Role of the CYR1G2066A mutation in ethanol tolerance of sake yeast Kyokai no. 11.

Sake yeast Kyokai no. 11 (K11) is an ethanol-tolerant mutant of Kyokai no. 7 (K7) and produces a higher ethanol concentration in the sake mash than K7. A previous study revealed that stress-induced genes under the control of STRE elements were upregulated in K11. To elucidate the causal mutation responsible for ethanol tolerance, we compared the genome sequences of the ethanol-tolerant mutants (K11 and K7AT2, a newly isolated ethanol-tolerant mutant of K7) with that of their parental strain, K7. We identified a shared loss of heterozygosity region in the left arm of chromosome X in both mutants. We focused on CYR1 in this region, as it encodes adenylate cyclase, which negatively regulates expression of STRE-regulated genes through the upregulation of protein kinase A. Nucleotide 2066 of CYR1 was changed from G/A (amino acids Arg/His) in K7 to A/A (amino acids His/His) in K11 and K7AT2. When the plasmid containing CYR12066G was introduced into K11 or K7AT2, the stress tolerance of the transformants decreased to the level of K7, whereas the introduction of CYR12066A had a minimal effect. Consistently, disruption of the CYR12066G allele in K7 increased stress tolerance, whereas disruption of CYR12066A decreased stress tolerance. Furthermore, when CYR1 in a laboratory haploid strain was disrupted and either the CYR12042A or CYR12042G allele of S288C (corresponding to K7CYR12066) was introduced into the disruptant, the transformants with CYR12042A showed higher stress tolerance than those with CYR12042G. We concluded that the CYR1G2066A mutation was responsible for ethanol tolerance in K11.