Tetsuyoshi Suizu

Methods for sporulation of industrially used sake yeasts

Industrially used sake yeasts (Saccharomyces cerevisiae) such as Kyokai no. 7 and no. 9 hardly sporulate under any of the nutritional conditions examined to date. However, through acquisition of either sensitivity to ethionine or the ability to grow at 35°C in the presence of β-alanine, these sake yeasts were found to regain the ability to form asci with 4 viable spores at high frequency. These methods for the mutant-selection may facilitate the genetics of industrially used yeasts and their breeding by hybridization.

Cysteine Protease Inhibitors Produced by the Industrial Koji Mold, Aspergillus oryzae O-1018

Aspergillus oryzae O-1018 (FERM P-15834) separated from industrial koji for brewing sake was found to produce five papain-inhibitory compounds in the culture supernatant. The five isolated inhibitors were named CPI-1 to CPI-5, and their structures were elucidated by spectroscopic analyses and chemical degradation. We determined the structures of CPI-2, CPI-3 and CPI-4 as 4-amino-1-[[N- [(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleucyl] amino]butane, 5-amino-1-[[N-[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleucyl]amino]pentane and N (8)- [N-[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleu-cyl]spermidine, respectively. We also confirmed by a degradation experiment that CPI-1 consisted of L-trans-epoxysuccinic acid, L-tyrosine and spermidine, and that CPI-5 was composed of L-trans-epoxysuccinic acid, L-phenylalanine and spermidine. Although CPI-4 was identified as kojistatin A,(1)) the other CPIs seemed to be novel compounds. All CPIs were cysteine protease-specific inhibitors with appreciable selectivity toward cathepsin B and L. The inhibition potency of CPIs against cysteine proteases was as high as or higher than that of E-64. In particular, CPI-2, -3 and -4 were ten times more effective than E-64 toward cathepsin B and L, and CPI-1 and -5 were about 100 times more inhibitory than E-64 toward cathepsin L.

Highly Efficient Sporulation Induced by Glutathione or Glutathione Thiol Esters in Sake (Kyokai No. 7) and a Wild-Type Yeast

Abstract When vegetative cells of Saccharomyces cerevisiae 4011 and a sake yeast (S. cerevisiae), Kyokai no. 7, were incubated in a sporulation medium, glutathione in the cells decreased with the increase in sporulation and was lost by the time the sporulation reached a maximum. The sporulation efficiency varied depending on the cellular glutathione content, and was lowered with a decrease of the level. Glutathione and its thiol esters significantly stimulated the sporulation of both strains when these chemicals were added to sporulation medium. The activities of glycolytic bypass enzymes, which are responsible for the synthesis of glutathione thiol ester, increased in both yeast strains during incubation in the sporulation medium. These results suggest that glutathione was converted into glutathione thiol ester and that the ester functioned as a stimulator of yeast sporulation.

On the importance of calcium and magnesium ions in yeast sporulation

Abstract Irrespective of the nutritional conditions, the sporulation frequency of wild and industrially used yeasts on agar or agarose plates has been found to vary from one experiment to another. An analysis of agar- and agarose-extracts by ion-exchange column chromatography proved that the amount of calcium and/or magnesium ions contained in the agar was a factor in the fluctuation of sporulation frequency. Furthermore, these two cations enhanced the formation of four-spored asci. When calcium or magnesium ions were added to a nutrition-deprived medium solidified with agarose containing no detectable calcium and magnesium ions, wild and industrially used sake yeasts efficiently sporulated with a frequency of 10–40%. A strictly controlled sporulation condition suitable for the analysis of meiosis and sporulation of yeast cells was constructed by using calcium and/or magnesium ions and highly purified agarose.

Induction of yeast sporulation by lysine-related compounds and glutathione in nutrition-rich conditions

Abstract In addition to dl -lysine (Kawado et al. , J. Ferment. Bioeng., 76, 391–394, 1993), dl -2-aminoadipate was found to induce sporulation in a wild-type yeast of Saccharomyces cerevisiae 4011 growing in a nutrition-rich medium without potassium acetate. In the presence of the acid at 10 mM, the sporulation frequency of the yeast cells increased in parallel with the growth and reached maximum (26%) in the stationary phase. Despite the expression of a meiosis inducer gene, IME1 , no sporulation was induced unless dl -lysine or dl -2-aminoadipate was included in the nutrition-rich medium. By using dl -2-aminoadipate in combination with glutathione, sporulation was also induced in industrially used sake yeasts growing in the nutrition-rich medium without potassium acetate.

Introduction of a feed back resistant α-isopropylmalate synthase gene of Saccharomyces cerevisiae into sake yeast

Abstract A mutant LEU4 gene (LEU4fbr-2), responsible for both the overproduction of iso-amyl alcohol in yeast and the phenotype of yeast resistant to 5,5,5-trifluoro- dl -leucine (TFL), was isolated from a TFL-resistant mutant of Saccharomyces cerevisiae F-7. The single copy number of LEU4fbr-2 complemented the leucine auxotrophy of S. cerevisiae HB190 (a, leu4, leu5), and also transformed it to TFL-resistant. Leucine-insensitive α-isopropylmalate synthase activity was detected in the crude extract of the Leu+ transformant. Also sake yeast Kyokai no. 7 (K-7) was transformed by the LEU4fbr-2 gene to TFL-resistant. The resulting transformants produced 3∼30-fold higher levels of iso-amyl alcohol (approx. 50∼475 ppm) in shaking cultures, while in static cultures the increase in productivity was only 2.5-fold compared with that of recipient strain K-7. The isolated LEU4fbr-2 gene may be useful as a positive selectable marker for the transformation of industrial yeast.

Yeast Sporulation Induced by Lysine during Growth in Nutrition-Rich Conditions

Abstract Sporulation of the yeast Saccharomyces cerevisiae 4011 and other strains of S. cerevisiae was induced by d - or l -lysine during growth in a nutrient-rich medium. Potassium acetate was not required for the sporulation. The effects of other d - or l -amino acids on sporulation, if any, were negligible or very slight. Although the sporulation efficiency varied depending on the yeast strain, lysine concentration, pH of the medium and incubation period, the results obtained indicated that nutritional starvation is not a prerequisite for the sporulation of yeast cells, and that lysine or a certain cellular molecule involved in the metabolism of the amino acid has the ability to cause meiosis and sporulation in yeast cells.

Analysis of lysine-dependent yeast sporulation: a decrease in cyclic AMP is not required for initiation of meiosis and sporulation in Saccharomyces cerevisiae

Cells of the yeast Saccharomyces cerevisiae sporulated in nutrient-rich medium containing L-lysine. Sporulation was specific to the presence of L-lysine and was initiated when the cellular content of this basic amino acid reached approximately 0.2-0.5 mmol (g cells)-1, at early stationary phase. The formation of asci was most efficient at pH 7.0 and 50-100 mM L-lysine; in these optimum conditions, the sporulation frequency reached about 60% after 5 d incubation. The L-lysine-dependent sporulation system in nutrient-rich conditions was distinct from the currently used potassium-acetate-dependent system in nutrient-deficient conditions. Analysis of the L-lysine-dependent system indicated that, prior to entrance into meiosis and/or sporulation processes, the yeast cells change in shape, their pool sizes for L-cysteine and glutathione alter, and they synthesize a protein with a molecular mass of 15 kDa. A low level of cAMP was not required for the entrance into meiosis and/or sporulation.