Analysis of the Role of Mitochondria of Sake Yeast during ... · drial structure of brewery yeast can be observed throughout the brewing of sake, the Japanese traditional rice wine

AGri-Bioscience Monographs, Vol. 3, No. 1, pp. 1–12 (2013) www.terrapub.co.jp/onlinemonographs/agbm/

Received on May 26, 2012Accepted on July 22, 2013Online published on

November 12, 2013

Keywords• mitochondrial morphology• pyruvate• breeding• alcoholic fermentation• fermentation ability

© 2013 TERRAPUB, Tokyo. All rights reserved.doi:10.5047/agbm.2013.00301.0001

AbstractMitochondrion is an organelle necessary for oxidative respiration. During industrial fer-mentation, brewery yeasts are exposed to long periods of hypoxia; however, the struc-ture, role, and metabolism of mitochondria of brewery yeast during hypoxia have notbeen studied in detail. Our recent studies, for the first time, elucidated that the mitochon-drial structure of brewery yeast can be observed throughout the brewing of sake, theJapanese traditional rice wine. As sake brewing proceeded, mitochondria of sake yeastchange their morphology, which is coupled with an increase in malate production. On thebasis 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 aninhibitor of mitochondrial pyruvate transport; and (2) modification of malate and succi-nate production by manipulating mitochondrial activity. These approaches provide newand practical methods to improve industrial fermentation technologies of sake brewing.

Analysis of the Role of Mitochondria of SakeYeast during Sake Brewing and Its Applicationsin Fermentation Technologies

Hiroshi Kitagaki1,2*, Haidong Tan3 and Lahiru Niroshan Jayakody1,2

1Department of Environmental Sciences, Faculty of Agriculture Saga University 1 Honjo-cho, Saga 840-8502, Japan2Department of Biochemistry and Applied Biosciences United Graduate School of Agricultural Sciences Kagoshima University Korimoto 1-21-24, Kagoshima 890-8580, Japan3Biotechnology Department Dalian Institute of Chemical Physics CAS. Dalian 116023, China*e-mail: [email protected]

charification of starch by enzymes produced by A.oryzae and ethanol fermentation by sake yeast in themash tank.

2. Sake yeast : A facultative anaerobe

Since sake yeast belongs to Saccharomycescerevisiae (Akao et al. 2011), it is a facultative anaer-obe, just as S. cerevisiae and can grow both with andwithout molecular oxygen. During respiration, namely,in the presence of oxygen and in the absence offermentable sugars, sake yeast uses mitochondria toobtain energy through oxidative phosphorylation. Incontrast, during fermentation, namely, in the absenceof molecular oxygen and in the presence of fermentablesugars, sake yeast mainly obtains energy by substrate-level phosphorylation. This is accomplished by re-

1. Sake brewing process

Sake is the Japanese traditional rice wine going backmore than 1300 years. First, polished and steamed riceis inoculated with mold Aspergillus oryzae. The fer-mented rice is called “koji”, and used as a source ofglycolytic, proteolytic and lypolytic enzymes. Koji ispitched into the mash tank together with steamed riceand yeast to form moto, the starter culture of yeast. Ina traditional brewing process, lactic acid bacteria areallowed to proliferate to decrease pH of moto. In amodern brewing process from the early 1900s, lacticacid is added to moto prior to yeast addition, which isnot accompanied by proliferation of lactic acid bacte-ria. The formed moto, koji and rice are pitched into themash tank in a three-step amplification process to formmoromi. Moromi is allowed to cause simultaneous sac-

2 H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013

doi:10.5047/agbm.2013.00301.0001 © 2013 TERRAPUB, Tokyo. All rights reserved.

pressed gene expression under high concentrations offermentable sugars (Gascón and Lampen 1968) and inthe absence of molecular oxygen (Plattner et al. 1970).Since S. cerevisiae is Crabtree-effect positive, it pro-duces ethanol even in the presence of oxygen and glu-cose rather than the tricarboxylic acid (TCA) cycle-related constituents (González Siso et al. 2012).

3. Hypoxia and yeast mitochondria

As described above, genes involved in oxidative res-piration are repressed in the presence of fermentablesugars and in the absence of oxygen. Similar to thisregulation, several genes are known to be induced inresponse to hypoxia. In contrast to upregulation of themitochondrial genes in response to molecular oxygenand low concentrations of glucose, under hypoxia,genes such as TIR1, ECM22, MGA2, COX5B, MET16,MET3, MET1, MET5, and HEM13 are induced throughtranscription factors such as UPC2, ROX1, TPA1,SUT1, SUT2, and IXR1 (González Siso et al. 2012). Insome fungi, nitrate and nitrite are used as electron ac-ceptors to produce mitochondrial electron potential,which leads to ATP synthesis through complex V(Takaya 2009). In Saccharomyces cerevisiae, cyto-chrome oxidase has been reported to reduce nitrite,which produces nitric oxide (NO) and mitochondrialelectron potential (Castello et al. 2008), although thehomolog of the nitrate reductase gene is not containedin the genome of S. cerevisiae. The role of these reac-tions during industrial fermentation is of significantinterest for the development of fermentation technolo-gies.

4. Effect of oxygen availability on yeast cell con-stituents

The existence of oxygen has great impact on not onlythe physiology of yeast cells but also the constituentsof yeast cells. This is because molecular oxygen is re-quired for the synthesis of many biological moleculessuch as unsaturated fatty acids, ergosterol, and hemein yeast (Ernst and Tielker 2009). Heme regulates thetranscription of genes involved in oxidative growththrough transcriptional regulator Hap1 together withthe CCAAT-binding complex Hap2/3/4/5. The regula-tion of these transcription factors is accomplished bythe transcriptional regulation of HEM13, which en-codes the enzyme coproporphyrinogen III oxidase, arate-limiting enzyme in heme synthesis. Furthermore,yeast dynamically alters their cell wall proteins accord-ing to oxygen availability. Specifically, yeast expressesa cell wall protein Tir1 only under hypoxic conditions(Kitagaki et al. 1997), whereas Rox1 represses thehypoxic expression of TIR1. Rox1 is a specific DNA-binding protein that recognizes the promoter of a di-

verse number of hypoxic genes and prevents their ex-pression in aerobic conditions together with other com-ponents, such as Mot3 and Ssn6/Tup1 (Kwast et al.2002). Oxygen availability also affects SO2 synthesisthrough cardiolipin synthesis in the mitochondria(Samp 2012). These molecules are considered to haveimportant roles under alcoholic fermentation.

5. Existence of molecular oxygen affects direc-tions of biochemical reactions

Molecular oxygen completely changes the directionof reactions that occur in living cells. Many reactionsinvolving oxidation and reduction occur in the cells,and these reactions use NADH/NAD+, NADPH/NADP+, and FADH2/FAD as cofactors. However, sincebiochemical reactions are driven by their Gibbs freeenergy, the direction of the biochemical reactions isdetermined by the concentrations of the substrates andproducts. Gibbs energy is given by the constant be-low;

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, cand d stand for the moles of A, B, C, and D, respec-tively, in the equilibrium.

In an environment with oxygen, where oxidative res-piration occurs in the electron transport chain of themitochondria, NADH, NADPH, and FADH2 are oxi-dized to NAD+, NADP+, and FAD, respectively, andNADH/NAD+, NADPH/NADP+, and FADH2/FAD ra-tios decrease. Therefore, in the presence of oxygen,biochemical reactions that use NADH, NADPH, andFADH2 as cofactors rarely occur, and those that useNAD+, NADP+, and FAD as a cofactor occur predomi-nantly. In contrast, in the absence of oxygen, becauseNADH, NADPH, and FADH2 are not oxidized toNAD+, NADP+, and FAD, respectively, NADH/NAD+,NADPH/NADP+, and FADH2/FAD ratios increase.Biochemical reactions that use NAD+, NADP+, andFAD as cofactors rarely occur unless they are coupledwith biochemical reactions with minus Gibbs free en-ergy. In contrast, reactions which use NADH, NADPH,and FADH2 as cofactors occur predominantly. There-fore, the presence of molecular oxygen determines thedirection of biochemical reactions by changing thecofactor balance.

6. Reactive oxygen species

Reactive oxygen species (ROS) are molecules de-rived from molecular oxygen gas. Since these mol-ecules are radicals and only have one electron in the

H. Kitagaki et al. / AGri-Biosci. Monogr. 3: 1–12, 2013 3


molecular orbital, they can easily remove one electronfrom other molecules. Most intracellular ROS are de-rived from superoxide anion (O2

–•), which is gener-ated by one electron reduction of O2 (Cadenas et al.1977). Superoxide anion is converted into hydrogenperoxide by superoxide dismutases (SODs) (Boveriset al. 1972). Eight sites in the mitochondria are in-volved in the generation of superoxide anion (Boveriset al. 1972). Within these sites, site IIIQo on complexIII and glycerol-3-phosphate dehydrogenase can re-lease superoxide anion into the intermembrane spaceof mitochondria. Since most molecules can pass theouter membrane of mitochondria, superoxide anion isconsidered to diffuse into the cytosol and cause vari-ous radical reactions. During hypoxia, reactive nitro-gen species are generated instead of ROS (Plattner etal. 1970).

7. Roles of yeast mitochondria during alcoholicfermentation

Most industrial alcoholic fermentations are per-formed in an environment with a limited concentra-tion of molecular oxygen. For example, during sakebrewing, after the initial stage, the concentration ofoxygen decreases to lower than 5 ppb (Nagai et al.1992). Therefore, during the fermentation, yeast cellsare exposed to long and extreme anaerobiosis. More-over, since sugars as the source of carbon biomass areconverted into ethanol, sugars are present at high con-centrations throughout the fermentation. In addition,many researchers have described that genes encodingmitochondrial proteins are subject to glucose repres-sion (Trumbly 1992) and oxygen upregulation (TerLinde and Steensma 2002). These regulations only

Fig. 1. Mitochondrial structures of yeast during sake brewing. Sake was brewed by mixing 60 g dried pre-gelatinized rice, 23g dried pre-gelatinized koji, 200 mL water, 45 µL 90% lactic acid, and 200 OD

600 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 fluores-cent microscope. GFP indicates fluorescence the image of GFP obtained by fluorescence microscopy and DIC indicates theimage of differential interference contrast microscopy. Reprinted from J. Biosci. Bioeng., 104, Kitagaki and Shimoi, Mito-chondrial dynamics of yeast during sake brewing, 227–230, 2007, with permission from Elsevier.

3 daysethanol0.3%

4 daysethanol1.3%

7 daysethanol6.1%

9 daysethanol8.8%

Cells incubated

in synthetic medium

DIC GFP DIC GFP

10 daysethanol10.1%

12 daysethanol12.5%

14 daysethanol14.6%



occur when mitochondria are functional, irrespectiveof retrograde response (Kitagaki et al. 2009). There-fore, under the condition of low oxygen concentrationand high glucose concentration, yeast cells neither re-spire and nor develop mitochondria.

Considering the above regulations, many studieshave documented that the mitochondria do not have asignificant role in alcoholic fermentation (O’Connor-Cox et al. 1996). For example, some researchers re-ported that rhodamine-stained mitochondria are notobserved during alcoholic fermentation, and thus pro-posing that mitochondrial structures are not present(Lloyd et al. 1996). Based on experiments involvingthe addition of chloramphenicol, an inhibitor of mito-chondrial protein synthesis, it appears that protein syn-thesis in the mitochondria does not play a major rolein alcoholic fermentation (Lodolo et al. 1995). There-fore, research has focused on non-respiratory oxygenconsumption pathways, which mainly consist of thesterol synthesis in yeast (Rosenfeld et al. 2003).

However, there are several studies, which imply that

the mitochondria play a role during fermentation. Forexample, it has been reported that bongkrekic acid, aninhibitor of the ATP-ADP translocation system of themitochondrial inner membrane, protracts fermentation(O’Connor-Cox et al. 1993). In addition, azide, an in-hibitor of cytochrome c oxidase, inhibits fermentationperformance (O’Connor-Cox et al. 1993; Lodolo et al.1999). It was also shown that respiratory-deficientmutants of wine yeasts achieve about 10% and 18%improvement in their glucose-to-ethanol conversionefficiency compared to their respective parent strains(Ooi and Lankford 2009). Together, these results sug-gest the potential role of yeast mitochondria duringalcoholic fermentation.

8. Mitochondrial structure of sake yeast duringsake brewing

Despite the potential significance of mitochondriaduring alcoholic fermentation, mitochondrial structuresof brewery yeasts during alcoholic fermentation have

1 day

6 days

(A)

17 days

Sake yeast

wild type

Sake yeastfis1 disruptant

(B)

(C)

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 sakeyeast 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 malateproduction in sake yeast, 675–678, 2008, with permission from Elsevier.



not been elucidated. To investigate the structure ofyeast mitochondria during alcohol brewing,mitochondrially targeted green fluorescent protein(mito-GFP), which enables observation of the dynamicstructure of mitochondria, was introduced into the sakeyeast. Mito-GFP contains the first 69 amino acids ofsubunit 9 of the F0 ATPase of Neurospora crassamatrix-targeting sequence and GFP under thetriosephosphate isomerase promoter and has been con-firmed to localize to the mitochondria (Okamoto andShaw 2005). Using this method, yeast mitochondrialstructures can be monitored throughout the sake brew-ing process in a real-time manner (Fig. 1) (Kitagakiand Shimoi 2007). As a result, it turned out that in theearly stage of sake brewing, yeast mitochondria arefilamentous, forming long tubules. As brewing pro-ceeds, the tubules start to fragment and are convertedinto dotted structures in the later phase of sake brew-ing.

9. Mitochondrial morphology and organic acidproduction profile

Mitochondrial morphology of yeast is regulated byseveral mitochondrial proteins. Among these proteins,Dnm1, a dynamin-related GTPase, was first identifiedas an essential regulator of mitochondrial fission inyeast (Bleazard et al. 1999). Deletion in DNM1 resultsin a networked structure of mitochondria. The samemitochondrial morphology is caused by deletion ofFIS1 (McNiven et al. 2000), a 17-kDa integral proteinlocalized at the outer mitochondrial membrane. Fis1-contains a single transmembrane domain with aprotein-protein interaction domain facing the cytosol,which is responsible for mitochondrial fragmentationduring vegetative growth. Kitagaki and his colleagues(Kitagaki et al. 2008) investigated whether mitochon-drial morphology affects metabolism during sake brew-ing by disrupting the FIS1 gene (K7 haploid fis1 ::natMX4) of sake yeast. Furthermore, a sake yeast mu-tant deleted in FIS1 gene with visualized mitochon-dria was also generated. Sake was brewed using themutant strain, and its mitochondrial morphology wasinvestigated. Mitochondria remained networkedthroughout brewing, indicating that Fis1 regulates mi-tochondrial morphology during sake brewing (Figs. 2A,B). Moreover, the content of malic acid increased infis1 relative to the wild-type strain (Fig. 2C). Theseresults indicate that structural change of brewery yeastmitochondria plays a role in the metabolism of organicacids during alcoholic fermentation and that inhibitionof mitochondrial fragmentation during alcoholic brew-ing leads to higher production of malate. Malate isfound in apples and is the compound responsible forthe “tartness” of sour apple flavoring. Thus, increas-ing the malate content during alcohol fermentation isfavorable and this strategy provides a novel approach

for breeding brewery yeast (Kitagaki et al. 2008;Kitagaki and Kitamoto 2013).

As described below, Kitagaki and his colleagues havealso found that a decrease in mitochondrial activityincreases malate productivity in sake yeast (Motomuraet al. 2012). Therefore, it can be hypothesized that fis1mutation causes a decrease in mitochondrial activity,and subsequently increases malate concentration.

10. Breeding of a low pyruvate-producing sakeyeast strain by targeting mitochondrial trans-port

Sake contains the highest concentration of ethanolamong the brewed alcohol beverages in the world.Recently, the consumption of sake in Japan has de-creased, probably because of the too high concentra-tion of ethanol contained in sake. Due to the recentconsumer’s demand of low-ethanol beverages in Ja-pan, lowering the content of ethanol of sake is desired.However, when low-ethanol sake is manufactured byfiltering the mash at a low concentration of ethanol,an off-flavor, diacetyl is formed.

Pyruvate

Thiamine pyrophosphate

ylide

Thiamine pyrophosphate

pyruvate

Hydroxyethyl-Thiamine

pyrophosphate carboanion

Pyruvate

Acetolactate

Fig. 3. Biosynthesis pathway of α-acetolactate from pyru-vate. Reprinted with permission from Abstracts of the 63thMeeting of the Society for Biotechnology, Japan, 63,Kitagaki, Mitochondrial transport-targeted breeding of a lowpyruvate sake yeast, 102, Fig. 3, 2011, the Society forBiotechnology, Japan.



Diacetyl is a butter-like critical off-flavor (Inoue etal. 1968), which has a very low detection threshold(0.15 mg/l), contained in beverages. Diacetyl is formedfrom α-acetolactate by non-enzymatic oxidative decar-boxylation during storage of alcoholic beverages. α-Acetolactate is synthesized from pyruvate by the path-way as follows; C2 carbon of pyruvate is attacked bythiamine pyrophosphate ylid, carboxylic carbon isdecarboxylated to form hydroxyethyl-thiamine pyro-phosphate carboanion, and hydroxyethyl-thiamine py-rophosphate carboanion attacking the C2 carbon ofpyruvate to form α-acetolactate (Fig. 3) . α-Acetolactate is used for the biosynthesis of leucine andvaline (Lewis and Weinhouse 1958). During sake brew-ing, the concentration of α-acetolactate correlates wellwith that of pyruvate (Sato et al. 1981). Pyruvate isconverted to acetaldehyde (Dohi et al. 1974) and ac-etate (Akamatsu et al. 2000; Goto-Yamamoto and Dang2006), which are known to impart unpleasant flavors,during manufacturing of sake. Keeping low pyruvatelevels has been thought to be critical for reduction ofthese off-flavors during sake brewing and, therefore,the concentration of pyruvate during sake brewing hasgenerally been utilized as an index to determine thetiming of the process of filtering the mash in the sakebrewing industry. Therefore, researchers have tried toaddress this issue by breeding sake yeast mutants withlow pyruvate productivity. For example, a mutant re-

sistant to 2-deoxyglucose was isolated as a pyruvate-underproducing sake yeast. A sake yeast mutant whichoverexpresses the gene encoding Jen1 (Tsuboi et al.2003), which imports pyruvate across the plasma mem-brane (Akita et al. 2000), was constructed. A sake yeastmutant resistant to a pyruvate analog (Fukuda et al.1998) was isolated as a pyruvate-underproducing sakeyeast. However, there was no practical sake yeast whichunderproduces pyruvate because of its deterioration infermentation ability.

Based on our findings of the mitochondrial structureof sake yeast during sake brewing as described above,it is possible to conduct the breeding strategy to de-crease pyruvate concentration by absorbing pyruvateinto the mitochondria, or enhancing the turnover ofpyruvate in mitochondria during alcoholic fermenta-tion. Although genes encoding pyruvate transportershave recently been identified (Bricker et al. 2012), uti-lization of genetically modified organisms for bever-ages is still open for discussion. Therefore, we at-tempted to obtain a mutant which has an increasedmitochondrial pyruvate transport by natural occurrenceor mutagenesis. It has been hypothesized that mutantsresistant to an inhibitor of mitochondrial pyruvatetransport should have a fortified pyruvate transport intomitochondria or alter pyruvate metabolism (Fig. 4).

Kitagaki and his colleagues isolated mutants resist-ant to ethyl α-transcyanocinnamate, an inhibitor of mi-

Fig. 4. Breeding strategy of low pyruvate-producing sake yeast. Pyruvate formed by glycolysis is converted to acetaldehydeby decarboxylation and to α-acetolactate which leaks through the cytoplasmic membrane and is further converted to diacetylby oxidative decarboxylation. Mutants resistant to ethyl α-transcyanocinnamate, an inhibitor of mitochondrial pyruvate trans-porter, are expected to fortify pyruvate transport to decrease these off-flavor substances.

Pyruvate

Diacetyl(Off-flavor)

Starch

Glucose

Sake yeast

Glucose

Glycolysis

Mitochondria

Oxidative decarboxylation

acetolactate

H

Acetaldehyde

Leak

Decarboxylation



0.3

0.4

0.5

0.6

0.7

0.8

Time (d)

**

**

****

(A) Brewing in a mash tank Brewing in a bottle(B)

(C)

Fig. 5. Pyruvate levels of sake brewed with strains resistant to ethyl α-transcyanocinnamate on laboratory, pilot and factoryscales. (A) Laboratory scale test. Sake brewing was performed as described in Fig. 1. The results are mean values, and barsrepresent standard errors for six independent brewing experiments from respective precultures. Open circles represent pyru-vate levels for Kyokai No. 7, and closed squares represent those for TCR7 (n = 6; **p < 0.005, unpaired one-tailed Student’st-test). Reprinted with permission from Biosci. Biotech. Biochem. 74(4), Horie et al., Breeding of a low pyruvate-producingsake yeast by isolation of a mutant resistant to ethyl alpha-transcyanocinnamate, an inhibitor of mitochondrial pyruvatetransport, 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 rectanglesrepresent those of TCR7. (C) Factory scale test. Sake was brewed on a factory scale (total 1 ton rice). F-4 is a sake yeastappropriate for brewing of ginjo-type sake. F-4 TCR is a mutant of F-4 resistant to ethyl α-transcyanocinnamate. Diamondsrepresent pyruvate levels of F-4 and rectangles represent those of F-4 TCR. Reprinted with permission from Journal ofBrewing Society of Japan, 106(5), Hirata et al., Brewing characteristics of sake yeast resistant to an inhibitor of mitochondrialpyruvate transport on a pilot scale, 323–331, Fig. 2, 2011, Brewing Society of Japan.

tochondrial pyruvate transport (Paradies and Ruggiero1988), and quantified pyruvate concentrations in sakebrewed with the mutants (Horie et al. 2010). Some ofthe sake mash brewed with the resistant mutants con-tained lower concentration of pyruvate relative to thatof the parent strain Kyokai No. 7 (data not shown). Toelucidate the mechanism more precisely, sake brew-ing profiles of a mutant, designated TCR7, were in-vestigated.

The mutant, TCR7, had a fermentation ability simi-lar to the wild type sake yeast (statistically not signifi-

cant). Sake brewed with TCR7 contained a significantlylower concentration of pyruvate (30% lower than theparent strain). However, the other organic acids werenot significantly altered, indicating that sake brewingwith TCR7 decreased pyruvate levels without signifi-cantly altering the taste of sake relative to the parentstrain. TCR7 also produced lower levels of pyruvateduring sake brewing on a pilot-scale (total 12 kg rice)(Fig. 5B) (Hirata et al. 2011). Furthermore, a sake yeastmutant resistant to ethyl α-transcyanocinnamate wasobtained from a sake yeast F-4, which is a progeny of



Kyokai No. 9, a strain appropriate for brewing of ginjosake, and was designated F-4 TCR. F-4 TCR producedlow levels of pyruvate on a factory-scale (total 1 tonrice) (Fig. 5C) without affecting the fermentation abil-ity (Sasaki et al. 2011) relative to its parent strain (F-4). Indeed, α-acetolactate, the direct precursor ofdiacetyl, produced by F-4 TCR was decreased to lessthan 20% relative to the parent strain on a factory scalewithout affecting final ethanol concentration (Table 1)(Sasaki et al. 2011). Therefore, it was concluded thatthis approach is a practical method for breeding brew-ery yeast strains.

11. Control of yeast mitochondrial activity affectsmalic and succinic acid production

Sake yeasts are exposed to various conditions affect-ing the mitochondrial state in the sake industry. Forexample, the size of the fermentation tank affects the

surface area of the mash, leading to different oxygenavailability. Further, temperature of the raw materials,such as water and rice, affects oxygen solubility forsake yeast. Furthermore, availability of oxygen is highfor yeast that localizes to the surface of the mash, whileit is low for yeast cells near the bottom of the tank.However, the involvement of the mitochondrial statesof sake yeast in the production profile of organic acidshas not been investigated in detail.

Therefore, we investigated the effect of the mitochon-drial activity of sake yeast on organic acid productionprofile during sake brewing. Two propagation condi-tions were designed: one is “respirative condition”where yeast cells were cultured in a non-fermentablecarbon source (3% w/v glycerol) with aerobic shakingof the flask, and the other is “fermentative condition”where yeast cells were cultured in a glucose-containing medium (10% w/v glucose) in a static cul-ture.

First, mitochondrial morphology of the sake yeastin these conditions were investigated. Exposure to thefermentative condition caused the mitochondria to ex-hibit long tubule structures, with little branches elon-gating along the surface of the cell (Fig. 6A). In con-trast, exposure to the respirative conditions caused themitochondria to be distributed all over the cell withshort filamentous and branched structures or clumpystructures (Fig. 6B). To examine the influence of themitochondrial activity on organic acid production, theorganic acid profiles of these yeasts were investigatedafter alcoholic fermentation for 48 h. Respirative sakeyeast produced significantly increased citric acid

F-4 is a sake yeast appropriate for brewing of ginjo-typesake. F-4 TCR is a mutant resistant to ethyl α-transcyanocinnamate.

Table 1. Sake brewing characterist ics of ethyl α-tanscyanocinnamate-resistant mutant and its parent sakeyeast strain.

F-4 F-4 TCR

Ethanol (v/v%) (23 days) 17.70 17.93

α-Acetolactate (mg/l) (9 days) 0.28 0.05

(B) Respirative sake yeast(A) Fermentative sake yeast

DIC GFP DIC GFP

Fig. 6. Mitochondrial morphology of sake yeast in respirative and fermentative conditions. (A) Morphology of mitochondriaof fermentative sake yeast. Sake yeast with visualized mitochondria was cultured anaerobically in a synthetic medium con-taining 10% glucose as a sole carbon source. (B) Morphology of mitochondria of respirative sake yeast. Sake yeast withvisualized mitochondria was cultured aerobically in a synthetic medium containing 3% glycerol as a sole carbon source. Yeastmitochondria were observed under a fluorescent microscope. GFP indicates the fluorescence image of GFP obtained by fluo-rescence microscopy and DIC indicates the image of differential interference contrast microscopy. Reprinted with permissionof John Wiley & Sons, Inc. from Journal of the Institute of Brewing, 118(1), Motomura et al., Mitochondrial activity of sakebrewery yeast affects malic and succinic acid production during alcoholic fermentation, 22–26, Fig. 1, 2012, Wiley-Liss,Inc., a Wiley Company.



(123%), decreased pyruvic acid (63.0%), decreasedmalic acid (74.6%), increased succinic acid (180%) andincreased acetic acid (129%) during the alcoholic fer-mentation phase, relative to that of fermentative yeast(Fig. 7A).

The role of mitochondrial state of yeast cells on thesechanges were investigated with the compoundcarbonylcyanide p-trifluoromethoxyphenylhydrazone,a specific uncoupler of mitochondrial membrane po-tential . When carbonylcyanide p-trifluoromethoxyphenylhydrazone was added to theculture, 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 tothose observed upon the transfer from the respirativeto fermentative condition. These results suggest thatthe mitochondrial state during the propagation stage isresponsible for the difference in the organic acid pro-duction between the respirative and fermentative yeast.

To develop a technology to alter the organic acidprofile by regulating mitochondrial activity, respirativeyeast cells were exposed to a medium containing 10%(w/v) of glucose, incubated as a static culture, and then

0

50

100

150

200

250

300

350

400

450

Citric acid Pyruvic acid Malic acid Succinic acid Lactic acid Acetic acid

Co

nce

ntr

atio

n (

mg

/l)

Fermentation

Respiration

*

*

**(A)

0

100

200

300

400

500

600


Co

nce

ntr

atio

n (

mg

/l)

Respiration-FCCP

Respiration+FCCP**

(B)

0

100

200

300

400

500

600


Co

nce

ntr

atio

n (

mg

/l)

Respiration

Transition to fermentation (12 h)

Transition to fermentation (24 h)

**

*

(c)

Fig. 7. Organic acid production profiles of sake yeast in respirative and fermentative conditions. (A) Organic acid profiles ofrespirative and fermentative sake yeasts. Statistical significance of difference was evaluated by unpaired one-tailed Student’st-test (*p < 0.05, **p < 0.01, n = 3). Reprinted with permission of John Wiley & Sons, Inc. from Journal of the Institute ofBrewing, 118(1), Motomura et al., Mitochondrial activity of sake brewery yeast affects malic and succinic acid productionduring alcoholic fermentation, 22–26, Fig. 2, 2012, Wiley-Liss, Inc., a Wiley Company. (B) Organic acid profiles of respirativesake 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 withpermission of John Wiley & Sons, Inc. from Journal of the Institute of Brewing, 118(1), Motomura et al., Mitochondrialactivity 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 respirativeto fermentative conditions. Organic acids are measured after additional incubation for 12 and 24 h after a change in culturecondition. Statistical significance of difference was evaluated by Dunnet’s multiple comparison test (*p < 0.05, n = 8). Re-printed with permission of John Wiley & Sons, Inc. from Journal of the Institute of Brewing, 118(1), Motomura et al., Mito-chondrial 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.



used for alcoholic fermentation. When yeast cells wereexposed to these conditions, where mitochondrial ac-tivity decreased, for 12 h and 24 h, production of malicacid increased significantly (Fig. 7C), while the pro-duction of succinic acid decreased significantly com-pared to the untreated cells (24 h cultivation). Theseresults indicated that the physiological state of sakeyeast can be converted from the respirative conditionto the fermentative condition by incubating yeast cellsunder static conditions for 12 h or 24 h in a mediumcontaining 10% glucose. Taken together, these resultsallow us to propose a new scheme of organic acid pro-duction in brewery yeast (Fig. 8). In fermentative sakeyeast cells, a major pool of pyruvate in the cytosol isnot transported into the mitochondria, but remains inthe cytosol and reduced to lactate or carboxylated to

oxaloacetate. Oxaloacetate is then reduced to malate,which is driven by accumulated NADH. In respirativesake yeast cells, a major pool of pyruvate in the cy-tosol is transported to mitochondria, where it is con-verted aerobically to acetyl-CoA, or succinic acid byaldol-condensation with oxaloacetate.

These results indicate, for the first time, that pro-duction profiles of organic acids in brewery yeast canbe manipulated by changing the mitochondrial state ofyeast (Motomura et al. 2012).

12. Concluding remarks

Through these studies, the structures and roles ofyeast mitochondria during sake brewing was first elu-cidated. These findings led to the establishment of

Fig. 8. A schematic diagram of the production pathways of organic acids by fermentative and respirative yeasts. (A) Morphol-ogy of mitochondria of fermentative sake yeast. Sake yeast with visualized mitochondria was cultured anaerobically in asynthetic medium containing 10% (w/v) of glucose as a sole carbon source. (B) Morphology of mitochondria of respirativesake yeast. Sake yeast with visualized mitochondria was cultured aerobically in a synthetic medium containing 3% glycerolas a sole carbon source. (C) Pathway of organic acid production by fermentative sake yeast. Major pool of pyruvate in thecytosol is not transported to mitochondria, remains in the cytosol and reduced to lactate or carboxylated to oxaloacetate andreduced to malate driven by accumulated NADH. (D) Pathway of organic acid production by respirative sake yeast. Majorpool of pyruvate in the cytosol is transported to mitochondria, cleaved aerobically to acetyl-CoA, enters into TCA cycle andconverted 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 duringalcoholic fermentation, 22–26, Fig. 5, 2012, Wiley-Liss, Inc., a Wiley Company.

mitochondriaAcetic acid

Pyruvic acid

Acetyl-CoA

Citric acid

cis-aconitic acid

Isocitric acid

Oxalosuccinic acid

2-oxoglutaric acid

Succinyl-CoA

Succinic acid

Fumaric acid

Malic acid

Oxaloacetic acid

mitochondria

glucose

Oxaloacetic acid

Malic acid

gluconeogenesisLactic acid

(A) Fermentative sake yeast (B) Respirative sake yeast

glucose

Pyruvic acidOxaloacetic acid

Acetyl-CoA

cis-aconitic acid

Isocitric acid

Oxalosuccinic acid

2-oxoglutaric acid

Succinyl-CoA

Succinic acid

Fumaric acid

Malic acid

Oxaloacetic acid Citric acid

(C) (D)



novel and practical fermentation technologies such ashigh malate production, low pyruvate production andmanipulation of malate and succinate. These resultsindicate that metabolisms and events that occur in mi-tochondria will be promising targets for breeding ofsake yeasts.

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Analysis of the Role of Mitochondria of Sake Yeast during ... · drial structure of brewery yeast can be observed throughout the brewing of sake, the Japanese traditional rice wine