STUDIES ON THE SPORULATION OF SAKE YEAST - MONOGRAPH

Defective sporulation of industrially used sake yeasts (Saccharomyces cerevisiae) such as Kyokai no. 7 and no. 9 is one of the most troubles ome problems inhibiting the breeding-by hybridization and/or genetic a nalysis-of yeast strains with improved characteristics for sake making . In order to clarify the cause of defective sporulation in industrial ly used sake yeasts, and thereby facilitate their molecular breeding, the sporulation-defective properties of Kyokai no. 7 were analyzed in nutfition-controlled media and compared with a wild-type diploid yeast (strain 4011) as a control. An analysis of the sporulation process of strain 4011 in a nutrient-starved medium indicated that Ca2+ glutathi one (GSH) and some other biomolecules were able to enhance sporulation . Of these, Ca2+ and GSH were prerequisites for sporulation. The basic amino acid D-, L-lysine was found to induce sporulation in cells grow ing in a nutrient-rich medium. An analysis of the sporulation of strai n 4011 and Kyokai no. 7 in a lysine-dependent system showed no signifi cant increase in IME1 transcript and a decrease in cAMP levels through out the sporulation, thus suggesting that at least a decreased level o f cAMP is not required for entry into meiosis. Although the formation of 4-spored asci and spore viability were low, the addition of GSH, it s analogues, and other biomolecules to the nutrition-starved medium ap parently improved the sporulation of Kyokai no. 7. This result indicat ed that the defective sporulation of Kyokai no. 7 was mainly due to me tabolic imbalance, and not to any serious genetic disorder upstream of the signal transduction pathway. The effects on sporulation of metabo lic properties such as ethionine-resistance, inability to utilize beta -alanine at 35 degrees C, and loss of acid phosphatase activity, all o f which are inherent characteristics of Kyokai no. 7, were thus invest igated. The elimination of any one of these properties was found to re store the sporulation of Kyokai no. 7. For example, a mutation with et hionine-sensitivity showed a marked increase in the formation of 4-spo red asci (80%) and in the viability of the spores (over 25%). These fi ndings were applicable to other Kyokai strains apart from no. 7, and w ill be utilized as a general method for the molecular breeding and gen etic analysis of industrially important brewery yeasts.