Rapid Evolution of Ethanol Tolerance in Yeast by Selection in a Turbidostat

Increasing ethanol tolerance of the yeast Saccharomyces cerevisiae is of key economic importance for the use of ethanol as a liquid fuel or fuel supplement. To this end, we have evolved a population of S. cerevisiae W303-1A strain in a turbidostat under the selection pressure of ethanol, and have succeeded in increasing the proliferation rate of this strain in the presence of 8% ethanol from 0.04h-1 to 0.32h-1. To uncover the genetic basis for this adaptation, we have sequenced the genomes of representative clones, which were isolated from the evolved population. Our premise was that the laboratory evolution procedure would select for clones carrying several genetic alterations that are together responsible for the significant phenotype change. Unexpectedly, each clone carried a single mutation that was responsible for the tolerant phenotype. We estimated that about 79% of the tolerant clones contained one of the three mutations replacing the stop-codon in ssd1-d for an amino acid-encoding codon. About 16% contained one of two mutations in UTH1, and about 5% did not contain mutations in either SSD1 or UTH1. Interestingly, none of the investigated clones harbored mutations in the Ras/cAMP/MSN2/4 pathway, known to be responsible for the tolerance to various stresses and shocks. We further show that the mutations in SSD1 and UTH1 increased tolerance of the cell wall to digesting enzymes and discuss the possibility that the stability of the cell wall was a major factor in increased tolerance to ethanol.