| Summary: | Wine fermentation presents a unique environment in which strains of the budding yeast Saccharomyces cerevisiae have evolved with superior tolerance to a multitude of stressors. Ethanol toxicity has one of the greatest impacts in reducing cellular viability and metabolic function and thus poses a threat in causing slow, stuck and incomplete fermentations. In pursuit of optimizing commercial strains of S. cerevisiae, identification of genes involved in ethanol tolerance has been of recent interest. Genomic resources such as the S288C deletion collection and microarray analysis have been widely utilized and have provided a foundation, albeit incomplete, for understanding ethanol toxicity in yeast. As a new approach, the recently developed molecular barcoded yeast open reading frame (MoBY-ORF) library, in which all S. cerevisiae genes along with native promoter and terminator sequences have been cloned into barcoded high copy 2μ plasmid vectors, has been utilized. Both the S288C laboratory strain and the M2 wine strain of S. cerevisiae were transformed with the MoBY ORF library and genes were identified by quantitation of molecular barcodes after 48 hours in 12% ethanol stressed library pools. Five genes were highly ranked in both S288C and M2 screens, two of which, RCN1 and RSA3, improved tolerance to high (16-21% v/v) ethanol toxicity over 1-3 hour incubation periods in both strain backgrounds. RCN1 is a regulator of the stress signalling protein calcineurin whereas RSA3 has a role in ribosome maturation. Additional fitness advantages conferred upon overproduction of RCN1 and RSA3 include increased resistance to cell wall degradation, heat, osmotic and oxidative stress. Neither RCN1 nor RSA3 over-expression in M2 during model fermentations of synthetic wine medium significantly increased the fermentation rate or final ethanol yield. Regulation of calcineurin and ribosomal assembly processes during ethanol stress, however, may still be key targets for improving tolerance to the stressful conditions of wine fermentation.
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