酿酒酵母SNF4基因敲除缺失菌株的构建

Construction of Saccharomyces cerevisiae mutant with knockout of SNF4 gene

摘要:

构建一株酿酒酵母SNF4基因缺失菌株并研究其对乙醇产量的影响。扩增带有SNF4基因上下游同源序列和Kanr筛选标记的SNF4基因敲除组件，转化到酿酒酵母YS2获得阳性克隆子，然后将质粒pSH65转到阳性克隆子中，半乳糖诱导pSH65表达Cre酶切除Kanr筛选标记，获得SNF4等位基因完全缺失菌株YS2-△SNF4。发酵实验结果表明，缺失菌株YS2-△SNF4乙醇产量较出发菌株提高了7.57％。利用Cre-LoxP系统，成功构建了SNF4等位基因完全缺失菌株并提高乙醇产生量。

Abstract:

Construction and ethanol production effects of SNF4 gene knockout in Saccharomyces cerevisiae were described in this paper. For knockout of SNF4 gene in S. cerevisiae YS2, a PCR-amplified disruption cassette was used, encoding the short flanking homologous regions to the SNF4 gene and Kanr as selectable marker. The SNF4 gene disruption cassette was transformed into S. cerevisiae YS2 through LiAc/SS Carrier DNA/PEG. The positive transformants were grown on G418 plates and verified by PCR. The Kanr marker was rescued by transforming plasmid pSH65 into positive transformants and inducing expression of Cre recombinase in galactose-containing medium. Lastly, the YS2-△SNF4 strain, in which SNF4 allele gene were completely knocked out, was obtained by repeating the same procedure. The result of anaerobic fermentation showed that ethanol production of the SNF4 gene knockout strain had increased by 7.57 percent as compared with the original strain YS2. The experiment indicated ethanol production could be improved significantly with the approach of SNF4 gene knockout by Cre-LoxP system.