Strees Tolerance of Baker Yeast Cells, Stress Protective Molecules and Genes Involved in Stress Tolerance

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Biotechnol. Appl. Biochem. (2009) 53, 155–164 (Printed in Great Britain) doi:10.1042/BA20090029 155

MINIREVIEW

Stress-tolerance of baker’s-yeast (Saccharomyces cerevisiae)cells: stress-protective molecules and genes involvedin stress tolerance

Jun Shima*1 and Hiroshi Takagi†

*National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan, and †Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan

During the fermentation of dough and the production

of baker’s yeast (Saccharomyces cerevisiae), cells are

exposed to numerous environmental stresses (baking-

associated stresses) such as freeze–thaw, high sugarconcentrations, air-drying and oxidative stresses.

Cellular macromolecules, including proteins, nucleic

acids and membranes, are seriously damaged under

stress conditions, leading to the inhibition of cell

growth, cell viability and fermentation. To avoid lethal

damage, yeast cells need to acquire a variety of stress-

tolerant mechanisms, for example the induction of

stress proteins, the accumulation of stress protectants,

changes in membrane composition and repression of

translation, and by regulating the corresponding gene

expression via stress-triggered signal-transduction

pathways. Trehalose and proline are considered to

be critical stress protectants, as is glycerol. It is

known that these molecules are effective for providing

protection against various types of environmental

stresses. Modifications of the metabolic pathways of

trehalose and proline by self-cloning methods have

significantly increased tolerance to baking-associated

stresses. To clarify which genes are required for stress

tolerance, both a comprehensive phenomics analysis

and a functional genomics analysis were carried out

under stress conditions that simulated those occuring

during the commercial baking process. These analyses

indicated that many genes are involved in stress

tolerance in yeast. In particular, it was suggested thatvacuolar H+-ATPase plays important roles in yeast cells

under stress conditions.

Introduction

Baker’s yeast (mostly strains of Saccharomyces cerevisiae)

is an essential ingredient in bakery products produced by

fermentation [1,2]. Around the world, about 2 million tons

of baker’s yeast are produced (based on 30% dry weight) per

year [3,4]. The function of baker’s yeast in bread making can

be summarized as follows: (i) to increase dough volume by

gas generation during fermentation; (ii) to develop structure

and texture in the dough; and (iii) to add a distinctive flavour

to the dough [5]. Baker’s yeast is produced in the form of

cream yeast (an aqueous suspension containing approx. 20%dry weight of cells), compressed yeast or dried yeast. The

compressed yeast is manufactured by partial dehydration

and contains approximately 30 % dry weight of cells. In Japan,

most baker’s yeasts are produced as cream or compressed

yeasts. However, dried yeast, which contains less than 5%

water, is imported from other countries and used in home

baking and bakery shops owing to the convenience of its

storage and delivery to Japan.

During the fermentation of dough and the production

of baker’s yeast, yeast cells are exposed to numerous

environmental stresses, including freeze–thaw, high sugar

concentrations and air-drying (baking-associated stresses)

[3]. In addition, the yeast cells encounter such stresses in a

multiple and sequential manner (for example, freeze–thaw

plus high sugar concentrations) [3]. It is believed that, by

undergoing freeze–thaw and air-drying treatments, yeast

cells are exposed to oxidative stress [3,6,7]. In general,

micro-organisms show some ability to adapt to environ-

mental stresses. Yeast cells also need to acquire a variety

of stress-adaptation mechanisms, for example, induction of

stress proteins, accumulation of stress protectants, changes

in membrane composition and repression of translation,

and by regulating the corresponding gene expression via

stress-triggered signal transduction pathways. Under se-

vere stress conditions, however, the fermentation ability of yeast is rather limited. To develop the commercial ferment-

ation and growth of baker’s yeast, it is necessary to construct

yeast strains with a higher tolerance to various stresses. The

Key words: baker’s yeast (Saccharomyces cerevisiae), baking-associated stress,

functional genomics, phenomics, stress protectant, stress tolerance.

Abbreviations used: AZC, azetidine-2-carboxylate; GK, γ -glutamyl kinase;

GPR, γ -glutamyl phosphate reductase; HS, high sugar; IC 50 , 50 % inhibitory

concentration; LS, low sugar; ORF, open reading frame; OTA, ornithine

transaminase; P5C , 1-pyrroline-5-carboxylate; PO, proline oxidase; ROS,

reactive oxygen species; SOD, superoxide dismutase; V-ATPase, vacuolar

H+-ATPase.1 To whom correspondence should be addressed (email [email protected]).

C 2009 Portland Press Ltd

www.babonline.org

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156 J. Shima and H. Takagi

Scheme 1 Process of baker’s-yeast production and bread making and the

environmental stresses associated with such processes

present minireview focuses on the mechanisms of tolerance

to environmental stresses to which baker’s-yeast cells are

exposed during industrial processes and the construction

of stress-tolerant baker’s-yeast strains. We describe stress-

tolerance mechanisms based on stress protectants, which

include trehalose and proline, and clarify, by post-genomic

analysis, which genes are required for stress tolerance.

Baking-associated environmental

stresses

During the bread-making process, baker’s yeast cells are

exposed to various types of environmental stresses in

a multiple and sequential manner [3]. We pay particular

attention to the baking-associated stresses freeze–thaw, high

sugar concentrations stress and air-drying, because yeast

cells are considered to be susceptible to damage from such

stresses during the commercial processes that baker’s yeast

undergoes (Scheme 1).

Frozen-dough baking is one of the key technologiesof bread making because it improves labour conditions

for bakers and allows for the supply of oven-fresh bakery

products to consumers. Because baker’s-yeast cells suffer

from freeze–thaw injuries in frozen-dough baking processes,

the fermentation ability of yeast cells after freeze–thaw

dramatically decreases [8,9]. Freeze–thaw injuries to yeast

cells may depend on many factors, including the genetic

background of the yeast strains, the physiological condition

of the yeast cells and the freezing conditions, such as the

length of the freezing period and the speed of freezing.

During frozen-dough baking, yeast cells are frozen in a

process that subjects them to low temperature, ice- crystal

formation in the cells and dehydration [10,11]. Freezing

can cause not only deleterious damage to the cell wall

and membrane but also denaturation of functional pro-

teins and DNA, thus decreasing cell viability. Changes in cellphysiology due to the onset of fermentation might weaken

freeze–thaw tolerance [12–14], possibly due to activation

of the cyclic AMP pathway [15]. A decrease in freeze–thaw

tolerance is strongly related to trehalose degradation, and

the levels of intracellular trehalose affect the tolerance of

baker’s yeast to freeze–thaw stress (for further details, see

below) [12,16]. In commercial frozen-dough processes, pre-

fermentation before freezing is desirable, because bread

made from pre-fermented frozen dough has the proper

texture and taste [8,17]. It is known that the freeze–

thaw tolerance of yeast is also determined by the genetic

characteristics of the strains. Some yeast strains with higherfreeze–thaw tolerance have been isolated from natural

sources and constructed by gene manipulation [16,18–20].

The freezing period is a critical parameter for freeze–thaw

injury; prolonged storage of the frozen dough damages yeast

cells greatly, owing to the growth of ice crystals [21,22].

Recently, the involvement of oxidative stress in freeze–

thaw injury to yeast cells has been analysed using mutants

defective in antioxidant functions [23–25]. Park et al. [26]

reported that superoxide anions and free radicals were

generated in yeast (S. cerevisiae) cells during aerobic freeze–

thaw and that cytoplasmic Cu,Zn-SOD (Cu,Zn-superoxide

dismutase) is required for tolerance to freeze–thaw stress.

Also, an oxidative burst during freeze–thaw has been

considered to lead to oxidative damage to many cellular

molecules, including proteins, lipids and DNA through the

generation of ROS (reactive oxygen species) [27]. Oxidative

stress is considered to cause serious injury to yeast cells

during freeze–thaw in addition to the physical damage caused

by ice nucleation and dehydration.

Yeasts used in breadmaking are exposed to different

sugar conditions during the dough-fermentation processes

[3,28]. Dough can be classified into lean or sweet dough

based on the sugar concentrations contained in the

dough. Lean dough contains no sugar (non-sugar dough) or

small amounts of sugar [below 5 % (w/w) of flour]. In general,sweet dough (high-sugar dough) contains up to 40 % sucrose

(w/w of flour). Such high sucrose concentrations exert

severe osmotic stress on yeast cells [29]. Yeast strains that

show higher tolerance to high concentrations of sucrose

are desirable for sweet-dough fermentation [3,28,30]. By

contrast, because there is little sucrose in non-sugar dough,

the yeast must use maltose derived from the flour [28,30].

Yeast strains that have a higher maltose-utilization ability

are required for non-sugar dough fermentation [30]. In

the modern baking industry, HS (high sugar; high-sucrose-

tolerant) and LS (low suger; maltose-utilizing) yeasts have




Stress tolerance of baker’s-yeast cells 157

been developed by using breeding techniques, and are

currently used commercially [28,30]. Baker’s-yeast cells

may change their level of metabolic activity, such as their

carbon metabolism and nitrogen metabolism, as well as

their stress response to environmental conditions [30,31].An understanding of the molecular basis behind the meta-

bolic adaptation that occurs under different sugar conditions

is needed to develop yeast strains that have tolerance to

high-sucrose stress and have maltose-utilization ability.

During the production of dried yeast, yeast cells are

also exposed to air-drying stress [32,33]. In breadmaking,

the distinct advantages of using dried yeast, such as active

dried yeast (dry matter 92–94%) and instant dried yeast

(dry matter 94–96 %), include their greater stability and

lower moisture content, both of which reduce both storage

and transport costs [34]. To gain greater advantages from

dried yeast, it must retain a higher fermentation abilityand flavour formation after the air-drying process. During

the drying process using cell dryers such as fluidized beds

or Roto-Louver® dryers [33], yeast cells are exposed

to air-drying stress. In general, owing to the flow of

hot air during these processes, the temperature of the

yeast cells is relatively high, namely about 37 ◦C. Air-drying

stress is considered to be a complex environmental stress

composed of dehydration and heat stresses. Dehydration

generated by drying processes, such as air-drying and

evaporation, causes severe damage to yeast cells, particularly

to the cellular membrane and proteins [35]. Dehydration

promotes the generation of ROS, which induce lipid

peroxidation, protein denaturation and nucleic acid damage

[36–38]. Therefore the ROS-scavenging systems mediated

by glutathione, catalase and SOD, for example, are believed

to confer tolerance to dehydration [39,40].

Trehalose as a stress protectant

Many research groups have analysed key factors and their

roles in the stress tolerance of S. cerevisiae. For instance,

heat-shock proteins function in the disaggregation and disas-

sembly of proteins denatured by environmental stress [41].

A variety of antioxidant enzymes, including SOD, catalase,and glutathione peroxidase, detoxify ROS by the direct

disproportion, decomposition or reduction of ROS under

oxidative-stress conditions [3]. In S. cerevisiae, it is known

that glycerol [42] and trehalose function as major stress

protectants and that the synthesis of glycerol or trehalose is

induced by many stress conditions at the transcriptional level

[43]. Owing to the ability of these compounds to prevent

the influx of excess salts into the cell or irreversible cell

dehydration, the osmotic imbalance after hyper- or hypo-

osmotic shock can be rapidly restored. Panadero et al.

[44] suggested that the heterologus expression of type I

Scheme 2 Metabolic pathway of trehalose in S. cerevisiae

Genes encoding particular metabolic enzymes are shown in parentheses

antifreeze peptide GS-5 from the polar fish grubby sculpin

( Myoxocephalus aenaeus) in baker’s yeast increased freeze–

thaw tolerance. It was also reported that overexpression of

the calcineurin target CRZ1 provided freeze–thaw tolerance

[45]. In response to osmotic or desiccation stress, proline

is accumulated in many bacterial and plant cells as an

osmoprotectant [46,47]. However, under various stresses,

yeast cells induce glycerol or trehalose synthesis, but do not

show an increase in proline [43]. In the present minireview,

we focus on trehalose and proline as stress protectants that

can provide tolerance to baking-associated stress.

Trehalose (α-D-glucopyranosyl α-D-glucopyranoside) is

a non-reducing disaccharide of glucose. This sugar is widely

distributed in various organisms, including bacteria, fungi,

plants, insects and invertebrates [48]. In S. cerevisiae,

trehalose is thought to be an important reserve carbo-

hydrate in the cell. However, the most significant function

of trehalose is in providing protection to proteins and lipids

included in the membrane structure against different kinds

of stress conditions, such as heat and freeze–thaw [48,49].

In S. cerevisiae, trehalose may constitute as much as 15 % of its dry weight when growing in a stress environment [3].

It has been pointed out that there is a strong correlation

between trehalose content and stress tolerance [12].

Cellular levels of trehalose are controlled by an

enzymatic balance between its synthesis and degradation

(Scheme 2) [48]. The synthesis of trehalose is catalysed

by a trehalose-synthesis protein complex composed of

four proteins. Tps1 synthesizes trehalose 6-phosphate

by the condensation of glucose 6-phosphate and UDP-

glucose. Trehalose is then generated by dephosphorylation

of trehalose 6-phosphate by Tps2. Tps3 and Tsl1, which







Scheme 3 Metabolic pathway of proline in S. cerevisiae

Genes encoding particular metabolic enzymes are shown in parentheses

to various environmental changes, including temperature,

ethanol, oxidation, pH and osmolarity [79,80]. Recently,

the correlation between gene-expression profiles and

intracellular contents of glycerol, trehalose, and proline were

determined under various stress conditions [43]. When

yeast cells were exposed to osmotic stress, the expression

of GPD1, encoding glycerol-3-phosphate dehydrogenase,

was induced, leading to glycerol accumulation. In the

presence of ethanol, the rapid induction of TPS2 resulted

in trehalose accumulation. By contrast, the expression of

genes involved in proline metabolism and the cellular level

of proline did not change during exposure to these stresses

[43]. These results suggest that yeast cells do not accumulate

proline in response to osmotic or ethanol stress.

It was shown that a put1-disruptant yeast in minimal

medium supplemented with external proline accumulated

higher levels of proline in the cells and conferred a

higher tolerance to freeze–thaw and dehydration stresses

[73]. Enhancement of the biosynthetic activity is also

important for the intracellular accumulation of proline. AZC

(azetidine-2-carboxylate), which is a toxic four-membered

ring analogue of proline, is transported into yeast cells

through proline transporters [81]. Once inside a cell, AZCcompetes with proline for incorporation into nascent

proteins, resulting in protein misfolding, which inhibits cell

growth [82]. Overproduction of proline dilutes the effect

of AZC. To increase cellular proline accumulation, AZC-

resistant mutants have been isolated from the put1-deficient

strain [69]. This mutant was recently found to carry an

allele of PRO1 encoding GK and to have a single amino

acid replacement at position 154 (aspartic acid replaced

by asparagine) [72]. Yeast cells expressing the mutant

GK accumulated proline and showed a drastic increase

in cell viability after freezing at −20 ◦C compared with

cells harbouring wild-type PRO1. The D154N mutant of

GK [IC50 (50 % inhibitory concentration)= 32 mM] showed

lower sensitivity to feedback inhibition than did the wild-type

enzyme (0.5 mM) and thermostability. These characteristics

lead to a higher level of proline accumulation [83,84].A high level of freeze–thaw tolerance clearly correlated

with the accumulation of proline in yeast cells. Such

a proline-accumulating laboratory strain was found to

be more tolerant to various stresses, including freezing,

dehydration, hydrogen peroxide and ethanol, than the wild-

type strain [69,71,72,74,84,85]. To improve the enzymatic

properties of GK, PCR random mutagenesis in PRO1

was employed [83]. Several mutant GKs that, owing to

extreme desensitization to inhibition, enhanced the ability

to synthesize proline, were successfully isolated. Further-

more, yeast cells expressing I150T and N142D/I166V

mutant GKs were found to be more tolerant to freezingstress than cells expressing the D154N mutant.

Recently we constructed self-cloning diploid baker’s-

yeast strains by disrupting PUT1 and replacing the wild-type

PRO1 with the pro1 (D154N) or pro1 (I150T ) allele [43]. The

resultant baker’s-yeast strains accumulated higher levels of

intracellular proline (3–6 % dry weight) compared with that

of the wild-type strain (0.20 %). As expected, the proline-

accumulating strains retained their higher fermentation

abilities in the frozen doughs at the same levels as those

observed before freezing, although that of the parent strain

fell to approx. 80 % of the prefreezing level. Therefore,

proline-accumulating baker’s yeast is considered to be

suitable for frozen-dough baking. It is noteworthy that

the combination of proline and trehalose might further

contribute to the enhancement of tolerance to baking-

associated stresses.

Phenomics approach to the

identification of genes required for

stress tolerance to baking-associated

stresses

In addition to analyses of cell-protective molecules, theidentification of novel genes which determine the tolerance

to baking-associated stress were attempted. Our group at-

tempted to find the genes for stress tolerance by two differ-

ent approaches, namely phenomics and functional genomics,

under freeze–thaw, high-sugar-concentration and air-drying

stress conditions [6,7,31,86–89]. The data for phenomics

and functional genomics under baking-associated stress

conditions are available at our web site (http://nfri.naro.

affrc.go.jp/english/Useful/yeast/index.pdf). A yeast deletion-

mutant collection should be a powerful tool for determining

gene function by analysing the phenotype of mutants lacking





Figure 1 Schematic view of overlap in genes required for each baking-

associated stress tolerance

The numbers in parentheses are the gene numbers determined by phenomics

analyses. The genes encoding the components of V-ATPase are included in thearea where the three circles overlap.

the gene (phenomics) [90–92]. An international consortium

has carried out the systematic deletion of all of the ORFs

(open reading frames) of S. cerevisiae by using a PCR-

mediated gene-deletion strategy [91]. Analysis using this

deletion-mutant collection relies on the number of genes

whose mutations that affect components of an important

pathway show a phenotype of sensitivity or tolerance

[90,92,93]. Identification of the genes required to cope

with high-sugar-concentrations, freeze–thaw and air-drying

stresses was carried out using phenomics [6,7,88]. The

complete deletion-strain collection of diploid S. cerevisiae

was used in these analyses because commercial baker’s

yeasts are generally diploid.

The screening identified 273 strains with high sucrose-

sensitivity, 58 strains with freeze–thaw-sensitivity and 278

strains with air-drying-sensitivity [6,7,88] (Figure 1). These

deleted genes were classified on the basis of their cellular

function and the localization of their gene products. A

total of 30 strains were sensitive to all the environmental

stresses tested. As described below, the deletion strains

of the genes encoding V-ATPase (vacuolar H+-ATPase) and

proline metabolism were included in the 30 strains. Otherthan such genes, the deletion strains of genes encoding

heat-shock response and vacuolar protein sorting were

included. The deletion strains of genes encoding trehalose

metabolism were sensitive to high-sucrose stress and air-

drying stress, but not to freeze–thaw stress, in this assay.

The deletion strains of the genes involved in trehalose

did not show sensitivity to freeze–thaw stress, because

we used exponential-phase cells (no-accumulation condition

for trehalose) in this assay. Interestingly, we found that

V-ATPase function is required for tolerance to all of the

baking-associated stresses tested. In yeast, V-ATPases are

responsible for the acidification of vacuoles, endosomes

and the late Golgi apparatus and contribute to cellular

pH homoeostasis [94,95]. The deletion mutant of STV1,

encoding a Golgi-type subunit, did not show sensitivity to

baking-associated stresses, suggesting that acidification of vacuoles may be important for stress tolerance (A. Ando,

H. Takagi and J. Shima, unpublished work). This V-ATPase−

(vma) phenotype is characterized by a distinct pattern of

pH- and calcium-sensitive growth, metal-ion-sensitivity and

an inability to grow on non-fermentable carbon sources

[95]. The phenomics approach using the yeast deletion-

mutant collection has indicated that the loss of V-ATPase

activity has unexpectedly diverse consequences [94]. As

Kane [94] summarized, the deletion strains of the gene

encoding V-ATPase showed multiple phenotypes. These

previous findings, together with our own results, suggest

that V-ATPase has highly critical roles in stress tolerance toenvironmental stresses.

Cross-sensitivity of the high-sucrose-sensitive mutants

to high concentrations of NaCl and sorbitol wasdetermined.

Among the 273 sucrose-sensitive deletion mutants, 269

showed cross-sensitivities to sorbitol or NaCl [88].

However, four mutants involved in purine metabolism

(ade5,7 , ade6, ade8 and pde2) were specifically sensitive to

high sucrose concentrations. In the presence of high-sucrose

stress, the intracellular contents of ATP in the ade mutants

were at least two-fold lower than that of the wild-type cells,

suggesting that depletion of ATP is a factor in sensitivity to

high-sucrose stress [88]. The genes involved in freeze–thaw-

stress-sensitivity were then classified on the basis of their

cellular function and on the localization of their products [6].

The results showed that the genes required for tolerance

to freeze–thaw stress were frequently involved in vacuole

functions and cell-wall biogenesis. The cross-sensitivity of

the freeze–thaw-sensitive mutants to oxidative stress and

to cell-wall stress was analysed, because these stresses are

considered to be environmental stresses closely related

to freeze–thaw stress. The results showed that defects in

the functions of V-ATPase conferred sensitivity to oxidative

stress and to cell-wall stress. However, defects in gene

products involved in cell-wall assembly conferred sensitivity

to cell-wall stress, but not to oxidative stress. These resultsshowed the presence of at least two different mechanisms

of freeze–thaw injury: oxidative stress generated during the

freeze–thaw process and defects in cell-wall assembly [6].

The genes involved in air-drying-sensitivity were classified

on the basis of their cellular function and on the localization

of their gene products [7]. The results showed that the genes

required for air-drying tolerance were frequently involved in

mitochondrial functions and V-ATPase. Intracellular pH was

monitored to determine the role of vacuolar acidification

in air-drying-stress tolerance. The results showed that

intracellular acidification was induced during air-drying and





that this acidification was amplified in a deletion mutant

of the VMA2 gene encoding a component of V-ATPase,

suggesting that V-ATPase helps to maintain intracellular pH

homoeostasis, which is affected by air-drying stress [7].

Mitochondrial membrane potential under air-drying stressconditions was assessed using MitoTracker® to determine

the effects of air-drying stress on mitochondria, and the

results showed that mitochondria were extremely sensitive

to air-drying stress. These data strongly suggested that a

mitochondrial function is required for tolerance to air-

drying stress. We also analysed the correlation between

oxidative-stress- and air-drying-stress sensitivities. It has

been suggested that oxidative stress is a critical determinant

of sensitivity to air-drying stress, although ROS-scavenging

systems are not necessary for air-drying stress tolerance

[7].

The genes identified by phenomics analyses might beimportant for tolerance to baking-associated stresses, and,

as such, should be target genes in future research into

molecular modification for the breeding of yeast strains

tolerant to such stresses.

Functional-genomics approach to the

identification of genes required for

baking-associated stress tolerance

Global studies of yeast cells via functional-genomics

approaches using DNA microarray profiling are a promising

tool for the analysis of metabolic adaptation [96–99].

Because laboratory yeast has been the model organism in

the development of DNA microarray techniques, metabolic

adaptation and stress tolerance under laboratory conditions

have been extensively studied [100–103]. Global gene-

expression analyses of laboratory yeast have frequently

been performed, but only a few studies have analysed

gene-expression profiles during fermentation of commercial

strains such as brewer’s [104,105] and wine [97,106–109]

yeasts. Because the fermentation of beer and wine takes

longer than that of bread dough, ranging from several days

to weeks, the gene-expression profile of brewer’s yeast maybe different from that of baker’s yeast.

Because information on gene expression during ferm-

entation simulating dough fermentation had not been previ-

ously obtained, our group first attempted to determine the

gene-expression pattern to obtain insights at the molecular

level into the rapid adaptation mechanisms of baker’s yeast

using a liquid fermentation medium, which simulates actual

dough baking [31]. The onset of fermentation caused drastic

changes in gene-expression profiles within 15 min. Genes

involved in the tricarboxylic acid cycle were down-regulated

and genes involved in glycolysis were up-regulated, indicating

a metabolic shift from respiration to fermentation. Genes

involved in ethanol production (the PDC genes and ADH1),

glycerol synthesis (GPD1 and HOR2) and low-affinity hexose

transport (HXT1 and HXT3) were up-regulated at the

beginning of fermentation. The genes involved in amino acid(e.g. arginine) metabolism and vitamin (e.g. riboflavin and

thiamin) biosynthesis were subsequently up-regulated after

30 min. Interestingly, the genes involved in the unfolded-

protein-response pathway were also subsequently up-

regulated. These results will provide the scientific basis for

genomic responses to various stresses during commercial

fermentation processes.

As described above, HS and LS yeasts have recently

been used in the baking industry. It is known that sugar

utilization and high-sucrose tolerance differ significantly

between HS and LS yeasts [3]. The gene-expression profiles

of HS and LS yeasts were analysed under different sucroseconditions to determine their basic physiology [87]. The

clustering analysis clearly showed that the gene-expression

patterns of LS yeast differed from those of HS yeast.

Quality threshold clustering was used to identify the gene

clusters containing up-regulated genes (cluster 1) and down-

regulated genes (cluster 2) under high-sucrose conditions.

Clusters 1 and 2 contained numerous genes involved

in carbon and nitrogen metabolism respectively. The

expression level of the genes involved in the metabolism of

glycerol and trehalose, which are known to be osmoprotec-

tants, was higher in LS yeast than in HS yeast under sucrose

concentrations of 5–40 %. No clear correlation between the

expression level of the genes involved in the biosynthesis of

such osmoprotectants and the intracellular contents of the

osmoprotectants was found [87].

Singh et al. analysed the transcriptional response of a

laboratory yeast strain during dehydration and rehydration

processes under minimal-glucose conditions [110]. To

gain insight into the physiology of yeast cells during the

commercial production of dried yeast, we obtained gene-

expression profiles under air-drying stress [86]. In a different

way from Singh et al. [110], we analysed gene expression

in commercial baker’s yeast under conditions that simulate

conditions used in dried-yeast production. To simulate the

dried-yeast production process, a compressed yeast productwas used as a model. The compressed yeast was

propagated by fed-batch cultivation using molasses as the

medium, and the cells in the compressed yeast were in

the quiescent condition. Therefore, the transcriptional res-

ponse of cells in compressed yeast products might signifi-

cantly differ from that in laboratory-yeast cells in culture

medium. Up- or down-regulated genes in yeast cells exposed

to air-drying were clarified by using gene clustering and func-

tional categorization. K-means clustering suggested that the

genes involved in protein folding, such as heat-shock proteins

and in the proteasome, were transiently up-regulated at





early stages. It was clarified that the genes involved in fatty

acid metabolism were continuously up-regulated.

The data obtained by functional genomics under

baking-associated stresses are important as probes for

determining cell physiology during commercial processes.The information on gene expression under baking-asso-

ciated stress conditions should be useful for the design of a

stress-tolerant baker’s yeast. We are currently attempting

to determine the effects of freeze–thaw stress on gene

expression by DNA microarray analysis.

Conclusions and future perspectives

Commercial baker’s yeasts are exposed to various

environmental stresses, including high sugar concentrations,

freeze–thaw and air-drying stress. Yeast cells have a widevariety of strategies for adapting to these environmental

changes. If stress levels are higher than the level to which

yeast cells can adapt, however, the cells’ fermentation

abilities are greatly restricted. In terms of applied aspects,

stress tolerance is the key for yeast cells. To improve

the fermentation or production process of yeast products,

baker’s yeast with higher stress tolerance should be

developed. Trehalose and proline are important molecules

in the stress tolerance of baker’s-yeast cells. In fact,

as described in the present minireview, the engineering

of trehalose and proline metabolism is a promising

approach to the development of stress-tolerant yeast strains.

However, the detailed molecular mechanisms of the pro-

tective functions of such molecules are poorly understood.

Stress tolerance mechanisms have been widely studied

by many research groups. Several important genes and mo-

lecules involved in tolerance have been identified. However,

there is a strong possibility that unknown mechanisms still

exist in yeast cells. Our group attempted to find novel

genes involved in stress tolerance using both phenomic

and functional-genomic approaches. In these screening

methods, we found many genes that make important

contributions to stress tolerance. Next, we plan to analyse

the functions of the genes identified by the screening

and attempt to construct novel stress-tolerant strains bygene modification. Other commercial yeasts, such as those

used to produce grape wine and sake (Japanese rice wine)

were exposed to environmental stress. During wine and

sake fermentation, high-sugar-stress tolerance may be an

important characteristic for yeasts. Our data involved in

high-sugar-stress tolerance may be useful for breeding of

yeasts for the fermentation of alcoholic drinks. Deletion

strains for the genes encoding V-ATPase are highly sensitive

to all of the baking-associated stresses, which suggests that

V-ATPase should be a key part of the machinery involved in

the construction of stress-tolerant strains. Improvement

in V-ATPase functions would be an interesting subject for

further research. Although the integration of data on pheno-

mics and functional genomics is difficult at present, data from

both types of analysis provide an important basis for the

construction of novel stress-tolerant baker’s-yeast strains.

Acknowledgements

We thank Dr Akira Ando and Dr Toshihide Nakamura

(National Food Research Institute, Tsukuba, Ibaraki, Japan)

for critical comments on the manuscript before its

submission.

Funding

Part of the work described was supported by the Program

for Promotion of Basic Research Activities for Innovative

Biosciences (PROBRAIN).

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Received 22 January 2009/4 March 2009; accepted 9 March 2009

Published on the Internet 29 May 2009, doi:10.1042/BA20090029


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