CRISPR/Cas9介导的酿酒酵母ADH2基因中断及反义RNA干扰GPD1的表达

CRISPR/Cas9 Mediated ADH2 Gene Disruptionin Saccharomyces Cerevisiae and Antisense RNA Interference in GPD1 Expression

CRISPR/Cas9是一个简单、高效的用于靶向目的基因和无标记的基因组工程的工具。本文通过构建酿酒酵母沉默组件PGK-SGPD1-CYC1,使甘油-3-磷酸脱氢酶I(Glycerol-3-phosphate dehydrogenase,GPD1)基因在PGK强启动子、CYC1终止子在特定区域内进行干扰和表达。应用CRISPR/Cas9基因编辑技术,在中断乙醇脱氢酶Ⅱ(alcohol dehydrogenase Ⅱ,ADH2)基因的同时,定点敲入GPD1基因的反义干扰组件,从而特定地干扰GPD1的表达。采用高效的酵母化学转化法将反应组件敲入酿酒酵母Y1H中,CRISPR/Cas9介导的同源重组效率达43.48%,由此获得了ADH2基因中断和GPD1反义干扰的酿酒酵母突变株。发酵实验结果表明,酿酒酵母突变菌株SG1-1与出发菌株Y1H相比,乙醇产率提高了9.07%,甘油产率下降了12.05%,乙酸产率下降了12.30%,结果表明通过中断ADH2基因及插入GPD1反义干扰组件,既能够中断ADH2基因的功能,减少乙醇转化为乙醛,同时也能在一定程度上干扰GPD1基因的表达,提高乙醇产率。

CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we constructed the silencing component PGK-SGPD1-CYC1 of Saccharomyces cerevisiae to interfere the glycerol 3-phosphate dehydrogenase1 （GPD1） geneand express in the specific region of PGK promoter and CYC1 terminator. Using CRISPR/Cas9 technology, while interrupting the alcohol dehydrogenase II （ADH2） gene, the target site were knocked into the antisense interference component of the GPD1 gene, thus interfering with the expression of GPD1. Through high-efficiency yeast transformation, the components were transformed into Y1H, CRISPR/Cas9 mediated recombination efficiencies of 43.40% were achieved, thus mutant strains with ADH2 gene interruption and GPD1 antisense interference were obtained. Fermentation test shows that the ethanol yield of the mutant strain SG1-1 was 9.07% higher than the wild type, and the yield of glycerol and acetate were decreased by 12.05% and 12.30%, respectively. Results showed that the antisense interference of the GPD1 and interruption of the ADH2 can not only interrupt the function of ADH2 gene, and reduce the conversion of ethanol into acetaldehyde, but also effectively interfere with GPD1 expression in engineered yeast strains, thus improving the yield of ethanol.