69NISR Research GRANT
2005 Young Investigator Research Grant

Molecular Evolutionary Analysis of High Sugar Tolerance ofWine Yeasts and Its Application for Breeding Industrial Yeasts
Youji NAKAGAWA
University of Yamanashi, Interdisciplinary Graduate School of Medicine and Engineering

Research aims
Technologies for enhancing the high sugar tolerance ofSaccharomyces cerevisiae have not been established in spiteof the importance of such tolerance in the fermentation industry. We have found that wine yeast strains, which havebeen selected and have evolved over many years to adapt tofermenting grape musts containing a high sugar concentration, are tolerant to high sugar stress, that the overexpression of Sed1p, a cell wall mannoprotein, confers high sugar
tolerance to S. cerevisiae, and that the length of the SED1
coding regions differs among strains working under different industrial conditions. The research aims of this study
are to elucidate the mechanism of the high sugar tolerance
mediated by SED1, and to develop a technology for enhancing the high sugar tolerance of industrial yeasts using
the high-sugar-tolerance mechanisms of wine yeasts.
Methods
The high sugar tolerance of S. cerevisiae cells was assayed by spotting cells on synthetic dextrose (SD) agar
plates containing 50% (w/v) glucose, unless otherwise indicated. Intracellular glycerol concentration was determinedby enzymatic assay (Glycerol UV-method, Boehringer
Mannheim). The assay for Zymolyase resistance of S. cerevisiae cells was performed by periodically measuring thedecrease in the optical density at 660 nm (OD660) of cellsuspensions containing 40 m g/ml of Zymolyase 20T (Seikagakukogyou).
Results
A high sugar concentration causes high osmotic stress byaltering water activity. The HOG pathway is the mostprominent signalling pathway for responding to high osmotic stress in S. cerevisiae. To investigate whether highsugar tolerance induced by SED1 overexpression requires
other factors regulated by the HOG pathway, we constructed a D hog1 strain in which HOG1 encoding the mitogen-activated protein kinase of the HOG pathway isdeleted, and analyzed the effect of SED1 overexpression inthe D hog1 strain. The result showed that SED1 overexpression in the D hog1 strain did not improve the high sugar tolerance, indicating that Hog1p is required for the high sugar
tolerance induced by SED1 overexpression, suggesting that
other factors regulated by the HOG pathway are essential
for such tolerance.

S. cerevisiae cells induce the intracellular accumulationof glycerol as a compatible solute in response to high osmotic stress. To investigate whether the high sugar tolerance induced by SED1 overexpression is due to the change
in intracellular glycerol concentration, we measured the intracellular glycerol concentration of the cells overexpressing SED1 and control cells. There was no significant difference in intracellular glycerol concentration between thecells overexpressing SED1 and the control cells. This indicates that the high sugar tolerance induced by SED1 overexpression is not due to a change in intracellular glycerolconcentration.
To elucidate the role of SED1 in the high sugar tolerance,
we constructed the D sed1 strain and analyzed the effect ofSED1 disruption on such tolerance. There was little difference in the growth on the SD medium containing 50%(w/v) glucose between the wild-type and D sed1 cells fromthe exponential-phase culture. In contrast, the growth of thewild-type cells from the stationary-phase culture was significantly better than that of the D sed1 cells from the stationary-phase culture on the SD medium containing 50%(w/v) glucose. These results indicate that Sed1p is required
for the high sugar tolerance of stationary-phase S. cerevisiae cells.
Sed1p has recently been shown to function in ribosomes
and mitochondria, as well as in the cell wall. To examine
the association between the high sugar tolerance induced bySED1 overexpression and the cell wall structure, the wildtype, SED1-overexpressing, and D sed1 cells were treatedwith Zymolyase, a mixture of cell wall-digesting enzymes.
The resistance to Zymolyase was enhanced by SED1 overexpression and reduced by SED1 disruption, corresponding
to the high sugar tolerance. Furthermore, the observation of
the cells by scanning electron microscopy revealed that theSED1-overexpressing cells have a rough surface in contrast
to the control cells with a smooth surface. These results
suggest that a change in cell wall structure is involved in
the high sugar tolerance induced by Sed1p.
To clarify whether the high sugar tolerance induced by
Sed1p depends on its function in the cell wall, we constructed a disruptant of KRE6, which is required for the incorporation of Sed1p into the cell wall, and analyzed thehigh sugar tolerance of D kre6 cells overexpressing SED1.
The result showed that D kre6 cells overexpressing SED1
exhibit no high sugar tolerance, indicating that Sed1p incorporated into the cell wall is required for such tolerance.
SED1 is characterized by many length and sequencepolymorphisms within both the species S. cerevisiae and70

the genus Saccharomyces. However, no evidence indicatingthat these polymorphisms have physiological significancewas obtained. We hypothesized that SED1 polymorphisms
are involved in the high sugar tolerance mentioned above.
Therefore, we sequenced the SED1 coding regions of thewine yeast strain OC-2 and the laboratory yeast strain
W303 and compared the primary structure of Sed1p between both strains. The result showed that the Sed1p of
W303 had two 51-amino-acid repeat units containing threecysteine residues. On the other hand, there were three repeat units in the Sed1p of OC-2. This difference in thenumber of cysteine residues in Sed1p might affect the highsugar tolerance through disulfide bond formation.

Conclusion
The high sugar tolerance induced by SED1 overexpression is dependent on Hog1p, but is not due to a change inintracellular glycerol concentration. Sed1p is required forthe high sugar tolerance of stationary-phase S. cerevisiae
cells. SED1 overexpression modifies the cell wall structure.
Incorporation of Sed1p into the cell wall is required for thehigh sugar tolerance induced by SED1 overexpression.
There are two and three 51-amino-acid repeat units containing three cysteine residues in the Sed1p proteins of the laboratory yeast strain W303 and the wine yeast strain OC-2,
respectively.