A sparkling-type draft cloudy sake (Japanese rice wine), AWANAMA, was recently developed using high hydrostatic pressure (HHP) treatment as a non-thermal pasteurization method. This prototype sake has a high potential market value, since it retains the fresh taste and flavor similar to draft sake while avoiding over-fermentation. From an economic point of view, a lower pressure level for HHP pasteurization is still required. In this study, we carried out a genome analysis of a pressure-sensitive (piezosensitive) mutant strain, a924E1, which was generated by UV mutagenesis from a laboratory haploid Saccharomyces cerevisiae strain, KA31a. This mutant strain had a deletion of the COX1 gene region in the mitochondrial DNA and had deficient aerobic respiration and mitochondrial functions. A metabolomic analysis revealed restricted flux in the TCA cycle of the strain. The results enabled us to use aerobic respiration deficiency as an indicator for screening a piezosensitive mutant. Thus, we generated piezosensitive mutants from a Niigata-sake yeast strain, S9arg, which produces high levels of ethyl caproate but does not produce urea and is consequently suitable for brewing a high-quality sake. The resultant piezosensitive mutants showed brewing characteristics similar to the S9arg strain. This study provides a screening method for generating a piezosensitive yeast mutant as well as insight on a new way of applying HHP pasteurization.