Comparative metabolome analysis of sake yeast with enhanced fermentation performance in sake fermentation conditions

Japanese sake is fermented with specific strains of budding yeast Saccharomyces cerevisiae. Sake yeasts can allow the ethanol concentration of sake to exceed 20 % without distillation. While the genetic mutations responsible for these exceptional properties have been investigated, the underlying metabolism has not been fully explored. It is because yeast cells cultured in sake mash are difficult to collect for metabolome analysis. This study aimed to clarify the metabolic differences of K701 sake yeast and the X2180 diploid laboratory strain when cultured under sake fermentation conditions. To find an alternative medium that mimics sake fermentation and is applicable to measurements of intracellular metabolome, we compared three liquid media, including SD2 medium (synthetic dextrose medium containing 2 % glucose), SD20 medium (synthetic dextrose medium containing 20 % glucose and 1.8 % lactic acid) and pseudo-sake medium (a supernatant of saccharified rice supplemented with 1.8 % lactic acid). Culture profile data demonstrated that the pseudo-sake medium successfully reproduced the metabolic traits of K701 observed in sake mash. Targeted metabolome analysis of yeast cells cultured in the pseudo-sake medium revealed that levels of glycolytic metabolites, such as glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and fructose-1,6-bisphosphate (FBP), were significantly higher with K701. Based on metabolite concentration data, we inferred that K701 cells had a higher ATP regeneration rate. Calculation of differential Gibbs free energy changes revealed that the glucokinase reaction was upregulated in K701. The present study has, for the first time, revealed the metabolism of K701 sake yeast responsible for its exceptional fermentation ability under sake fermentation conditions.