Appl Microbiol Biotechnol (2004) 66: 108–114
DOI 10.1007/s00253-004-1624-4
APPLIED MICRO BIAL AND CELL PHYSIOLOGY
Shingo Izawa . Machiko Sato . Kumio Yokoigawa .
Yoshiharu Inoue
Intracellular glycerol influences resistance to freeze stress
in Saccharomyces cerevisiae: analysis of a quadruple mutant
in glycerol dehydrogenase genes and glycerol-enriched cells
Received: 23 February 2004 / Revised: 2 April 2004 / Accepted: 7 April 2004 / Published online: 4 May 2004
# Springer-Verlag 2004
Abstract Glycerol is well known as a cryoprotectant
similar to trehalose. However, there is little information
about the effects of intracellular glycerol on the freeze-
thaw stress tolerance of yeast. Through analysis of a
quadruple-knockout mutant of glycerol dehydrogenase
genes (ara1Δ gcy1Δ gre3Δ ypr1Δ) in Saccharomyces
cerevisiae, we revealed that the decrease in glycerol
dehydrogenase activity led to increased levels of intracel-
lular glycerol. We also found that this mutant showed
higher tolerance to freeze stress than wild type strain
W303-1A. Furthermore, we demonstrated that intracellu-
lar-glycerol-enriched cells cultured in glycerol medium
acquire tolerance to freeze stress and retain high leavening
ability in dough even after frozen storage for 7 days. These
results suggest the possibility of using intracellular-
glycerol-enriched cells to develop better frozen dough.
Introduction
Frozen dough technology has been used in the baking
industry to improve labor conditions for bakers and to
supply oven-fresh bakery products to consumers. One of
the serious drawbacks of this technology is a significant
reduction in leavening activity during freeze storage.
Freeze stress affects the viability and activity of yeast
cells. To develop better baker’s yeasts for frozen dough,
mechanisms of freeze-thaw stress tolerance in Saccharo-
myces cerevisiae have been investigated with great
interest. The growth phase and the prefermentation period
S. Izawa . Y. Inoue (*)
Laboratory of Molecular Microbiology, Graduate School of
Agriculture, Kyoto University,
Uji, Kyoto, 611-0011, Japan
e-mail: inoue@food2.food.kyoto-u.ac.jp
Tel.: +81-774-383773
Fax: +81-774-333004
M. Sato . K. Yokoigawa
Department of Food Science and Nutrition, Nara Women’s
University,
Nara, 630-8506, Japan
before freezing have been reported to affect freeze-thaw
stress tolerance (Lewis et al. 1993; Hino et al. 1990; Park
et al. 1997). It has also been reported that several cellular
factors affect freeze-thaw stress tolerance, including
intracellular trehalose levels (Hino et al. 1990; Shima et
al. 1999; Hirasawa et al. 2001), heat shock protein levels
(Kaul et al. 1992), the lipid composition of the cell
membrane (Murakami et al. 1996), respiratory capacity
(Park et al. 1997), expression of aquaporin (Tanghe et al.
2002), and certain amino acids (Takagi et al. 1997; Shima
et al. 2003).
Glycerol is well known as a cryoprotectant similar to
trehalose (Coutinho et al. 1988), and is widely used in
laboratories as a stabilizer of enzymes. Exogenous glyc-
erol is a less toxic cryoprotectant for yeast cells (Lewis et
al. 1993), thus glycerol is also used as a frozen storage
solution for such cells. In contrast with trehalose, curiously
enough, little information is available on the effects of
intracellular glycerol on the freeze stress tolerance of
yeast. As shown in Fig. 1, in S. cerevisiae, glycerol is
biosynthesized by the reduction of dihydroxyacetone
phosphate (DHAP) to glycerol 3-phosphate (G3P) by
NAD+-dependent glycerol-3-phosphate dehydrogenase
(GPDH) followed by dephosphorylation of G3P to
glycerol by glycerol-3-phosphate phosphatase (G3Pase)
(Albertyn et al. 1994; Ansell et al. 1997; Påhlman et al.
2001). The genes GPD1 and GPD2 encode isozymes of
GPDH (Larsson et al. 1993; Eriksson et al. 1995), and
GPP1 and GPP2 encode G3Pase (Påhlman et al. 2001). It
has been reported that deletion of both GPD1 and GPD2
results in a total shutdown of the glycerol synthesis
pathway and no glycerol is synthesized (Ansell et al. 1997;
Nissen et al. 2000). On the other hand, in addition to
passive diffusion of glycerol, yeast cells can take up
extracellular glycerol via the transporters Gup1 and Gup2
(Sutherland et al. 1997; Holst et al. 2000), and export
intracellular glycerol via the Fps1 channel (Luyten et al.
1995; Sutherland et al. 1997; Tamas et al. 1999; Remize et
al. 2001).
Glycerol is converted to dihydroxyacetone by NADP+-
dependent glycerol dehydrogenase (GDH) in yeast
 109

Materials and methods

Yeast strains and media

The S. cerevisiae strains used in this study are listed in

Fig. 1 Pathways involved in glycerol metabolism in Saccharomy-
ces cerevisiae. Glycerol is formed from dihydroxyacetone phosphate
(DHAP) in a two-step reaction catalyzed by glycerol-3-phosphate
dehydrogenase (GPDH) (Gpd1, Gpd2) and glycerol-3-phosphate
phosphatase (G3Pase) (Gpp1, Gpp2). Additionally, yeast cells can
take in glycerol via transporters Gup1 and Gup2. Glycerol is
converted to dihydroxyacetone (DHA) by NADP+-dependent glyc-
erol dehydrogenase (GDH) (Ara1, Gcy1, Gre3, Ypr1). GAP
Glyceraldehyde 3-phosphate, G3P glycerol 3-phosphate
(Fig. 1). S. cerevisiae contains at least four genes encoding
GDH: ARA1, GCY1, GRE3, and YPR1 (Oechsner et al.
1988; Kuhn et al. 1995; Nakamura et al. 1997; Kim et al.
1998). It is expected that dysfunction of GDH causes the
accumulation of intracellular glycerol. In order to
determine the effect of GDH on intracellular glycerol
levels, we constructed and analyzed knockout mutants of
GDH genes. Through analysis of such a GDH-mutant
(ara1Δgcy1Δgre3Δypr1Δ quadruple knockout mutant),
we revealed that decreased activity of GDH caused
increased levels of intracellular glycerol and increased
freeze-thaw tolerance. Furthermore, we found that dough
prepared with intracellular-glycerol-enriched cells cultured
in glycerol medium leavened well after frozen storage,
indicating the possibility of using intracellular-glycerol-
enriched cells to develop better frozen dough.
Table 1. Since it has been reported that freeze tolerance is
higher in stationary phase cells than in log-phase cells
(Park et al. 1997), all cells used in this study were cultured
until stationary phase. Cells were cultured in SD minimal
medium (2% glucose, 0.67% yeast nitrogen base w/o
amino acids, pH 5.5) or SG medium (0.5% glucose, 3%
glycerol, 0.67% yeast nitrogen base w/o amino acids,
pH 5.5) with appropriate amino acids and bases at 28°C
with reciprocal shaking (120 rpm) in Erlenmeyer flasks.
GDH-gene disruption
Genes encoding GDH were disrupted by the one-step PCR
method (Lorenz et al. 1995). The DNA fragment (1.2 kb)
encoding the ara1Δ∷TRP1 region was amplified using
the plasmid pRS314 as a template and 5′-
ACTGGGTTTGGGGACAGCAAATCCTCACGAAAA-
GTTTCGGTGATGACGGTGAAAACCT-3′ and 5′-
ATGGCTGCAAATGGCTCATCGATGAATCTCACTG-
GTATTTCACACCGCAGGCAAGTGCA-3′ as primers.
The DNA fragment (3.0 kb) encoding the gcy1-
Δ∷LEU2 region was amplified using the genomic DNA
of a gcy1Δ∷LEU2 mutant (Oechsner et al. 1988) as a
template and primers 5′-TTTATCTTCCAAGGACAT-
GACC-3′ and 5′-CAATCATACGAGAAACACGCAA-3′.
The DNA fragment (1.8 kb) encoding the gre3Δ∷cgHIS3
region was amplified using the plasmid pSHB1805
(Kitada et al. 1995) as a template and primers 5′-
GTTGCGACTACGGCAACGAAAAGGAAGTTGGTG-
AAGGTACAGCTATGACCATGATTACG-3′ and 5′-
GATTTTGGAATGACGGCAATGCCTCTCTCTGAGT-
TGCCCATATGTGCTGCAAGGCGATTAA-3′.
The DNA fragment (1.2 kb) encoding the ypr1-
Δ∷URA3 region was amplified using the plasmid
YEp24 as a template and primers 5′-
AGAAGAAGTTGGCAGGGCTATTAAAGAATTCCG-
GAGTCCCTAGTCCGTGGAATTCTCATG-3′ and 5′-
GATCCCCACTTCATATCAACGACTCTCTTTGTAC-
CATGCTCATTACGACCGAGATTCCC-3′.

Table 1 Yeast strains used in this study

W303-1A MATa ade2-1 ura3-1 his3–1 trp1-1 leu2-3, 112 can1-100
W303-1B MATα ade2-1 ura3-1 his3-1 trp1-1 leu2-3, 112 can1-100
ara1Δ W303-1A, except for ara1Δ∷TRP1
gcy1Δ W303-1A, except for gcy1Δ∷LEU2
gre3Δ W303-1A, except for gre3Δ∷cgHIS
ypr1Δ W303-1A, except for ypr1Δ∷URA3
ara1Δgcy1Δypr1Δ W303-1A, except for ara1Δ∷TRP1 gcy1Δ∷LEU2 ypr1Δ∷URA3
ara1Δgcy1Δgre3Δypr1Δ W303-1A, except for ara1Δ∷TRP1 gcy1Δ∷LEU2 gre3Δ∷cgHIS ypr1Δ∷URA3
gpd1Δgpd2Δ W303-1A, except for gpd1Δ∷URA3 gpd2Δ∷LEU2
110
These DNA fragments were introduced into the W303-
1A and W303-1B strains to construct each knockout
mutant. Disruption of each gene was confirmed by PCR.
Double, triple, and quadruple mutants of GDH genes were
constructed by crossing each mutant followed by tetrad-
dissection analysis.
Freeze-tolerance test
Cells were cultured in 50 ml SD medium at 28°C to
stationary phase, then collected and washed once with
distilled water. After dilution with 10 mM potassium
phosphate buffer (pH 7.0) to an optical density (OD) at
610 nm of 1.0, 100 μl cell suspension (approximately
2×106 cells) was stored in a 1.5 ml sample tube at −20°C.
Frozen cells were thawed at 25°C, and cell survival was
monitored by diluting in the same buffer and plating
aliquots on YPD plates (2% glucose, 2% peptone, 1%
yeast extract, 2% agar, pH 5.5).
Preparation of frozen dough and analytical techniques
The preparation of frozen dough was described previously
(Murakami et al. 1996). Dough was cut into pieces of 70 g
and frozen at −20°C for 7 days. Dough-leavening ability
was measured by monitoring gas production at 30°C with
a Fermograph (AF-1000, ATTO, Tokyo, Japan) as
reported previously (Nakagawa and Ouchi 1994). GDH
activity was assayed by the method of de Vries et al.
(2003). Intracellular glycerol was measured using a kit (F-
kit Glycerin; Roche, Mannheim, Germany).
Northern blotting analysis
Northern blotting analysis was performed as previously
described (Izawa et al. 1999). Probes were generated by
random labeling each DNA fragment with [α-32P]dCTP
using a kit (Random Primer DNA Labeling Kit Ver. 2;
Takara, Kyoto, Japan). The DNA fragments were
amplified by PCR using the following primers: for
FPS1, 5′-CAAGTACGCTCGAGGGTACA-3′ and 5′-
CAGGCTGAGTTCATGTCAAC-3′; for GUT1, 5′-
ATGTGGTTTGAGTTGTGGCC-3′ and 5′-TGCAATA-
GAAGGTGGAGAGG-3′; for GPD1, 5′-CCCCCCTCCA-
CAAACACAAATA-3′ and 5′-CCTCGAAAAAAGTGG-
GGGAAAG-3′; for GPD2, 5′-GCTCCCCTTCCTTAT-
CAATGCT-3′ and 5′-GGGAGAGTGTCTATTCGTCA-
TC-3′; for GPP1, 5′-TCGTAAGTATCTCTTGATTGCC-
3′ and 5′-CGGAAATGGAGGGAAATCATAC-3′; and for
GPP2, 5′-CTAGTGTTTACCAGATCAGTGG-3′ and 5′-
GGATTACCATTTCAACAGATCG-3′.
Results
Intracellular glycerol levels elevated by GDH gene
deletion
We compared GDH activity between a wild-type strain
(W303-1A) and its isogenic quadruple mutant strain
(ara1Δgcy1Δgre3Δypr1Δ). As shown in Fig. 2, the
GDH activity in the quadruple mutant was less than 15%
of that in the wild-type. A triple mutant, ara1Δgcy1-
Δypr1Δ, also showed decreased GDH activity compared
with wild-type cells, but retained higher activity than the
quadruple mutant. These results indicate that GDH is
encoded mainly by ARA1, GCY1, GRE3, and YPR1 in S.
cerevisiae. Since GDH activity was not completely
abolished in the quadruple mutant, yeast cells presumably
possess other GDH isozymes and/or yet to be identified
enzyme(s) with GDH activity.
We next investigated the effects of the deletion of GDH
genes on the expression of other genes encoding glycerol-
metabolizing enzymes. GPDH and G3Pase are involved in
glycerol biosynthesis (Fig. 1), and are encoded by the
genes GPD and GPP, respectively (Larsson et al. 1993;
Eriksson et al. 1995; Påhlman et al. 2001). On the other
hand, GUT1 encodes glycerol kinase, which phosphor-
ylates glycerol to generate G3P (Pavlik et al. 1993), and
the glycerol transporter encoded by FPS1 is involved
mainly in glycerol efflux (Luyten et al. 1995; Tamas et al.
1999). Both Gut1 and Fps1 function to decrease the level
of intracellular glycerol. Northern blotting analysis
revealed no significant difference in the expression of
any of these genes between the wild type and the
quadruple mutant (data not shown). This result suggests
that the quadruple disruption of GDH genes has little
effect on other steps that increase or decrease the level of
intracellular glycerol.
Fig. 2 GDH activity in ara1Δgcy1Δgre3Δypr1Δ cells. Cells
cultured in SD medium were collected and washed twice with
distilled water, and cell-free extract was prepared. Results are
expressed as the mean ± SD of three independent experiments

We further investigated the effect of decreased GDH
activity on intracellular glycerol levels. As shown in
Table 2, the quadruple mutant showed significantly higher
levels of intracellular glycerol than wild-type cells. A
slight increase in the level of intracellular glycerol was
observed in the triple mutant. These results suggest that
decreased GDH activity leads to the accumulation of
intracellular glycerol. Taken together with the results of
Northern blotting analysis, the increased level of intracel-
lular glycerol in the quadruple mutant seems to be caused
mainly by repression of the reaction catalyzed by GDH.
Deletion of GDH genes leads to increased resistance
to freeze stress
To estimate the effect of intracellular glycerol on viability
under freeze stress, we determined the freeze tolerance of
the GDH mutants. As shown in Fig. 3, wild-type cells
showed a sharp decline of viability during storage at
−20°C. On the other hand, the number of viable cells of
the quadruple mutant decreased gradually during the
storage period. The survival rate of the quadruple mutant
was apparently higher than that of the wild type, indicating
that the quadruple disruption of GDH genes improved the
tolerance to freeze stress. We also investigated the freeze
tolerance of the single, double, and triple GDH gene
mutants. No apparent difference in tolerance was observed
between the wild type, single mutants, and double mutants
(data not shown). The survival rate of the triple mutant
(ara1Δgcy1Δypr1Δ) was slightly higher than that of the
wild type but lower than that of the quadruple mutant.
These results seems reasonable, since single and double
mutants of GDH genes showed no significant increase in
intracellular glycerol contents (data not shown) and the
ara1Δgcy1Δypr1Δ cells showed a slight increase
(Table 2). These results suggest that the increased level
of intracellular glycerol caused by the disruption of GDH
genes enhanced the freeze tolerance of yeast cells. On the
other hand, the GPDH-deficient mutant (gpd1Δgpd2Δ)
was sensitive to freeze stress compared to the wild type
(Fig. 3). We confirmed that the gpd1Δgpd2Δ mutant
contains decreased levels of intracellular glycerol
(Table 2). It is likely that decreased levels of intracellular
glycerol partly affect the sensitivity to freeze stress of
gpd1Δgpd2Δ cells. However, the difference in the freeze
tolerance between the gpd1Δgpd2Δ mutant and wild type
Table 2 Intracellular glycerol levels in various yeast strains. Cells
cultured in SD medium were collected and washed twice with
distilled water, and the intracellular glycerol contents were mea-
sured. Results are expressed as the mean ± SD of three independent
experiments
Strain Intracellular glycerol (μmol/mg cells)
Wild type 31±2.4
ara1Δgcy1Δypr1Δ 34±3.2
ara1Δgcy1Δgre3Δypr1Δ 50±4.4
gpd1Δgpd2Δ 3.2±1.8
111
was only slight. This indicates that glycerol is not the only
factor to confer freeze stress resistance on yeast cells.
Other cellular factors, such as trehalose, presumably
contribute to maintenance of freeze stress tolerance.
Intracellular-glycerol-enriched cells are tolerant of
freeze stress
Above, we have shown a possible correlation between
intracellular glycerol and resistance to freeze stress. We
further tested whether intracellular-glycerol-enriched cells
acquire resistance to freeze stress. Glycerol is taken in by
yeast cells not only by specific transporters such as Gup1
and Gup2 but also by passive diffusion (Luyten et al.
1995; Sutherland et al. 1997; Holst et al. 2000). Thus, we
cultured cells under glycerol-rich conditions to increase
levels of intracellular glycerol. Wild type cells (W303-1A)
were cultured in SG medium at 28°C until stationary
phase. As expected, cells grown in SG medium showed
levels of intracellular glycerol approximately 3- to 4-fold
higher than cells in SD medium (Fig. 4a). The quadruple
mutant (ara1Δgcy1Δgre3Δypr1Δ) grown in SG medium
to stationary phase had levels of intracellular glycerol
almost identical to those of wild type cells (Fig. 4a). No
significant effect of decreased GDH activity was observed
on intracellular glycerol content when cells were grown in
SG medium to stationary phase. Such intracellular-glyc-
erol-enriched cells were remarkably tolerant to freeze
stress (Fig. 4b). Both the wild type and the quadruple
mutant showed almost identical tolerance to freeze stress,
and their tolerances to freeze stress were higher than that
of ara1Δgcy1Δgre3Δypr1Δ cells grown in SD medium
(Figs. 3, 4b). This result strongly supports the idea that
intracellular glycerol plays a role in the resistance to freeze
Fig. 3 Freeze stress tolerance in various yeast strains. Freeze stress
tolerance of cells was determined by measuring viability at −20°C
for 7 days. Percent survival was expressed relative to the initial
viability prior to freezing. Results are representative of three
independent experiments. Open circles Wild-type cells, closed
circles ara1Δgcy1Δgre3Δypr1Δ, triangles ara1Δgcy1Δypr1Δ,
squares gpd1Δgpd2Δ
112
stress, and clearly indicates that increased levels of
intracellular glycerol confer improved freeze stress toler-
ance on yeast cells.
Intracellular-glycerol-enriched cells showed improved
leavening ability in frozen/thawed dough
To determine whether intracellular-glycerol-enriched cells
are useful for the frozen dough method, we measured the
leavening ability of wild-type cells (W303-1A) in dough
after frozen storage. Figure 5 shows the amount of CO2
Fig. 4 Effect of cultivation in SG medium on the level of
intracellular glycerol. a Wild type cells (W303-1A) cultured in SD
or SG medium and ara1Δgcy1Δgre3Δypr1Δ cells cultured in SG
medium were collected and washed twice with distilled water, and
intracellular glycerol contents were measured. Results are expressed
as the mean ± SD of three independent experiments. Lanes: 1 Wild-
type cells cultured in SD medium, 2 wild-type cells in SG medium,
3ara1Δgcy1Δgre3Δypr1Δ cells in SG medium. b Freeze stress
tolerance of cells was determined as described in Fig. 3. Circles
Wild-type cells cultured in SD medium, triangles wild-type cells
cultured in SG medium, squares ara1Δgcy1Δgre3Δypr1Δ cells in
SG medium
evolved in 5 h from frozen/thawed dough and non-frozen
dough. Before freezing, there was a slight difference in
gassing power between the glycerol-enriched cells cul-
tured in SG medium and the normal cells cultured in SD
medium. However, a marked difference was observed in
frozen/thawed dough. The intracellular-glycerol-enriched
cells retained high gassing power after storage at −20°C
for 7 days, whereas normal cells lost significant gassing
power during storage (Fig. 5). The difference in gassing
power between the intracellular-glycerol-enriched cells
and normal cells seems to reflect their viability after frozen
storage (Fig. 4b).
Discussion
We demonstrated here that disruption of four GDH genes
(ARA1, GCY1, GRE3, and YPR1) led to decreased activity
of GDH and increased levels of intracellular glycerol
(Fig. 2, Table 2). Northern blotting analysis indicated that
the decrease in GDH activity seems not to affect other
steps of glycerol metabolism (data not shown). These
results clearly suggest that blocking the dissimilation of
glycerol is an effective approach to increase the level of
intracellular glycerol along with activation of the biosyn-
thesis of glycerol. Our quadruple mutant still retained
GDH activity (Fig. 2) and the YJR096w gene has recently
been reported as a candidate encoding glycerol dehydro-
genase (Träff et al. 2002). A quintuple mutation of GDH
genes and YJR096w, or the combined mutation of GDH
Fig. 5 Gas production of yeast in frozen/thawed dough. White
bread dough (70 g) was prepared using glycerol-enriched and
normal wild-type cells (W303-1A) cultured in SD or SG medium.
After storage at −20°C for 7 days, the dough was thawed at 30°C;
remaining gassing power was measured and compared with that of
non-frozen samples. Results are representative of four independent
experiments. Open squares Non-frozen dough with normal cells
cultured in SD medium, open circles non-frozen dough with
glycerol-enriched cells cultured in SG medium, closed squares
frozen/thawed dough with normal cells cultured in SD medium,
closed circles frozen/thawed dough with glycerol-enriched cells
cultured in SG medium

and other enzymes that dissimilate glycerol, such as Gut1,
might lead to a much greater accumulation of glycerol.
We also found that the quadruple mutant (ara1-
Δgcy1Δgre3Δypr1Δ) showed more resistance to freeze-
thaw stress than the wild-type cells (Fig. 3). On the other
hand, gpd1Δgpd2Δ cells showed lower levels of intra-
cellular glycerol and were more sensitive to freeze stress
compared with wild-type cells (Table 2, Fig. 3). These
findings suggest that intracellular glycerol plays an
important role in the tolerance to freeze stress. This idea
was supported by analysis of intracellular-glycerol-en-
riched cells (Figs. 4, 5), which retained higher gassing
power in frozen/thawed dough than normal cells (Fig. 5).
Our data suggest that glycerol can function as quite a
useful cryoprotectant even inside the cell. There is also the
possibility that intracellular glycerol indirectly affects the
resistance to freeze stress. Since several mutant strains
defective in glycerol metabolism exhibit changes in lipid
composition (Sutherland et al. 1997; Toh et al. 2001),
levels of intracellular glycerol may affect lipid composi-
tion of membranes. It has been reported that the lipid
composition of yeast affects the tolerance to freeze stress
(Murakami et al. 1996). Thus, the effects of increased
intracellular glycerol on freeze tolerance may arise partly
from changes in lipid composition. Additionally, Siderius
et al. (2000) have proposed that intracellular glycerol
functions as a thermoprotectant by affecting signal trans-
duction, in an as yet unexplained fashion. It is also likely
that intracellular glycerol affects tolerance to freeze-thaw
stress via signal transduction in the response.
Increased intracellular glycerol has no effect on final
bread quality in terms of flavor, color, and texture (Myers
et al. 1998). On the other hand, it has been reported that an
increased level of intracellular glycerol has several other
benefits for the shelf life of wet yeast products and for the
leavening activity in sweet dough containing high levels of
sugars (Myers et al. 1998). One of the authors of the
present study also reported a close correlation between the
intracellular glycerol level and the fermentation ability
(Hirasawa and Yokoigawa 2001). In this study, the
intracellular-glycerol-enriched cells also showed a slight
improvement in the gassing power before freezing (Fig. 5).
Here, we used laboratory yeast strains, and such strains
generally have low fermentation ability compared with
commercial baker’s yeast strains. Therefore, a much more
obvious effect on glycerol enrichment is expected in the
case of commercial strains, and thus this method would be
a useful technique for the baking industry.
S. cerevisiae accumulates intracellular glycerol as an
osmolyte through the high osmolarity glycerol signaling
pathway under osmotically stressed conditions (Schuller et
al. 1994; Lewis et al. 1995). It has also been reported that
the fps1Δ mutant and overexpression of the GPD1 or
GPD2 gene caused an increase in intracellular glycerol
levels (Michnick et al. 1997; Remize et al. 1999, 2001;
Toh et al. 2001). Therefore, pretreatment of yeast cells
with osmotic stress, and the genetic modification of
glycerol metabolism are presumably effective ways to
acquire freeze tolerance in yeast. However, for applica-
113
tions in the baking industry, these methods seem onerous
and not suited to win the admiration of consumers. On the
other hand, cultivation in SG medium to stationary phase,
the method used in this study, is a relatively simple way to
increase intracellular glycerol, and one which could be
applicable to commercial yeast strains.
Acknowledgements We are grateful to Dr. W. Bandlow
(gcy1Δ∷LEU2) and Dr. S. Harashima for providing yeast strains
and plasmids. We also thank Mr. T. Tanaka and Mr. T. Suzuki for
their technical support in the construction of yeast mutants. This
study was supported by the Iijima Memorial Foundation for the
Promotion of Food Science and Technology and Bio-oriented
Technology Research Advancement Institution (BRAIN).
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