Identification of genes required for growth under ethanol stress using transposon mutagenesis in Saccharomyces cerevisiae

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 7000 mutants created by transfor ming yeast cells with a transposon (mTn-lacZ/LEU2)-mutagenized genomic libr ary. 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 thes e genes by a PCR-directed disruption method and confirmed that the disrupta nts, like the ets mutants, were ethanol sensitive. Thus, these five genes a re 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 main ly beta -1,3- glucanase, indicating that the integrity of the cell wall pla ys an important role in ethanol tolerance in S. cerevisiae.