Journal of Bioscience and Bioengineering

VOL. 116 No. 1, 79e84, 2013

www.elsevier.com/locate/jbiosc

Competitive advantage and tolerance of selected shochu yeast in barley

shochu mash

Hideharu Takashita,1, * Emi Fujihara,1 Mihoko Furutera,1 Yasuhiro Kajiwara,1 Masahiko Shimoda,1
Masayoshi Matsuoka,2 Takahira Ogawa,2 Seiji Kawamoto,3 and Kazuhisa Ono3

Research & Development Laboratory, Sanwa Shurui Co., Ltd., 2231-1 Yamamoto, Usa, Oita 879-0495, Japan,1 Department of Applied Microbial Technology, Faculty of Biotechnology
and Life Science, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan,2 and Department of Molecular Biotechnology, Graduate School of
Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan3

Received 17 December 2012; accepted 16 January 2013

Available online 27 February 2013

A shochu yeast strain, Saccharomyces cerevisiae BAW-6, was previously isolated from Kagoshima yeast strain Ko, and
has since been utilized in shochu production. The BAW-6 strain carries pho3/pho3 homozygous genes in contrast to the
heterozygous PHO3/pho3 genes in the parental Ko strain. However, absence of the PHO3 gene per se cannot explain the
fermentation superiority of BAW-6. Here, we demonstrate the growth advantage of the BAW-6 strain over the Ko strain
by competitive cultivation in barley shochu preparation, where alcohol yield and nihonshudo of the former strain were
higher than those of the latter strain. In addition, the maximum growth rate of BAW-6 was less affected than that of Ko
by high Brix values of barley koji medium, suggesting that BAW-6 is less sensitive to growth inhibitory compounds
derived from barley or barley koji. The tolerance of BAW-6 to growth inhibitory compounds, cerulenin and diethyl-
stilbestrol (an HD-ATPase inhibitor), was also higher than that of other yeast strains. Consistent with BAW-60 s tolerance
to diethylstilbestrol in the presence of 8% ethanol (pH 4.5), HD-ATPase activity, but not transcription of its gene, was
higher in BAW-6 than in Ko. We conclude that the BAW-6 strain is associated with certain gene alterations other than
PHO3, such that it can maintain cellular ion homeostasis under conditions of ethanol stress during the latter phase of
fermentation.
Ó 2013, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Shochu yeast; Barley shochu; Cerulenin; Diethylstilbestrol; Hþ-ATPase]

The traditional Japanese liquor shochu is prepared from various
ingredients, such as barley, sweet potato, rice, and buckwheat. The
shochu koji used as a raw material for the first-stage fermentation
contains a large amount of citric acid produced by a shochu koji
mold, e.g., Aspergillus kawachii. The acidic conditions of the mash
prevent undesirable bacterial contamination. The yeast typically
used for shochu production is a homothallic diploid strain, which is
taxonomically classified as Saccharomyces cerevisiae. The shochu
production process involves two mash preparations: the first
intended for yeast proliferation and the second for fermentation of
the main ingredient. Typically, a portion of first mash is added to
another first mash, which is termed sashimoto and is repeated
throughout several months. After a two-month continuous culture
with initial use of the Kagoshima yeast strain (Ko), we isolated
a novel yeast strain from the barley shochu mash, which we named
BAW-6. The alcohol tolerance of BAW-6 was higher than that of its
parental strain Ko, and alcohol production in the latter phase of the
second mash, in which yeast cells are highly stressed by alcohol,
was considerably improved (1). Even though our initial selection
marker for BAW-6 was the lack of constitutive acid phosphatase
* Corresponding author. Tel.: þ81 (0) 978 33 3844; fax: þ81 (0) 978 33 5811.
E-mail address: takashita-h@kokuzo.co.jp (H. Takashita).
activity (PHO3 phenotype), the PHO5/3 chimeric gene resulting
from the deletion of a 1.9-kb region between the PHO5 and PHO3
tandem genes does not seem to be correlated with alcohol
production (2). However, the change in phenotype of BAW-6
suggests the possibility of mutations in other genes that influence
shochu production.
Recently, it was revealed that sake yeast exhibits high alcohol
productivity due to the repression of gene expression mediated by
the environmental stress-responsive transcription factors Msn2p
and Msn4p (3), and a loss-of-function mutation in the RIM15 gene,
which encodes a putative upstream activator of Msn2p and Msn4p
(4), resulting in a lack of stress response in the yeast. In sake
production, the sake yeast is grown in a batch culture and is
removed from the mash at the end of fermentation, whereas in the
shochu preparation process, the shochu yeast is recycled many
times, i.e., a small portion of shochu yeast culture is inoculated into
the next batch as a starter, which is repeated many times. Due to
the differences in environmental stress (fermentation temperature,
fermentation duration, alcohol concentration, organic acid
concentrations, and so forth) of sake and shochu mash, the high
alcohol productivity of BAW-6 might be associated with a mecha-
nism different from that in sake yeast. In this regard, a multi-drug
resistant mutant of sake yeast was reported to be proficient in

1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2013.01.005
80 TAKASHITA ET AL. J. BIOSCI. BIOENG.,

alcohol productivity (5,6). Moreover, Fukuda et al. isolated tricho-
thecin-resistant mutants from sake and shochu yeasts that showed
multi-drug resistance and superior alcohol productivity (7,8). Since
BAW-6 exhibits high alcohol productivity, this strain may also have
gained multi-drug resistance. In this study, we investigated the
characteristics of the BAW-6 strain in order to determine whether it
has a competitive advantage over Ko in barley shochu mash, and
examined the potential for drug resistance as one of its stress
tolerance traits.
MATERIALS AND METHODS
1 mM EDTA (pH 7.0)] and 0.17 volume formaldehyde (37% v/v). The denatured
RNA was then separated by gel electrophoresis [1.5% agarose, 1 MOPS, 0.16
volume formaldehyde (37% v/v)], and transferred to a nylon membrane (Hybond-
N; Amersham). The DNA primers ACT1F (50 -ACGGTCCCAATTGCTCGAGAGA-30 ) and
ACT1R (50 -GAAGACAGCACGAGGAGCGTC-30 ) were used to detect the expression of
the ACT1 gene, whereas PMA1F (50 -TGTTTCAGCTCATCAGCCAAC-30 ) and PMA1R
(50 -ACCAGCTTGGAATTCTTGAACG-30 ) were used to detect the expression of the
PMA1 gene. In order to synthesize the DNA probes for these genes, DNA
fragments were labeled by using PCR DIG labeling mix (Roche) and a Veriti
96-well thermal cycler (Applied Biosystems). Hybridization was carried out at
42 C with hybridization buffer [5 SSC, 50% formamide, 50 mM sodium
phosphate buffer (pH 7.0), 7% SDS, 2% blocking reagent, 0.1% lauroylsarcosine]. The
detection was performed with a DIG Luminescent Detection Kit (Roche), and RNA
bands were visualized using a luminescent image analyzer (LAS-1000; Fuji).

Strains The laboratory diploid yeast BY4947 was provided by the National
BioResource Project. The shochu yeast strain BAW-6 was preserved in our RESULTS AND DISCUSSION
laboratory following a previous study (1). Two other shochu yeast strains,
Kagoshima yeast (Ko) and SH-4 (RIB1019), were obtained from the Kagoshima
Federation of Brewers Association and the National Research Institute of Brewing, Competitive culture of Ko and BAW-6 strains in barley koji
respectively. medium The BAW-6 strain is a D.C. white isolate obtained
Media and growth conditions All the percentages of ingredients are
expressed as weight per volume (w/v). The conventional complete medium for yeast
culture is YPD (1% yeast extract, 2% polypeptone, 2% glucose). Synthetic dextrose (SD)
medium contains 2% glucose, and 0.67% yeast nitrogen base without amino acids. A
phosphate-rich medium contains 1% glucose, 0.2% polypeptone, 0.15% yeast extract,
0.1% KH2PO4, and 0.04% MgSO4$7H2O. Agar was added to a final concentration of 2% for
solid media. Barley koji medium was prepared by incubating 5 kg of barley koji at 55 C
for 7 h after adding 1 g of Amano 2 enzyme for shochu (Amano Enzyme, Aichi, Japan)
and 6 L of water. The solution of the incubated koji was then filtered through linen cloth,
and the filtrate was used as barley koji medium after Brix adjustment. Brix values were
measured using a portable refractometer (PAL-1; Atago). All the liquid stationary
cultures and agar plates were incubated at 30 C, unless otherwise specified.
Competitive culture assay The BAW-6 and Ko strains were grown in 10 ml
of barley koji medium at 30 C for 2 days, and 2  108 cells of each strain were added
to a 300 ml conical flask containing 117 g of barley koji and 120 ml of water. After
incubating at 25 C for 4e5 days, 1 ml of the culture was then used as a starter for
a new batch of fermentation. This successive cultivation was repeated 4 times,
and the culture was diluted and plated on phosphate-rich medium. After
incubation at 30 C for 2 days, yeast colonies on the plates were subjected to diazo
coupling (D.C.) staining for constitutive acid phosphatase (cAPase), as described
by Toh-e and Oshima (9). The D.C. staining method turned colonies of BAW-6
white (cAPase negative) and those of Ko brown (cAPase positive) on the
phosphate-rich medium. Nihonshudo and the alcohol content were measured
using routine methods.
Proliferative activity test in barley koji medium Barley koji media with
compounds of different concentrations were prepared with Brix values of 8, 12, or
18, glucose concentration of 7% or 10.5%, and citric acid concentration of 2% or 4%.
The pH of all the media was adjusted to 3. Maximum specific growth rate (mmax) was
calculated after cultivating each yeast strain at 30 C with 30 shakings per minute
(spm) in a temperature gradient incubator (TN-2612; Advantec).
Chemical tolerance assay Each of the yeast strains was cultivated in YPD
medium at 30 C for 24 h with constant shaking, and the culture was diluted to an
OD660nm of 0.2, equivalent to 1  106 cells/ml. Media were supplemented with
cerulenin, cycloheximide, clotrimazole and diethylstilbestrol to the final concen-
tration of 0.63, 1.25, 2.5, 5, 10, 20, 40, and 80 mg/ml in solid SD medium with or
without 8% ethanol (pH adjusted to 4.5). These supplemented SD plates were used
for the application of 10 ml/spot of diluted yeast culture and were incubated at 30 C
for 6 days. The minimum growth inhibitory concentration (MIC) was defined as the
lowest concentration of the chemical that inhibited the growth of each yeast strain
as judged by the naked eye.
Time course of yeast extracellular pH Cells in stationary phase were
collected after cultivation of each strain in 10 ml of YPD medium at 30 C overnight
with constant shaking. The collected cells were then washed 4 times at 4 C with
250 mM sorbitol solution and then diluted to an OD660nm of 1.2 with 250 mM sorbitol.
Ten milliliters of the diluted cell suspensions were incubated at 30 C for 5 min, and
their pH was adjusted to 6.0. After the pH became stable, 250 mM glucose was added
to the suspensions. The change in pH was monitored from the time when the glucose
was added.
Diethylstilbestrol tolerance culture test A pre-culture of yeast strains
grown in YPD medium was inoculated into YPD medium with 4% ethanol containing
0 to 20 mg/ml diethylstilbestrol, and the cultures were incubated at 30 C in
a temperature gradient incubator (TN-2612; Advantec) with constant shaking.
RNA extraction and northern blot analysis Cells were grown in YPD
medium, and were collected during both the logarithmic (OD660nm ¼ 0.2e0.3)
and the stationary growth phase (OD660nm ¼ 1.0). The yeast RNA was extracted FIG. 1. Changes in D.C. staining rate (A), alcohol content (B) and nihonshudo (C) in
following the method by Schmitt et al. (10) The extracted total RNA (10 mg/ml) barley fermentation mash of each sample of subculture. Symbols: closed circles, Ko;
was denatured at 65 C for 15 min in a solution containing 50% formamide, open circles, BAW-6; and closed triangles, mixed culture. Data are represented as the
1 MOPS [20 mM 3-(N-morpholino)-propanesulfonic acid, 5 mM sodium acetate, mean values of two experiments.
VOL. 116, 2013 COMPETITIVE ADVANTAGE AND TOLERANCE OF SHOCHU YEAST
from a repetitive-cultured shochu mash containing the Ko strain as
a starter and is associated with improved alcohol productivity (1).
Fig. 1A shows the number of D.C. brown phenotype colonies
formed by plating samples taken from each of the 4 cycles of
cultivation of Ko, BAW-6, and a 1:1 mixture of both strains.
Throughout the successive cultivation, Ko maintained a 100% D.C.
brown phenotype, whereas BAW-6 showed 100% D.C. white. The
initial ratio of the D.C. brown colonies in the mixed culture of Ko
and BAW-6 was 50%; however, the percentage of D.C. brown
colonies decreased as the cultivation was repeated. This decline
in the percentage of D.C. colonies can be attributed to the
competitive overgrowth of BAW-6 compared to Ko in the shochu
mash. Fig. 1B and C shows the alcohol content and nihonshudo,
respectively, of each culture measured on each of the final days of
the cycle. Both the alcohol content and nihonshudo of the mash of
BAW-6 culture were always higher than those of Ko. The mixed
culture showed intermediate values for alcohol content and
nihonshudo, which were closer to those of the BAW-6 culture
(Fig. 1B and C). A previous laboratory-scale shochu fermentation
showed that shochu mash using BAW-6 emitted more CO2 in the
latter half of the second fermentation than did one using Ko (1).
The higher CO2 production of BAW-6 is correlated with its higher
glucose utilization, resulting in higher alcohol yield. These results
confirmed that the previously reported BAW-6 advantage over Ko
could be successfully reproduced.
The influence of Brix, glucose, and citric acid concentrations
on yeast growth in barley koji medium We focused on the
composition of barley shochu mash in order to investigate the
reason why BAW-6 has a growth advantage over Ko. Table 1 shows
the maximum specific growth rate (mmax) of BAW-6 and Ko under
various conditions. Ko showed a higher mmax than BAW-6 in the
medium of Brix: 8, glucose: 7%, citric acid: 2% or Brix: 12,
glucose: 10.5%, citric acid: 2%. However, the growth of BAW-6
was less inhibited by an increase in Brix and had a higher mmax
than Ko, the proliferation of which was potently hindered. BAW-6
and Ko showed no tolerance to 4% citric acid in barley koji
medium. Thus, we assumed that BAW-6 grows faster than Ko
because BAW-6 is less sensitive than Ko to growth inhibitory
compounds that originate from barley or barley koji mold. One of
the thionins derived from barley is known to be a basic protein
with antimicrobial activity. The thionin found mainly in Triticae is
a 5-kDa peptide, and consists of 8 cysteine residues (11). The
tolerance of BAW-6 and Ko to thionin was measured; however, no
difference was observed between the two strains (data not
shown). Li et al. reported that proanthocyanidins extracted from
grapes during red wine fermentation inhibited the activity of Hþ-
ATPase during the initial phase of the fermentation, parallel to
a decrease in cell growth (12). It might be plausible that barley
thionin is decomposed by a protease produced by koji mold in
shochu mash, thus another antimicrobial compound such as
indigestible peptides or polyphenols may be present.
Chemical tolerance of laboratory and shochu
yeasts During the shochu preparation process, yeast strains are
exposed to many stress factors such as alcohol, high temperature,
TABLE 1. Maximum specific growth rate (mmax) of BAW-6 and Ko in the various
conditions of the barley koji medium (pH 3).
Brix Glucose (%) Citric acid (%) mmax (h1)
BAW-6 Ko
8 7 2 0.216  0.007 0.230  0.002
12 7 2 0.223  0.003 0.209  0.011
12 7 4 0.185  0.001 0.195  0.014
12 10.5 2 0.198  0.004 0.202  0.006
18 10.5 2 0.175  0.006 0.152  0.016
Data are represented as the mean values of two experiments.
81
and citric acid. It is therefore conceivable that BAW-6 is stress
tolerant, which enables the strain to proliferate in the shochu mash.
Table 2 shows the minimum growth inhibitory concentration (MIC)
of some anti-eukaryotic compounds. The MIC of cerulenin against
BAW-6 was 8 times higher than that against other yeast strains in
SD medium. Moreover, BAW-6 showed significant tolerance to
diethylstilbestrol in YPD medium with 8% ethanol (pH 4.5), while
other yeast strains did not.
Ichikawa et al. isolated three strains producing a high level of
caproic acid from 54 cerulenin-resistant mutants (13). It is known
that a mutation in FAS2-1250S results in the phenotypes of cer-
ulenin resistance and high productivity of caproic acid and ethyl
caproate (14). Further, the YCF1 and FLR1 genes that code for multi-
drug resistance transporters are known to be involved in the cer-
ulenin resistance of S. cerevisiae (15). BAW-6, on the other hand,
does not have a point mutation in FUS2-1250S or high ethyl cap-
roate-productivity (data not shown). Thus, we assume that another
mechanism in BAW-6 contributes to its cerulenin resistance, the
association of which with its fermentation characteristics in barley
shochu preparation remains unknown.
Focusing on diethylstilbestrol that specifically inhibits plasma
membrane Hþ-ATPase (16), Takahashi et al. isolated mutants from
a sake yeast strain, K-701. They then isolated a strain with higher
nihonshudo and alcohol content in the latter phase of fermentation
from 0.1 mM diethylstilbestrol-tolerant mutants (Takahashi, T.,
Kondo, S., Mizoguchi, T., Abstr. J. Brew. Soc. Japan, 102, p. 697, 2007).
The left panels in Fig. 2A and B show the tolerance of the test strains
in a spot dilution assay on YPD (pH not adjusted) and YPD with 8%
ethanol (pH adjusted to 4.5) agar plates, respectively, in the presence
of 0.1 mM diethylstilbestrol. BAW-6 showed higher tolerance against
diethylstilbestrol in YPD medium regardless of the presence of
ethanol at low pH. On the other hand, the tolerance of other shochu
strains, Ko and SH-4, to diethylstilbestrol was much lower than that
of BAW-6, and was even lower than that of the laboratory yeast
strain, BY4947. This indicates that the BAW-6 strain has a consistent
and high tolerance to diethylstilbestrol as compared with Ko, and
therefore exhibits higher alcohol production and glucose consump-
tion, rendering it more competitive in the barley shochu mash.
To examine the possibility that the diethylstilbestrol tolerance of
BAW-6 is due to vacuolar Hþ-ATPase activity, its inhibitor, bafilo-
mycin-A1 (17), was added to the test plates (Fig. 2A and B, right
panels). The site of bafilomycin inhibition has been localized to an
integral membrane proton channel domain (V0) (18,19) and the
inhibition requires residues of V0 subunit c (20,21). As shown in
Fig. 2, the presence of bafilomycin-A1 did not affect the tolerance of
BAW-6 to diethylstilbestrol. We therefore concluded that the
diethylstilbestrol tolerance of this strain is caused by a plasma
membrane Hþ-ATPase.
TABLE 2. Comparison of minimum growth inhibitory concentration (MIC) of various
drugs in laboratory yeast and shochu yeasts.
MIC (mg/ml)
Cerulenin Cycloheximide Clotrimazole Diethylstilbestrol
SD
BY4947 1.25 0.62 10 20
BAW-6 10 2.5 5 20
Ko 1.25 1.25 0.62 10
SH-4 1.25 1.25 2.5 20
YPD þ 8% ethanol (pH 4.5)
BY4947 5 0.62 10 40
BAW-6 10 1.25 10 80<
Ko 5 1.25 10 20
SH-4 2.5 1.25 20 20
The minimum growth inhibitory concentration (MIC) was defined as the lowest
concentration of the chemical that inhibited the growth of each yeast strain as
judged by the naked eye. Data are represented as the mean values of two experi-
ments. Twenty mg/ml diethylstilbestrol corresponds to 0.075 mM.
82 TAKASHITA ET AL.
FIG. 2. Resistance to diethylstilbestrol in laboratory and shochu yeasts on YPD and YPD
containing 8% ethanol (pH 4.5) agar plates in the absence and presence of bafilomycin-
A1. (A) 0.1 mM diethylstilbestrol-containing YPD agar plates and (B) 0.1 mM diethyl-
stilbestrol-containing YPD agar plus 8% ethanol plates (pH 4.5). Diluted yeast cultures
were applied as 10 ml/spots (from left to right as 1, 10, 102, 103 dilutions of
1  106 cells/ml) and were incubated at 30 C for 6 days before the photographs were
taken. Left panels, no other supplements; right panels, 0.2 mM bafilomycin-A1
supplementation.
Influence of diethylstilbestrol on yeast proliferation Yeast
strains were grown in liquid medium supplemented with diethyl-
stilbestrol with constant shaking. The growth rate was the highest
for Ko, followed by BAW-6 and BY4947 in YPD medium containing
4% ethanol; however, the addition of 20 mg/ml of diethylstilbestrol
caused Ko and BY4947 to exhibit a 35-h lag time before
FIG. 3. The effect of diethylstilbestrol concentration on the growth of laboratory yeast
and shochu yeasts in YPD medium containing 4% ethanol. Pre-cultures of yeast strains
in YPD medium were inoculated into YPD medium containing 4% ethanol and 0, 15, or
20 mg/ml of diethylstilbestrol, and incubated at 30 C in a temperature gradient incu-
bator with constant shaking.
J. BIOSCI. BIOENG.,
proliferation. In contrast, BAW-6 continued to grow slowly without
a lag time in the presence of the same concentration of the
compound (Fig. 3). When the concentration of diethylstilbestrol
was 15 mg/ml, the lag time of Ko was shortened to approximately
20 h, indicating that lag time of Ko increased in a concentration-
dependent manner but that the growth rate was not affected.
Therefore, it was assumed that Ko has a certain mechanism to
indirectly decrease the effect of diethylstilbestrol, by degrading
diethylstilbestrol or regenerating plasma membrane Hþ-ATPase,
whereas BAW-6 has a direct tolerance system related to plasma
membrane Hþ-ATPase that maintains strain growth.
HD-ATPase activity of Ko and BAW-6 Hþ-ATPase activity
was measured by constantly monitoring the pH of Ko and BAW-6
cell suspension in 250 mM sorbitol. Fig. 4 shows that BAW-6
decreased the pH more rapidly than Ko, indicating a higher the
Hþ-ATPase activity of BAW-6 compared to Ko. The initial rate of
[Hþ] increase due to BAW-6 was approximately 1.4 times higher
than that of Ko (Fig. 4, inset). Rosa et al. reported that 3e10% (v/v)
ethanol increased the in-vivo activity of the plasma membrane
Hþ-ATPase of S. cerevisiae (22). Furthermore, Mizoguchi et al.
demonstrated that cell growth in the presence of 8% ethanol
activates the yeast plasma membrane Hþ-ATPase, which
maintains ion homeostasis inside the cell even in the presence of
high concentrations of ethanol (23). The enhancement of ATPase
activity by Ca2þ was ascribed to alleviation of the inhibition of
ATPase activity by ethanol through protection of the membrane
structure (24). Therefore, it is assumed that the high activity of
plasma membrane Hþ-ATPase observed in BAW-6 improves the
ion homeostatic balance within the cells that are under ethanol
stress during the latter half of fermentation.
PMA1 gene expression in Ko and BAW-6 A molecular bio-
logical approach was adopted to investigate the plasma membrane
Hþ-ATPase activity in Ko and BAW-6. Although PMA1 and PMA2 are
the two structural genes for the membrane Hþ-ATPases in S. cer-
evisiae (25,26), expression of the PMA1 gene was examined in this
study because the activation of Hþ-ATPase by ethanol is dependent
solely on the PMA1 gene (27). As shown in Fig. 5, expression of the
PMA1 gene was comparable among the BY4947, Ko, and BAW-6
FIG. 4. Time course of yeast extracellular pH variations. Cells grown in YPD medium
were washed and suspended at an OD660nm of 1.2 in 250 mM sorbitol at 30 C, and the
pH was adjusted at 6.0. The pH of the suspension was measured following the addition
of 250 mM glucose at time zero. Data are represented as the mean values of triplicate
measurements. Symbols: closed circles, Ko; open circles, BAW-6.
VOL. 116, 2013
FIG. 5. Northern blot analysis of PMA1 and ACT1. Abbreviations: L, logarithmic growth
phase; S, stationary phase. Yeast RNAs were extracted and analyzed by northern
blotting as described in Materials and methods.
strains, although quantitative measurement showed slightly lower
(approximately 6%) transcript levels in BAW-6 compared with
BY4947 and Ko. The transcript levels were almost the same in
both logarithmic and stationary phases in all the strains. This
unexpected result indicated the presence of mechanism(s)
causing the increased Hþ-ATPase activity other than transcription
enhancement. It is known that the intracellular level of active
Pma1p is highly regulated at both transcription and post-
transcription modification steps by glucose (28). Another enzyme,
Ptk2p, localized in the cell membrane, is a yeast protein kinase
that activates Hþ-ATPase by phosphorylating serine residues in
the C-terminal region of the protein (29,30). Considering the fact
that BAW-6 exhibited higher glucose consumption as well as
higher Hþ-ATPase activity than Ko, it is possible that the PTK2
gene was highly expressed and/or Ptk2p activity was enhanced in
BAW-6.
In conclusion, we isolated a pho3/pho3 mutant strain of yeast
with superior fermentation traits (BAW-6) from shochu mash, in
which the majority of yeast cells were replaced by PHO3 strains
after repetitive cultivation of the initial Ko strain carrying hetero-
zygous PHO3/pho3 genes (1). Recently, we showed that the genetic
instability of cAPase in shochu and sake yeast strains originated from
a looping-out or loss of heterozygosity at the heteroallelic PHO3/pho3
locus (2). However, when shochu production tests were conducted
using a series of newly isolated yeast strains carrying different
genotypes of the pho3 locus, there was little correlation between the
genotype (PHO3/pho3 vs. pho3/pho3) and final alcohol content and/
or nihonshudo (2). Therefore, we inferred that BAW-6 probably
accumulates mutation(s) other than that at pho3 locus, leading to
superior fermentation properties. In this study, we showed that
BAW-6 competitively outgrows Ko in barley koji medium. In
addition, the BAW-6 strain exhibited higher tolerance than Ko to
cerulenin in SD medium and to diethylstilbestrol in YPD medium
(Table 2, Fig. 2). It is intriguing that a mutant with such phenotypes
spontaneously occurred in barley shochu mash. In support of the
diethylstilbestrol tolerance of BAW-6, this strain exhibited enhanced
plasma membrane Hþ-ATPase activity compared with the parental
Ko strain (Fig. 4).
Other workers have reported that a trichothecin-tolerant
mutant showed higher ethanol productivity and stronger tolerance
to organic acids (7,8). The trichothecin-tolerant mutant was resis-
tant to cycloheximide, cerulenin in addition to trichothecin,
but was susceptible to miconazole. Other workers have isolated
clotrimazole-tolerant mutant strains from sake yeast that exhibited
higher fermentation activity (5,6). As shown in Table 2, BAW-6 was
tolerant to cerulenin but not to clotrimazole. Thus, BAW-6 may be
similar to the trichothecin-tolerant mutant. In future studies, we
would like to investigate the mechanism(s) responsible for the
cerulenin tolerance in the BAW-6 strain derived from barley and
barley koji, which exhibits the higher ethanol yield and the resis-
tance to anti-bacterial compounds.
COMPETITIVE ADVANTAGE AND TOLERANCE OF SHOCHU YEAST 83
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