乙醇脱氢酶I基因敲除的酿酒酵母重组菌构建的初步研究

Preliminary Study on Deletion of Saccharomyces cerevisiae Alcohol Dehydrogeniase I Gene

本实验根据酿酒酵母乙醇代谢途径,构建一株低乙醇产量的酿酒酵母基因工程菌株,以满足人们对低醇啤酒的需要。利用抗性基因筛选基因敲除突变体的方法,通过引物L1和L2扩增潮霉素B基因(两翼与酿酒酵母同源),按常规醋酸锂法转化酵母细胞后,筛选标记与酵母adhI基因发生同源重组,得到一株ADHI酶活性降低的工程菌株。发酵实验结果表明,转化菌株乙醇含量平均值为1.8%(V/V),较原始菌株低了65%。说明转化菌株体内乙醇生成途径受到干扰。

The main purpose of this research is to construct a low alcohol producing strain according to the alcohol metabolic pathway of Saccharomyces cerevisiae, so as to satisfy the people who prefer to drink low-alcohol beer. Hygromycin B resistant gene was used to screen mutants with adh Ⅰ gene knocked out. After Hygromycin B resistant gene was amplified with primers L1 and L2 （the flanking fragments were complement with Saccharomyces cerevisiae gene）, it was transformed into yeast HDY- 01 by LiAc method and the alcohol dehydrogenase Ⅰ （ADH Ⅰ） in Saccharomyces cerevisiae was deleted through homologous recombination. A transformant was obtained with low ADH Ⅰ activity. The fermentation tests showed that the average alcohol content of the transformant is 1.8%（V/V）, 65% lower than the origin one. The alcohol metabolic pathway in this transformant is interfered.