Takahashi T, Shimoi H, Ito K
General Research Laboratories, Kiku-Masamune Sake Brewing Co. Ltd, Kobe, Japan.
Mol Genet Genomics. 2001 Aug;265(6):1112-9. doi: 10.1007/s004380100510.
The yeast Saccharomyces cerevisiae exhibits high ethanol tolerance compared with other microorganisms. The mechanism of ethanol tolerance in yeast is thought to be regulated by many genes. To identify some of these genes, we screened for ethanol-sensitive S. cerevisiae strains among a collection of mutants obtained using transposon mutagenesis. Five ethanol-sensitive (ets) mutants were isolated from approximately 7,000 mutants created by transforming yeast cells with a transposon (mTn-lacZ/LEU2)-mutagenized genomic library. Although these mutants grew normally in a rich medium, they could not grow in the same medium containing 6% ethanol. Sequence analysis of the ets mutants revealed that the transposon was inserted in the coding regions of BEM2, PAT1, ROM2, VPS34 and ADA2. We constructed deletion mutants for these genes by a PCR-directed disruption method and confirmed that the disruptants, like the ets mutants, were ethanol sensitive. Thus, these five genes are indeed required for growth under ethanol stress. These mutants were also more sensitive than normal cells to Calcofluor white, a drug that affects cell wall architecture, and Zymolyase, a yeast lytic enzyme containing mainly beta-1,3- glucanase, indicating that the integrity of the cell wall plays an important role in ethanol tolerance in S. cerevisiae.
与其他微生物相比，酿酒酵母表现出较高的乙醇耐受性。酵母中乙醇耐受性的机制被认为受许多基因调控。为了鉴定其中一些基因，我们在使用转座子诱变获得的一系列突变体中筛选对乙醇敏感的酿酒酵母菌株。从大约7000个通过用转座子（mTn-lacZ/LEU2）诱变的基因组文库转化酵母细胞而产生的突变体中分离出五个对乙醇敏感（ets）的突变体。尽管这些突变体在丰富培养基中正常生长，但它们在含有6%乙醇的相同培养基中无法生长。对ets突变体的序列分析表明，转座子插入到了BEM2、PAT1、ROM2、VPS34和ADA2的编码区。我们通过PCR定向破坏方法构建了这些基因的缺失突变体，并证实这些破坏体与ets突变体一样对乙醇敏感。因此，这五个基因确实是乙醇胁迫下生长所必需的。这些突变体对影响细胞壁结构的药物荧光增白剂和主要含有β-1,3-葡聚糖酶的酵母裂解酶溶菌酶也比正常细胞更敏感，这表明细胞壁的完整性在酿酒酵母的乙醇耐受性中起重要作用。
