiae strains were cultured in liquid medium at 32°C, glucose concentration 40g / L, pH=4.0 (no stress condition) and 37°C, glucose concentration 300g / L, pH=8.0 (stress condition). The cultured cells were subjected to transcriptomic sequencing, and two sets of transcriptome data were obtained. The two sets of data were compared, and the genes SOD1, SOD2, and GRX2 whose transcription level was higher under stress than under no stress were selected. In the reference genome GCF_000146045 .2_R64 found the sequence of 1000 bases upstream of the start codon ATG of the corresponding gene as the stress-driven promoters SOD1p, SOD2p, and GRX2p.
[0025] SOD1p, SOD2p, and GRX2p were cloned from the genome, respectively connected with the cyan fluorescent protein gene to form an expression cassette, and integrated into the chromosome of industrial Saccharomyces cerevisiae. The strains were cultured under the above-mentioned non-stress conditions and...
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