黄酒酵母一个新基因的鉴定及其对酵母抗逆性能的影响  

The Identification of a Novel Gene and Its Effect on Stress Tolerance in Saccharomyces cerevisiae Chinese Rice Wine Strain

作　　者：王明月 余雯雯 张璐 梁新乐[1] 李余动[1] Wang Mingyue;Yu Wenwen;Zhang Lu;Liang Xinle;Li Yudong(School of Food Science and Biotechnology,Zhejiang Gongshang University,Hangzhou 310018)

机构地区：[1]浙江工商大学食品与生物工程学院,杭州310018

出　　处：《中国细胞生物学学报》2018年第8期1334-1342,共9页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:31671836)资助的课题~~

摘　　要：该文鉴定了一个来自黄酒酵母菌株D2的新基因g5170,并研究了其对黄酒酵母环境胁迫耐受性与发酵性能的影响。生物信息学分析发现,此基因编码的蛋白质含有酰胺酶(amidase)保守功能域,并在YPD培养条件下有较高的基因表达量。利用Cre/lox P系统构建了基因g5170的敲除菌株,通过扩增带有g5170基因上下游同源序列和kanr筛选标记的基因敲除组件,并通过醋酸锂法转化到黄酒酵母菌株D2获得阳性克隆子,然后将质粒p SH65转到阳性克隆子中,半乳糖诱导p SH65表达Cre酶切除kanr筛选标记。重复此实验过程,最终获得两个g5170基因拷贝完全缺失菌株D2?g5170。梯度生长实验显示,与原始菌株相比,敲除菌株对高糖浓度、高温耐受性无明显变化,但对乙醇胁迫的耐受性显著降低,而过表达g5170的重组酵母菌株对乙醇的耐受性明显比对照菌株强。模拟黄酒发酵实验结果表明,敲除菌株D2?g5170的生长速率、乙醇产量及理化指标与原始菌株相似。因此,g5170基因可能与黄酒酵母适应乙醇胁迫有关,并有助于提高酵母的黄酒发酵性能。They identified a putative gene g5170 from Saccharomyces cerevisiae（S. cerevisiae）, a Chinese rice wine strain D2, and characterized its effects on stress tolerance and fermentation performance. A protein encoded by g5170 contains an amidase domain, and was over-expressed in growth culture in YPD medium. A knockout strain D2？g5170 is constructed by using Cre/lox P system. A disruption cassette of g5170 with a short flanking homologous regions to g5170 and kanr as selectable marker was amplified and transformed into S. cerevisiae through Li Ac/SS carrier DNA/PEG method. Positive transformants were screened on G418 plates and verified by PCR. The kanr marker was removed by transforming plasmid p SH65 into the positive transformants and inducing expression of Cre recombinase by galactose. The procedure was repeated to obtain the knockout strain D2？g5170, in which two g5170 copies were completely deleted. Compared to a wild type strain, mutant D2？g5170 showed no significant alterations of tolerance to heat stress and osmotic stress, but exhibited significant lower tolerance to ethanol stress. In contrast, over-expression of g5170 in S. cerevisiae resulted in enhanced toleranceto ethanol stress. Simulation of rice wine fermentation showed that the growth rate, ethanol production and other parameters of the mutant were similar to that of the wild strain. These results suggested that g5170 was associated with ethanol stress response, which might impact fermentation performance of S. cerevisiae in rice wine production.

关 键 词：基因敲除 新基因 乙醇胁迫 酿酒酵母 

分 类 号：TS261.11[轻工技术与工程—发酵工程]