Download Analysis of the Role of Mitochondria of Sake in Fermentation Technologies

AGri-Bioscience Monographs, Vol. 3, No. 1, pp. 1–12 (2013)
www.terrapub.co.jp/onlinemonographs/agbm/
Analysis of the Role of Mitochondria of Sake
Yeast during Sake Brewing and Its Applications
in Fermentation Technologies
Hiroshi Kitagaki1,2*, Haidong Tan3 and Lahiru Niroshan Jayakody1,2
1
Department of Environmental Sciences, Faculty of Agriculture
Saga University
1 Honjo-cho, Saga 840-8502, Japan
2
Department of Biochemistry and Applied Biosciences
United Graduate School of Agricultural Sciences
Kagoshima University
Korimoto 1-21-24, Kagoshima 890-8580, Japan
3
Biotechnology Department
Dalian Institute of Chemical Physics
CAS. Dalian 116023, China
*e-mail: [email protected]
Abstract
Mitochondrion is an organelle necessary for oxidative respiration. During industrial fermentation, brewery yeasts are exposed to long periods of hypoxia; however, the structure, role, and metabolism of mitochondria of brewery yeast during hypoxia have not
been studied in detail. Our recent studies, for the first time, elucidated that the mitochondrial structure of brewery yeast can be observed throughout the brewing of sake, the
Japanese traditional rice wine. As sake brewing proceeded, mitochondria of sake yeast
change their morphology, which is coupled with an increase in malate production. On the
basis of these insights, new fermentation technologies were developed. They include (1)
breeding of low pyruvate-producing sake yeast by isolation of a mutant resistant to an
inhibitor of mitochondrial pyruvate transport; and (2) modification of malate and succinate production by manipulating mitochondrial activity. These approaches provide new
and practical methods to improve industrial fermentation technologies of sake brewing.
1. Sake brewing process
Sake is the Japanese traditional rice wine going back
more than 1300 years. First, polished and steamed rice
is inoculated with mold Aspergillus oryzae. The fermented rice is called “koji”, and used as a source of
glycolytic, proteolytic and lypolytic enzymes. Koji is
pitched into the mash tank together with steamed rice
and yeast to form moto, the starter culture of yeast. In
a traditional brewing process, lactic acid bacteria are
allowed to proliferate to decrease pH of moto. In a
modern brewing process from the early 1900s, lactic
acid is added to moto prior to yeast addition, which is
not accompanied by proliferation of lactic acid bacteria. The formed moto, koji and rice are pitched into the
mash tank in a three-step amplification process to form
moromi. Moromi is allowed to cause simultaneous sac-
© 2013 TERRAPUB, Tokyo. All rights reserved.
doi:10.5047/agbm.2013.00301.0001
Received on
May 26, 2012
Accepted on
July 22, 2013
Online published on
November 12, 2013
Keywords
• mitochondrial morphology
• pyruvate
• breeding
• alcoholic fermentation
• fermentation ability
charification of starch by enzymes produced by A.
oryzae and ethanol fermentation by sake yeast in the
mash tank.
2. Sake yeast : A facultative anaerobe
Since sake yeast belongs to Saccharomyces
cerevisiae (Akao et al. 2011), it is a facultative anaerobe, just as S. cerevisiae and can grow both with and
without molecular oxygen. During respiration, namely,
in the presence of oxygen and in the absence of
fermentable sugars, sake yeast uses mitochondria to
obtain energy through oxidative phosphorylation. In
contrast, during fermentation, namely, in the absence
of molecular oxygen and in the presence of fermentable
sugars, sake yeast mainly obtains energy by substratelevel phosphorylation. This is accomplished by re-
2
H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013
pressed gene expression under high concentrations of
fermentable sugars (Gascón and Lampen 1968) and in
the absence of molecular oxygen (Plattner et al. 1970).
Since S. cerevisiae is Crabtree-effect positive, it produces ethanol even in the presence of oxygen and glucose rather than the tricarboxylic acid (TCA) cyclerelated constituents (González Siso et al. 2012).
verse number of hypoxic genes and prevents their expression in aerobic conditions together with other components, such as Mot3 and Ssn6/Tup1 (Kwast et al.
2002). Oxygen availability also affects SO 2 synthesis
through cardiolipin synthesis in the mitochondria
(Samp 2012). These molecules are considered to have
important roles under alcoholic fermentation.
3. Hypoxia and yeast mitochondria
5. Existence of molecular oxygen affects directions of biochemical reactions
As described above, genes involved in oxidative respiration are repressed in the presence of fermentable
sugars and in the absence of oxygen. Similar to this
regulation, several genes are known to be induced in
response to hypoxia. In contrast to upregulation of the
mitochondrial genes in response to molecular oxygen
and low concentrations of glucose, under hypoxia,
genes such as TIR1, ECM22, MGA2, COX5B, MET16,
MET3, MET1, MET5, and HEM13 are induced through
transcription factors such as UPC2, ROX1, TPA1,
SUT1, SUT2, and IXR1 (González Siso et al. 2012). In
some fungi, nitrate and nitrite are used as electron acceptors to produce mitochondrial electron potential,
which leads to ATP synthesis through complex V
(Takaya 2009). In Saccharomyces cerevisiae, cytochrome oxidase has been reported to reduce nitrite,
which produces nitric oxide (NO) and mitochondrial
electron potential (Castello et al. 2008), although the
homolog of the nitrate reductase gene is not contained
in the genome of S. cerevisiae. The role of these reactions during industrial fermentation is of significant
interest for the development of fermentation technologies.
4. Effect of oxygen availability on yeast cell constituents
The existence of oxygen has great impact on not only
the physiology of yeast cells but also the constituents
of yeast cells. This is because molecular oxygen is required for the synthesis of many biological molecules
such as unsaturated fatty acids, ergosterol, and heme
in yeast (Ernst and Tielker 2009). Heme regulates the
transcription of genes involved in oxidative growth
through transcriptional regulator Hap1 together with
the CCAAT-binding complex Hap2/3/4/5. The regulation of these transcription factors is accomplished by
the transcriptional regulation of HEM13, which encodes the enzyme coproporphyrinogen III oxidase, a
rate-limiting enzyme in heme synthesis. Furthermore,
yeast dynamically alters their cell wall proteins according to oxygen availability. Specifically, yeast expresses
a cell wall protein Tir1 only under hypoxic conditions
(Kitagaki et al. 1997), whereas Rox1 represses the
hypoxic expression of TIR1. Rox1 is a specific DNAbinding protein that recognizes the promoter of a di-
Molecular oxygen completely changes the direction
of reactions that occur in living cells. Many reactions
involving oxidation and reduction occur in the cells,
and these reactions use NADH/NAD + , NADPH/
NADP+, and FADH2/FAD as cofactors. However, since
biochemical reactions are driven by their Gibbs free
energy, the direction of the biochemical reactions is
determined by the concentrations of the substrates and
products. Gibbs energy is given by the constant below;
aA + bB ⇔ cC + dD
∆G = ∆G0 + RTln([C]c∗[D]d)/([A]a∗[B] b)
∆G0 stands for the standard Gibbs energy and a, b, c
and d stand for the moles of A, B, C, and D, respectively, in the equilibrium.
In an environment with oxygen, where oxidative respiration occurs in the electron transport chain of the
mitochondria, NADH, NADPH, and FADH2 are oxidized to NAD+, NADP+, and FAD, respectively, and
NADH/NAD+, NADPH/NADP +, and FADH2/FAD ratios decrease. Therefore, in the presence of oxygen,
biochemical reactions that use NADH, NADPH, and
FADH2 as cofactors rarely occur, and those that use
NAD+, NADP+, and FAD as a cofactor occur predominantly. In contrast, in the absence of oxygen, because
NADH, NADPH, and FADH 2 are not oxidized to
NAD+, NADP+, and FAD, respectively, NADH/NAD+,
NADPH/NADP +, and FADH 2/FAD ratios increase.
Biochemical reactions that use NAD+, NADP+, and
FAD as cofactors rarely occur unless they are coupled
with biochemical reactions with minus Gibbs free energy. In contrast, reactions which use NADH, NADPH,
and FADH2 as cofactors occur predominantly. Therefore, the presence of molecular oxygen determines the
direction of biochemical reactions by changing the
cofactor balance.
6. Reactive oxygen species
Reactive oxygen species (ROS) are molecules derived from molecular oxygen gas. Since these molecules are radicals and only have one electron in the
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H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013
DIC
DIC
GFP
Cells
incubated
in synthetic
medium
3
GFP
9 days
ethanol
8.8%
3 days
ethanol
0.3%
10 days
ethanol
10.1%
4 days
ethanol
1.3%
12 days
ethanol
12.5%
7 days
ethanol
6.1%
14 days
ethanol
14.6%
Fig. 1. Mitochondrial structures of yeast during sake brewing. Sake was brewed by mixing 60 g dried pre-gelatinized rice, 23
g dried pre-gelatinized koji, 200 mL water, 45 µL 90% lactic acid, and 200 OD600 units of laboratory yeast with visualized
mitochondria and incubated at 15°C for 14 d. Sake mash was sampled at different time points and observed under a fluorescent microscope. GFP indicates fluorescence the image of GFP obtained by fluorescence microscopy and DIC indicates the
image of differential interference contrast microscopy. Reprinted from J. Biosci. Bioeng., 104, Kitagaki and Shimoi, Mitochondrial dynamics of yeast during sake brewing, 227–230,  2007, with permission from Elsevier.
molecular orbital, they can easily remove one electron
from other molecules. Most intracellular ROS are derived from superoxide anion (O 2–•), which is generated by one electron reduction of O2 (Cadenas et al.
1977). Superoxide anion is converted into hydrogen
peroxide by superoxide dismutases (SODs) (Boveris
et al. 1972). Eight sites in the mitochondria are involved in the generation of superoxide anion (Boveris
et al. 1972). Within these sites, site IIIQo on complex
III and glycerol-3-phosphate dehydrogenase can release superoxide anion into the intermembrane space
of mitochondria. Since most molecules can pass the
outer membrane of mitochondria, superoxide anion is
considered to diffuse into the cytosol and cause various radical reactions. During hypoxia, reactive nitrogen species are generated instead of ROS (Plattner et
al. 1970).
7. Roles of yeast mitochondria during alcoholic
fermentation
Most industrial alcoholic fermentations are performed in an environment with a limited concentration of molecular oxygen. For example, during sake
brewing, after the initial stage, the concentration of
oxygen decreases to lower than 5 ppb (Nagai et al.
1992). Therefore, during the fermentation, yeast cells
are exposed to long and extreme anaerobiosis. Moreover, since sugars as the source of carbon biomass are
converted into ethanol, sugars are present at high concentrations throughout the fermentation. In addition,
many researchers have described that genes encoding
mitochondrial proteins are subject to glucose repression (Trumbly 1992) and oxygen upregulation (Ter
Linde and Steensma 2002). These regulations only
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H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013
4
(A)
Sake
yeast
wild type
(B)
Sake yeast
fis1 disruptant
(C)
1 day
6 days
17 days
Fig. 2. Inhibition of mitochondrial fragmentation during sake brewing causes high malate production in sake yeast. (A), (B)
Mitochondrial structures of yeasts during sake brewing. Sake brewing was performed as described in Fig. 1 except that (A)
wild-type sake yeast with visualized mitochondria and (B) fis1 ∆ sake yeast with visualized mitochondria were used. (C)
Organic acid profile of sake brewed with the strains. Dotted bars represent the contents of organic acids in wild type sake
yeast and hashed bars represent those in three independent transformants of sake yeast fis1 disruptant. Reprinted from J.
Biosci. Bioeng., 105, Kitagaki et al., Inhibition of mitochondrial fragmentation during sake brewing causes high malate
production in sake yeast, 675–678,  2008, with permission from Elsevier.
occur when mitochondria are functional, irrespective
of retrograde response (Kitagaki et al. 2009). Therefore, under the condition of low oxygen concentration
and high glucose concentration, yeast cells neither respire and nor develop mitochondria.
Considering the above regulations, many studies
have documented that the mitochondria do not have a
significant role in alcoholic fermentation (O’ConnorCox et al. 1996). For example, some researchers reported that rhodamine-stained mitochondria are not
observed during alcoholic fermentation, and thus proposing that mitochondrial structures are not present
(Lloyd et al. 1996). Based on experiments involving
the addition of chloramphenicol, an inhibitor of mitochondrial protein synthesis, it appears that protein synthesis in the mitochondria does not play a major role
in alcoholic fermentation (Lodolo et al. 1995). Therefore, research has focused on non-respiratory oxygen
consumption pathways, which mainly consist of the
sterol synthesis in yeast (Rosenfeld et al. 2003).
However, there are several studies, which imply that
the mitochondria play a role during fermentation. For
example, it has been reported that bongkrekic acid, an
inhibitor of the ATP-ADP translocation system of the
mitochondrial inner membrane, protracts fermentation
(O’Connor-Cox et al. 1993). In addition, azide, an inhibitor of cytochrome c oxidase, inhibits fermentation
performance (O’Connor-Cox et al. 1993; Lodolo et al.
1999). It was also shown that respiratory-deficient
mutants of wine yeasts achieve about 10% and 18%
improvement in their glucose-to-ethanol conversion
efficiency compared to their respective parent strains
(Ooi and Lankford 2009). Together, these results suggest the potential role of yeast mitochondria during
alcoholic fermentation.
8. Mitochondrial structure of sake yeast during
sake brewing
Despite the potential significance of mitochondria
during alcoholic fermentation, mitochondrial structures
of brewery yeasts during alcoholic fermentation have
doi:10.5047/agbm.2013.00301.0001 © 2013 TERRAPUB, Tokyo. All rights reserved.
H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013
not been elucidated. To investigate the structure of
yeast mitochondria during alcohol brewing,
mitochondrially targeted green fluorescent protein
(mito-GFP), which enables observation of the dynamic
structure of mitochondria, was introduced into the sake
yeast. Mito-GFP contains the first 69 amino acids of
subunit 9 of the F 0 ATPase of Neurospora crassa
matrix-targeting sequence and GFP under the
triosephosphate isomerase promoter and has been confirmed to localize to the mitochondria (Okamoto and
Shaw 2005). Using this method, yeast mitochondrial
structures can be monitored throughout the sake brewing process in a real-time manner (Fig. 1) (Kitagaki
and Shimoi 2007). As a result, it turned out that in the
early stage of sake brewing, yeast mitochondria are
filamentous, forming long tubules. As brewing proceeds, the tubules start to fragment and are converted
into dotted structures in the later phase of sake brewing.
9. Mitochondrial morphology and organic acid
production profile
Mitochondrial morphology of yeast is regulated by
several mitochondrial proteins. Among these proteins,
Dnm1, a dynamin-related GTPase, was first identified
as an essential regulator of mitochondrial fission in
yeast (Bleazard et al. 1999). Deletion in DNM1 results
in a networked structure of mitochondria. The same
mitochondrial morphology is caused by deletion of
FIS1 (McNiven et al. 2000), a 17-kDa integral protein
localized at the outer mitochondrial membrane. Fis1contains a single transmembrane domain with a
protein-protein interaction domain facing the cytosol,
which is responsible for mitochondrial fragmentation
during vegetative growth. Kitagaki and his colleagues
(Kitagaki et al. 2008) investigated whether mitochondrial morphology affects metabolism during sake brewing by disrupting the FIS1 gene (K7 haploid fis1 ::
natMX4) of sake yeast. Furthermore, a sake yeast mutant deleted in FIS1 gene with visualized mitochondria was also generated. Sake was brewed using the
mutant strain, and its mitochondrial morphology was
investigated. Mitochondria remained networked
throughout brewing, indicating that Fis1 regulates mitochondrial morphology during sake brewing (Figs. 2A,
B). Moreover, the content of malic acid increased in
fis1 relative to the wild-type strain (Fig. 2C). These
results indicate that structural change of brewery yeast
mitochondria plays a role in the metabolism of organic
acids during alcoholic fermentation and that inhibition
of mitochondrial fragmentation during alcoholic brewing leads to higher production of malate. Malate is
found in apples and is the compound responsible for
the “tartness” of sour apple flavoring. Thus, increasing the malate content during alcohol fermentation is
favorable and this strategy provides a novel approach
5
Thiamine
pyrophosphate
ylide
Pyruvate
Thiamine
pyrophosphate
pyruvate
HydroxyethylThiamine
pyrophosphate
carboanion
Pyruvate
Acetolactate
Fig. 3. Biosynthesis pathway of α -acetolactate from pyruvate. Reprinted with permission from Abstracts of the 63th
Meeting of the Society for Biotechnology, Japan, 63,
Kitagaki, Mitochondrial transport-targeted breeding of a low
pyruvate sake yeast, 102, Fig. 3,  2011, the Society for
Biotechnology, Japan.
for breeding brewery yeast (Kitagaki et al. 2008;
Kitagaki and Kitamoto 2013).
As described below, Kitagaki and his colleagues have
also found that a decrease in mitochondrial activity
increases malate productivity in sake yeast (Motomura
et al. 2012). Therefore, it can be hypothesized that fis1
mutation causes a decrease in mitochondrial activity,
and subsequently increases malate concentration.
10. Breeding of a low pyruvate-producing sake
yeast strain by targeting mitochondrial transport
Sake contains the highest concentration of ethanol
among the brewed alcohol beverages in the world.
Recently, the consumption of sake in Japan has decreased, probably because of the too high concentration of ethanol contained in sake. Due to the recent
consumer’s demand of low-ethanol beverages in Japan, lowering the content of ethanol of sake is desired.
However, when low-ethanol sake is manufactured by
filtering the mash at a low concentration of ethanol,
an off-flavor, diacetyl is formed.
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H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013
6
Starch
H
Decarboxylation
Acetaldehyde
Glucose
Glycolysis
Pyruvate
Glucose
Leak acetolactate
Oxidative
decarboxylation
Mitochondria
Diacetyl
(Off-flavor)
Sake yeast
Fig. 4. Breeding strategy of low pyruvate-producing sake yeast. Pyruvate formed by glycolysis is converted to acetaldehyde
by decarboxylation and to α-acetolactate which leaks through the cytoplasmic membrane and is further converted to diacetyl
by oxidative decarboxylation. Mutants resistant to ethyl α -transcyanocinnamate, an inhibitor of mitochondrial pyruvate transporter, are expected to fortify pyruvate transport to decrease these off-flavor substances.
Diacetyl is a butter-like critical off-flavor (Inoue et
al. 1968), which has a very low detection threshold
(0.15 mg/l), contained in beverages. Diacetyl is formed
from α-acetolactate by non-enzymatic oxidative decarboxylation during storage of alcoholic beverages. αAcetolactate is synthesized from pyruvate by the pathway as follows; C2 carbon of pyruvate is attacked by
thiamine pyrophosphate ylid, carboxylic carbon is
decarboxylated to form hydroxyethyl-thiamine pyrophosphate carboanion, and hydroxyethyl-thiamine pyrophosphate carboanion attacking the C2 carbon of
pyruvate to form α -acetolactate (Fig. 3). α Acetolactate is used for the biosynthesis of leucine and
valine (Lewis and Weinhouse 1958). During sake brewing, the concentration of α-acetolactate correlates well
with that of pyruvate (Sato et al. 1981). Pyruvate is
converted to acetaldehyde (Dohi et al. 1974) and acetate (Akamatsu et al. 2000; Goto-Yamamoto and Dang
2006), which are known to impart unpleasant flavors,
during manufacturing of sake. Keeping low pyruvate
levels has been thought to be critical for reduction of
these off-flavors during sake brewing and, therefore,
the concentration of pyruvate during sake brewing has
generally been utilized as an index to determine the
timing of the process of filtering the mash in the sake
brewing industry. Therefore, researchers have tried to
address this issue by breeding sake yeast mutants with
low pyruvate productivity. For example, a mutant re-
sistant to 2-deoxyglucose was isolated as a pyruvateunderproducing sake yeast. A sake yeast mutant which
overexpresses the gene encoding Jen1 (Tsuboi et al.
2003), which imports pyruvate across the plasma membrane (Akita et al. 2000), was constructed. A sake yeast
mutant resistant to a pyruvate analog (Fukuda et al.
1998) was isolated as a pyruvate-underproducing sake
yeast. However, there was no practical sake yeast which
underproduces pyruvate because of its deterioration in
fermentation ability.
Based on our findings of the mitochondrial structure
of sake yeast during sake brewing as described above,
it is possible to conduct the breeding strategy to decrease pyruvate concentration by absorbing pyruvate
into the mitochondria, or enhancing the turnover of
pyruvate in mitochondria during alcoholic fermentation. Although genes encoding pyruvate transporters
have recently been identified (Bricker et al. 2012), utilization of genetically modified organisms for beverages is still open for discussion. Therefore, we attempted to obtain a mutant which has an increased
mitochondrial pyruvate transport by natural occurrence
or mutagenesis. It has been hypothesized that mutants
resistant to an inhibitor of mitochondrial pyruvate
transport should have a fortified pyruvate transport into
mitochondria or alter pyruvate metabolism (Fig. 4).
Kitagaki and his colleagues isolated mutants resistant to ethyl α-transcyanocinnamate, an inhibitor of mi-
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H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013
(A)
(B)
0.8
Brewing in a mash tank
7
Brewing in a bottle
**
0.7
0.6
**
**
**
0.5
0.4
0.3
Time (d)
(C)
Fig. 5. Pyruvate levels of sake brewed with strains resistant to ethyl α -transcyanocinnamate on laboratory, pilot and factory
scales. (A) Laboratory scale test. Sake brewing was performed as described in Fig. 1. The results are mean values, and bars
represent standard errors for six independent brewing experiments from respective precultures. Open circles represent pyruvate levels for Kyokai No. 7, and closed squares represent those for TCR7 (n = 6; **p < 0.005, unpaired one-tailed Student’s
t-test). Reprinted with permission from Biosci. Biotech. Biochem. 74(4), Horie et al., Breeding of a low pyruvate-producing
sake yeast by isolation of a mutant resistant to ethyl alpha-transcyanocinnamate, an inhibitor of mitochondrial pyruvate
transport, 843–847, Fig. 2a,  2010, Japan Society for Bioscience, Biotechnology, and Agrochemistry. (B) Pilot scale test.
Sake was brewed on a pilot scale (total 12 kg rice). Diamonds represent pyruvate levels of Kyokai No. 7 and rectangles
represent those of TCR7. (C) Factory scale test. Sake was brewed on a factory scale (total 1 ton rice). F-4 is a sake yeast
appropriate for brewing of ginjo-type sake. F-4 TCR is a mutant of F-4 resistant to ethyl α-transcyanocinnamate. Diamonds
represent pyruvate levels of F-4 and rectangles represent those of F-4 TCR. Reprinted with permission from Journal of
Brewing Society of Japan, 106(5), Hirata et al., Brewing characteristics of sake yeast resistant to an inhibitor of mitochondrial
pyruvate transport on a pilot scale, 323–331, Fig. 2,  2011, Brewing Society of Japan.
tochondrial pyruvate transport (Paradies and Ruggiero
1988), and quantified pyruvate concentrations in sake
brewed with the mutants (Horie et al. 2010). Some of
the sake mash brewed with the resistant mutants contained lower concentration of pyruvate relative to that
of the parent strain Kyokai No. 7 (data not shown). To
elucidate the mechanism more precisely, sake brewing profiles of a mutant, designated TCR7, were investigated.
The mutant, TCR7, had a fermentation ability similar to the wild type sake yeast (statistically not signifi-
cant). Sake brewed with TCR7 contained a significantly
lower concentration of pyruvate (30% lower than the
parent strain). However, the other organic acids were
not significantly altered, indicating that sake brewing
with TCR7 decreased pyruvate levels without significantly altering the taste of sake relative to the parent
strain. TCR7 also produced lower levels of pyruvate
during sake brewing on a pilot-scale (total 12 kg rice)
(Fig. 5B) (Hirata et al. 2011). Furthermore, a sake yeast
mutant resistant to ethyl α-transcyanocinnamate was
obtained from a sake yeast F-4, which is a progeny of
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H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013
8
(A) Fermentative sake yeast
GFP
DIC
(B) Respirative sake yeast
DIC
GFP
Fig. 6. Mitochondrial morphology of sake yeast in respirative and fermentative conditions. (A) Morphology of mitochondria
of fermentative sake yeast. Sake yeast with visualized mitochondria was cultured anaerobically in a synthetic medium containing 10% glucose as a sole carbon source. (B) Morphology of mitochondria of respirative sake yeast. Sake yeast with
visualized mitochondria was cultured aerobically in a synthetic medium containing 3% glycerol as a sole carbon source. Yeast
mitochondria were observed under a fluorescent microscope. GFP indicates the fluorescence image of GFP obtained by fluorescence microscopy and DIC indicates the image of differential interference contrast microscopy. Reprinted with permission
of John Wiley & Sons, Inc. from Journal of the Institute of Brewing, 118(1), Motomura et al., Mitochondrial activity of sake
brewery yeast affects malic and succinic acid production during alcoholic fermentation, 22–26, Fig. 1,  2012, Wiley-Liss,
Inc., a Wiley Company.
Sake brewing characteristics of ethyl α tanscyanocinnamate-resistant mutant and its parent sake
yeast strain.
Table 1.
Ethanol (v/v%) (23 days)
α -Acetolactate (mg/l) (9 days)
F-4
F-4 TCR
17.70
0.28
17.93
0.05
F-4 is a sake yeast appropriate for brewing of ginjo-type
sake. F-4 TCR is a mutant resistant to ethyl α transcyanocinnamate.
Kyokai No. 9, a strain appropriate for brewing of ginjo
sake, and was designated F-4 TCR. F-4 TCR produced
low levels of pyruvate on a factory-scale (total 1 ton
rice) (Fig. 5C) without affecting the fermentation ability (Sasaki et al. 2011) relative to its parent strain (F4). Indeed, α -acetolactate, the direct precursor of
diacetyl, produced by F-4 TCR was decreased to less
than 20% relative to the parent strain on a factory scale
without affecting final ethanol concentration (Table 1)
(Sasaki et al. 2011). Therefore, it was concluded that
this approach is a practical method for breeding brewery yeast strains.
11. Control of yeast mitochondrial activity affects
malic and succinic acid production
Sake yeasts are exposed to various conditions affecting the mitochondrial state in the sake industry. For
example, the size of the fermentation tank affects the
surface area of the mash, leading to different oxygen
availability. Further, temperature of the raw materials,
such as water and rice, affects oxygen solubility for
sake yeast. Furthermore, availability of oxygen is high
for yeast that localizes to the surface of the mash, while
it is low for yeast cells near the bottom of the tank.
However, the involvement of the mitochondrial states
of sake yeast in the production profile of organic acids
has not been investigated in detail.
Therefore, we investigated the effect of the mitochondrial activity of sake yeast on organic acid production
profile during sake brewing. Two propagation conditions were designed: one is “respirative condition”
where yeast cells were cultured in a non-fermentable
carbon source (3% w/v glycerol) with aerobic shaking
of the flask, and the other is “fermentative condition”
where yeast cells were cultured in a glucosecontaining medium (10% w/v glucose) in a static culture.
First, mitochondrial morphology of the sake yeast
in these conditions were investigated. Exposure to the
fermentative condition caused the mitochondria to exhibit long tubule structures, with little branches elongating along the surface of the cell (Fig. 6A). In contrast, exposure to the respirative conditions caused the
mitochondria to be distributed all over the cell with
short filamentous and branched structures or clumpy
structures (Fig. 6B). To examine the influence of the
mitochondrial activity on organic acid production, the
organic acid profiles of these yeasts were investigated
after alcoholic fermentation for 48 h. Respirative sake
yeast produced significantly increased citric acid
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H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013
(A)
(B)
**
450
9
600
400
500
300
250
*
Fermentation
Respiration
200
Concentration (mg/l)
Concentration (mg/l)
350
400
Respiration-FCCP
300
Respiration+FCCP
**
200
150
100
50
100
*
0
Citric acid
0
Citric acid
Pyruvic acid
Malic acid
Succinic acid Lactic acid
Pyruvic acid
Malic acid
Succinic acid
Lactic acid
Acetic acid
Acetic acid
(c)
600
*
500
Concentration (mg/l)
400
Respiration
300
Transition to fermentation (12 h)
Transition to fermentation (24 h)
200
*
*
100
0
Citric acid
Pyruvic acid
Malic acid
Succinic acid
Lactic acid
Acetic acid
Fig. 7. Organic acid production profiles of sake yeast in respirative and fermentative conditions. (A) Organic acid profiles of
respirative and fermentative sake yeasts. Statistical significance of difference was evaluated by unpaired one-tailed Student’s
t-test (*p < 0.05, **p < 0.01, n = 3). Reprinted with permission of John Wiley & Sons, Inc. from Journal of the Institute of
Brewing, 118(1), Motomura et al., Mitochondrial activity of sake brewery yeast affects malic and succinic acid production
during alcoholic fermentation, 22–26, Fig. 2,  2012, Wiley-Liss, Inc., a Wiley Company. (B) Organic acid profiles of respirative
sake yeasts in the presence of mitochondrial potential uncoupler, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP).
Statistical significance of difference was evaluated by unpaired one-tailed Student’s t-test (**p < 0.01, n = 3). Reprinted with
permission of John Wiley & Sons, Inc. from Journal of the Institute of Brewing, 118(1), Motomura et al., Mitochondrial
activity of sake brewery yeast affects malic and succinic acid production during alcoholic fermentation, 22–26, Fig. 3, 
2012, Wiley-Liss, Inc., a Wiley Company. (C) Changes in organic acid profiles of sake yeasts upon transfer from respirative
to fermentative conditions. Organic acids are measured after additional incubation for 12 and 24 h after a change in culture
condition. Statistical significance of difference was evaluated by Dunnet’s multiple comparison test (*p < 0.05, n = 8). Reprinted with permission of John Wiley & Sons, Inc. from Journal of the Institute of Brewing, 118(1), Motomura et al., Mitochondrial activity of sake brewery yeast affects malic and succinic acid production during alcoholic fermentation, 22–26, Fig.
4,  2012, Wiley-Liss, Inc., a Wiley Company.
(123%), decreased pyruvic acid (63.0%), decreased
malic acid (74.6%), increased succinic acid (180%) and
increased acetic acid (129%) during the alcoholic fermentation phase, relative to that of fermentative yeast
(Fig. 7A).
The role of mitochondrial state of yeast cells on these
changes were investigated with the compound
carbonylcyanide p-trifluoromethoxyphenylhydrazone,
a specific uncoupler of mitochondrial membrane potential.
When
carbonylcyanide
ptrifluoromethoxyphenylhydrazone was added to the
culture, the production of malic acid significantly in-
creased (374%) and succinic acid decreased (87.9%,
although not statistically significant (p < 0.05)) (Fig.
7B). Notably, these changes were almost similar to
those observed upon the transfer from the respirative
to fermentative condition. These results suggest that
the mitochondrial state during the propagation stage is
responsible for the difference in the organic acid production between the respirative and fermentative yeast.
To develop a technology to alter the organic acid
profile by regulating mitochondrial activity, respirative
yeast cells were exposed to a medium containing 10%
(w/v) of glucose, incubated as a static culture, and then
doi:10.5047/agbm.2013.00301.0001 © 2013 TERRAPUB, Tokyo. All rights reserved.
H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013
10
(B) Respirative sake yeast
(A) Fermentative sake yeast
(C)
(D)
gluconeogenesis
glucose
Oxaloacetic acid
glucose
Lactic acid
Pyruvic acid
Pyruvic acid
mitochondria
Oxaloacetic acid
mitochondria
Malic acid
Acetyl-CoA
Oxaloacetic acid
Acetyl-CoA
Oxaloacetic acid
Citric acid
Citric acid
Malic acid
cis-aconitic acid
Fumaric acid
Acetic
acid
Malic acid
cis-aconitic acid
Fumaric acid
Succinic acid
Isocitric acid
Succinyl-CoA
Oxalosuccinic acid
Succinic acid
Isocitric acid
Oxalosuccinic acid
Succinyl-CoA
2-oxoglutaric acid
2-oxoglutaric acid
Fig. 8. A schematic diagram of the production pathways of organic acids by fermentative and respirative yeasts. (A) Morphology of mitochondria of fermentative sake yeast. Sake yeast with visualized mitochondria was cultured anaerobically in a
synthetic medium containing 10% (w/v) of glucose as a sole carbon source. (B) Morphology of mitochondria of respirative
sake yeast. Sake yeast with visualized mitochondria was cultured aerobically in a synthetic medium containing 3% glycerol
as a sole carbon source. (C) Pathway of organic acid production by fermentative sake yeast. Major pool of pyruvate in the
cytosol is not transported to mitochondria, remains in the cytosol and reduced to lactate or carboxylated to oxaloacetate and
reduced to malate driven by accumulated NADH. (D) Pathway of organic acid production by respirative sake yeast. Major
pool of pyruvate in the cytosol is transported to mitochondria, cleaved aerobically to acetyl-CoA, enters into TCA cycle and
converted to succinic acid. Reprinted with permission of John Wiley & Sons, Inc. from Journal of the Institute of Brewing,
118(1), Motomura et al., Mitochondrial activity of sake brewery yeast affects malic and succinic acid production during
alcoholic fermentation, 22–26, Fig. 5,  2012, Wiley-Liss, Inc., a Wiley Company.
used for alcoholic fermentation. When yeast cells were
exposed to these conditions, where mitochondrial activity decreased, for 12 h and 24 h, production of malic
acid increased significantly (Fig. 7C), while the production of succinic acid decreased significantly compared to the untreated cells (24 h cultivation). These
results indicated that the physiological state of sake
yeast can be converted from the respirative condition
to the fermentative condition by incubating yeast cells
under static conditions for 12 h or 24 h in a medium
containing 10% glucose. Taken together, these results
allow us to propose a new scheme of organic acid production in brewery yeast (Fig. 8). In fermentative sake
yeast cells, a major pool of pyruvate in the cytosol is
not transported into the mitochondria, but remains in
the cytosol and reduced to lactate or carboxylated to
oxaloacetate. Oxaloacetate is then reduced to malate,
which is driven by accumulated NADH. In respirative
sake yeast cells, a major pool of pyruvate in the cytosol is transported to mitochondria, where it is converted aerobically to acetyl-CoA, or succinic acid by
aldol-condensation with oxaloacetate.
These results indicate, for the first time, that production profiles of organic acids in brewery yeast can
be manipulated by changing the mitochondrial state of
yeast (Motomura et al. 2012).
12. Concluding remarks
Through these studies, the structures and roles of
yeast mitochondria during sake brewing was first elucidated. These findings led to the establishment of
doi:10.5047/agbm.2013.00301.0001 © 2013 TERRAPUB, Tokyo. All rights reserved.
H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013
novel and practical fermentation technologies such as
high malate production, low pyruvate production and
manipulation of malate and succinate. These results
indicate that metabolisms and events that occur in mitochondria will be promising targets for breeding of
sake yeasts.
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