Mechanism of high trehalose accumulation in a spore clone isolated from Shirakami kodama yeast

Abstract

The intracellular trehalose levels in Shirakami kodama yeast, a strain of Saccharomyces cerevisiae, isolated in 1997 from leaf mold in the Shirakami Mountains and since used as a commercial baker s yeast, are remarkably high, which presumably is related to its tolerance of freezing and drought conditions. We isolated a spore clone from Shirakami kodama yeast with about 1.7-fold higher intracellular trehalose levels than the parental strain and set out to elucidate how this spore clone can accumulate intracellular trehalose to such a high concentration. The gene for trehalose 6-phosphate synthase, TPS1, was duplicated in this spore clone. Both TPS1 genes contributed to the high level of intracellular trehalose as a 3.4-fold decrease resulted from the disruption of one of the two TPS1 genes. Both Msn2 and Msn4, which bind to stress responsive elements in the promoter region of TPS1, were required for production of high levels of trehalose. Furthermore, the neutral trehalase activity of this spore clone is about 3-fold less than that of the laboratory strain although the gene for neutral trehalase, NTH1, functioned normally. These findings indicate that two TPS1 genes and the low trehalase activity are associated with high trehalose accumulation in this spore clone. The wide range of stresses of which we found the spore clone to be tolerant makes this yeast very attractive for commercial application and for further research into the mechanisms underlying stress responses and trehalose metabolism.Key words: NTH1; TPS1; trehalase; trehalose; Shirakami kodama yeast IntroductionThe disaccharide trehalose is found in organisms as diverse as yeast and other fungi, bacteria, and a variety of plants and invertebrates, in which it accumulates significantly during adverse environmental conditions (Gaff, 1971;Thevelein, 1984;Singer and Lindqust, 1998;Zentella et al., 1999;Chen et al., 2002;Schluepmann et al., 2004). A number of studies have demonstrated a correlation between intracellular trehalose levels and the ability of Saccharomyces cerevisiae to survive various environmental stresses, such as starvation, desiccation, dehydration, osmotic and oxidative stress and extremes in temperature (Thevelein and Hohmann, 1995;Hounsa et al., 1998;Elbein et al., 2003;Herdeiro et al., 2006).In S. cerevisiae, the enzymes that catalyze the reactions of trehalose biosynthesis, a trehalose 6-phosphate (T6P) synthase encoded by TPS1 and a T6P phosphatase encoded by TPS2, are part of a complex in which two other, non-catalytic proteins, Tsl1 and Tps3, participate (Gancedo and Flores, 2004). Degradation of cytoplasmic trehalose is mainly catalyzed by neutral trehalases that work best at pH 6.8 7.0 (Londesborough and Varimo, 1984;App and Holzer, 1989) and are encoded by two duplicated genes, NTH1 and NTH2, which share 77% identity in their predicted amino acid sequences (Kopp et al., 1993(Kopp et al., , 1994Wolf and Lohan, 1994). Major trehalase activity for intracellular trehalose is conferred by NTH1, the expression of which is regulated by...