Journal of Bioscience and Bioengineering

VOL. xx No. xx, 1e10, 2017

www.elsevier.com/locate/jbiosc

REVIEW

Molecular mechanisms of the yeast adaptive response and tolerance to stresses

encountered during ethanol fermentation

Choowong Auesukaree1, 2, *

Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand1 and Center of Excellence on Environmental Health and Toxicology, CHE, Ministry of
Education, Bangkok 10400, Thailand2

Received 20 February 2017; accepted 16 March 2017

Available online xxx
During ethanol fermentation, yeast cells encounter various stresses including sugar substrates-induced high osmo-
larity, increased ethanol concentration, oxygen metabolism-derived reactive oxygen species (ROS), and elevated tem-
perature. To cope with these fermentation-associated stresses, appropriate adaptive responses are required to prevent
stress-induced cellular dysfunctions and to acquire stress tolerances. This review will focus on the cellular effects of
these stresses, molecular basis of the adaptive response to each stress, and the cellular mechanisms contributing to
stress tolerance. Since a single stress can cause diverse effects, including specific and non-specific effects, both specific
and general stress responses are needed for achieving comprehensive protection. For instance, the high-osmolarity
glycerol (HOG) pathway and the Yap1/Skn7-mediated pathways are specifically involved in responses to osmotic and
oxidative stresses, respectively. On the other hand, due to the common effect of these stresses on disturbing protein
structures, the upregulation of heat shock proteins (HSPs) and trehalose is induced upon exposures to all of these
stresses. A better understanding of molecular mechanisms underlying yeast tolerance to these fermentation-associated
stresses is essential for improvement of yeast stress tolerance by genetic engineering approaches.
Ó 2017, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Cellular response; Stress tolerance; Osmotic stress; Ethanol stress; Oxidative stress; Heat stress; Saccharomyces cerevisiae]

Due to the increasing demand for energy and the negative
environmental impacts of fossil fuels, the consumption of ethanol
as an eco-friendly alternative fuel has increased in many countries.
The budding yeast Saccharomyces cerevisiae is commonly selected
for industrial-scale bioethanol production because it offers several
advantages such as a highly efficient ethanol productivity and a
relatively high tolerance to various fermentation-associated
stresses (1). During ethanol fermentation, yeast cells are simulta-
neously and sequentially exposed to a number of stresses including
osmotic, ethanol, heat, and oxidative stresses (2). Upon pitching
yeast cells into a fermentation starter, yeast cells initially encounter
osmotic stress due to high concentrations of sugar substrates (2). At
the end of fermentation process, on the other hand, high ethanol
concentration is the most important stress that inhibits yeast
growth and metabolism, leading to a termination of ethanol pro-
duction (3). Particularly, when performing very high gravity (VHG)
fermentation using media containing more than 250 g L
1 sugar in
order to obtain a high yield of ethanol (>15% (v/v)), the adverse
effects of both stresses become more serious (4). Although ethanol
fermentation is normally performed under semi-anaerobic condi-
tions, oxygen (O2) is essential for the cultivation and propagation of
yeast cells prior to pitching them into fermentation tanks (2).
However, if the reduction of O2 in the mitochondrial electron
transport chain is incomplete, endogenous generation of reactive
* Corresponding author at: Department of Biology, Faculty of Science, Mahidol
University, Bangkok 10400, Thailand. Tel.: þ662 2015273; fax: þ662 3547161.
E-mail address: choowong.aue@mahidol.ac.th.
oxygen species (ROS), derivative forms of O2, will be enhanced,
leading to an induction of oxidative stress (5). Consistent with this
notion, ROS accumulation and oxidative damage have been
observed in the enological strains of S. cerevisiae during fermen-
tation in high-sugar-containing media that mimics the composition
of grape must (6). At an industrial scale, the fermentation tem-
perature, which is usually maintained by using water-cooling sys-
tems, can increase beyond an optimal range due to high
environmental temperature, especially during summer or in trop-
ical countries. High temperature is therefore another stress that
yeast cells potentially encounter during fermentation.
To cope with these fermentation-associated stresses, appro-
priate cellular responses are required for protecting yeast cells from
stress-induced cellular damages and acquiring tolerances against
these stresses. The understanding of the molecular basis of the
yeast adaptive response to various stresses present during
fermentation is important for successful construction of genetically
modified yeast strains with improved multiple stress tolerance,
which is a desirable trait for efficient bioethanol production. This
review will thus focus on the current understanding of the mo-
lecular mechanisms of the yeast response and tolerance to stresses
encountered during ethanol fermentation.
OSMOTIC STRESS: CELLULAR RESPONSES AND TOLERANCE
MECHANISMS
Osmotic stress induced by high sugar concentrations in the
fermentation starter is the primary stress that yeast cells encounter

1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2017.03.009

Please cite this article in press as: Auesukaree, C., Molecular mechanisms of the yeast adaptive response and tolerance to stresses encountered
during ethanol fermentation, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.009
2 AUESUKAREE
during the ethanol fermentation process (2). Upon being exposed
to osmotic shock, yeast cells rapidly lose intracellular water,
thereby resulting in a loss of turgor pressure followed by cell
shrinkage (7). The plasma membrane is therefore the primary
target for damage caused by hyperosmolarity. Supporting this idea,
the depolarization and permeabilization of the plasma membrane
were observed during hyperosmolarity-induced dehydration (8).
The common cellular mechanism involved in the equilibration of
osmolarity between intracellular and extracellular spaces is the
accumulation of compatible solutes, thereby increasing internal
osmolarity and restoring turgor pressure (9). In S. cerevisiae, glyc-
erol is the major compatible solute whose production is rapidly
induced in response to hyperosmolarity under the regulation of the
high-osmolarity glycerol (HOG) pathway, one of major yeast
mitogen-activated protein kinase (MAPK) pathways (Fig. 1) (10).
The HOG pathway contains two independent upstream
signaling branches, i.e., the SLN1 and SHO1 branches (10). The
osmosensor for the SLN1 branch is Sln1, which contains a cytosolic
histidine kinase domain and forms a phosphor-relay signaling
system together with the other two regulators, Ypd1 and Ssk1 (11).
Under normal osmotic conditions, Sln1 is active and autophos-
phorylates a conserved histidine residue, which in turn transfers
the phosphate to Ypd1 and eventually to Ssk1 (11). The phos-
phorylated Ssk1 is inactive and unable to activate the downstream
MAPK cascade (12). Upon hyperosmotic shock, the histidine kinase
activity of Sln1 is inhibited in response to changes in cellular turgor
pressure (13), resulting in an increased level of unphosphorylated
Ssk1. This allows the binding of Ssk1 to two MAPK kinase kinases
(MAPKKKs) Ssk2 and Ssk22 (12), triggering their autophosphor-
ylation and self-activation. Active Ssk2 and Ssk22 then phosphor-
ylate and activate the MAPK kinase (MAPKK) Pbs2, which in turn
phosphorylates and activates the MAPK Hog1 (11,12). On the other
FIG. 1. The osmotic stress signal transduction through the HOG pathway, which contains two upstream branches, the SLN1 and SHO branches.
Please cite this article in press as: Auesukaree, C., Molecular mechanisms of the yeast adaptive response and tolerance to stresses encountered
during ethanol fermentation, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.009
J. BIOSCI. BIOENG.,
hand, the SHO1 branch involves two putative osmosensors, i.e.,
mucin-like transmembrane glycoproteins Hkr1 and Msb2, thereby
further dividing into the HKR1 and MSB2 sub-branches that regu-
late the HOG pathway by different mechanisms (14,15). Sho1 is
thought to serve as a scaffold protein for recruiting some signaling
components of this pathway to the plasma membrane, including
the membrane-anchor protein Opy2, the MAPKKK Ste11, and the
MAPKK Pbs2, to the plasma membrane. In addition, Sho1 has also
been shown to function as an osmosensor in the HKR1 sub-branch
(16). In response to osmotic shock, Ste11 is phosphorylated by
Cdc42-activated Ste20 and/or Cla4 kinases, which then sequentially
phosphorylates and activates Pbs2 and Hog1 (17e19). Activated
Hog1 is rapidly imported into the nucleus to stimulate the
expression of osmotic stress-responsive genes by regulating the
activities of Hot1, Smp1, Msn1, Msn2, and Msn4 transcription ac-
tivators and the Sko1 transcription repressor (10).
The main target genes of the HOG pathway are glycerol
metabolism-related genes such as GPD1 and GPD2 encoding iso-
enzymes of NAD-dependent glycerol 3-phosphate dehydrogenase,
and GPP1 and GPP2 encoding glycerol-3-phosphate phosphatase
(Fig. 1) (10). The upregulation of these genes leads to an increased
production and accumulation of intracellular glycerol as an osmo-
lyte, which in turn restores the turgor pressure across the plasma
membrane (10). In addition to glycerol metabolism-related genes,
the expression of genes involved in the metabolisms of trehalose
and glycogen, which also function as compatible osmolytes, has
been shown to be upregulated in response to osmotic stress (20).
Based on transcriptome studies, the other major functional groups
of osmo-inducible genes include those involved in the protection
against oxidative damage and protein denaturation, such as genes
encoding antioxidants (e.g., CTT1, GRE3, TRX2, TTR1) and chaper-
ones (e.g., HSP12, HSP26, HSP42, HSP104) (21e23). In agreement
VOL. xx, 2017
with these findings, the general stress response transcription fac-
tors Msn2/Msn4, which are the transcription regulators of these
antioxidant and chaperone genes, have been shown to be under the
control of Hog1 (22).
Supporting the role of glycerol as a compatible osmolyte during
osmotic stress, it has been shown that the mutants lacking genes
encoding glycerol-synthesizing enzymes (e.g., Dgpd1, Dgpp1) and
components of the HOG pathway (e.g., Dpbs2, Dhog1) were hy-
persensitive to osmotic stress (24e26). Consistent with this idea,
our recent study revealed that the intracellular glycerol contents of
the multiple stress-tolerant yeast strains were correlated with their
growth under osmotic stress conditions, suggesting the important
role of glycerol in conferring enhanced osmotolerance to these
strains (27). In addition to its role in osmotolerance, glycerol is also
involved in protecting yeast cells against heat and oxidative
stresses (28,29). Siderius et al. (28) showed that the suppression of
osmosensitivity of hog1 mutant at 37 C was caused by an increased
accumulation of intracellular glycerol, suggesting that the ability to
control intracellular glycerol levels is important for proper osmotic
stress signaling at high temperatures. Pahlman et al. (29) found that
the Dgpp1Dgpp2 mutant was hypersensitive to the superoxide
anion generator, paraquat, and the expression of GPP2 was induced
upon paraquat exposure, suggesting the role of glycerol metabolism
in adaptation to oxidative stress. Contrarily, our recent study
showed that glycerol synthesis was greatly enhanced only after
exposure to osmotic stress induced by high concentrations of
glucose or sorbitol (27), suggesting that the major biological role of
glycerol is limited to osmoadaptation. In addition to glycerol, a
supplementary compatible solute trehalose also has an important
protective function for yeast survival under severe osmotic stress
conditions (30). However, hyperaccumulation of trehalose seems to
be insufficient to improve osmotolerance (30). Despite its potential
role in osmoadaptation, it is likely that trehalose has no role in
protection against glucose-induced osmotic stress during ethanol
FIG. 2. Deleterious effects of ethanol on yeast cells and cellular adaptive response to ethanol stress.
Please cite this article in press as: Auesukaree, C., Molecular mechanisms of the yeast adaptive response and tolerance to stresses encountered
during ethanol fermentation, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.009
YEAST RESPONSE AND TOLERANCE TO FERMENTATION STRESS 3
fermentation because the trehalose biosynthesis is inhibited under
high glucose conditions (27,31).
ETHANOL STRESS: CELLULAR RESPONSES AND TOLERANCE
MECHANISMS
As a natural byproduct of fermentation, ethanol represents
another important stress that yeast encounters. The increased
ethanol concentration in fermentation media can lead to a reduc-
tion in growth rate and cell viability, which in turn results in a
termination of the fermentation process (3). The cellular toxicities
of ethanol include an inhibition of glucose and amino acid uptake, a
reduction of glycolytic enzyme activity, and a disruption of mem-
brane integrity (Fig. 2) (3).
The main targets of ethanol in yeast cells are thought to be
cellular membranes, especially the plasma membrane (32). Ethanol
disturbs the plasma membrane through intercalating into the hy-
drophilic interior of lipid bilayer, resulting in a loss of membrane
integrity and an increase in membrane permeability (33,34). The
membrane-permeabilizing effect of ethanol then induces an
increased passive influx of ions, particularly protons, across the
plasma membrane, thereby triggering cytosolic acidification
(34,35).
To cope with this effect, the vacuolar Hþ-ATPase and the plasma
membrane Hþ-ATPase seem to play an important role in a recovery
from ethanol-induced cytosolic acidification through their func-
tions in pumping protons into the vacuole and out of cells,
respectively (Fig. 2) (34,36). Consistent with this idea, the recent
study on dynamic changes in cytosolic, vacuolar, and external pHs
of yeast cells during ethanol exposure revealed that the ethanol-
induced cytosolic acidification was recovered by vacuolar acidifi-
cation and proton extrusion from cells (34). In addition, a number
of vacuolar Hþ-ATPase genes have been shown to be required for
ethanol tolerance (24,37) and the expression of PMA1 and PMA2
4 AUESUKAREE
genes encoding the plasma membrane Hþ-ATPases was moderately
upregulated when grown in the presence of ethanol (38). Never-
theless, it seems that the ethanol-sensitive phenotype of mutants
lacking vacuolar Hþ-ATPase activity is not caused by a highly
acidified cytosol, but rather by elevated endogenous oxidative
stress (34).
In response to the membrane-perturbing effect of ethanol, the
alteration in plasma membrane compositions such as unsaturated
fatty acids (UFAs) and ergosterol is induced in order to increase
membrane integrity and to modify membrane fluidity (Fig. 2). The
levels of UFAs, especially palmitoleic (D9-cisC16:1) and oleic (D9-
cisC18:1) acids, have been shown to increase upon ethanol exposure
(39,40). These UFAs are catalyzed by the membrane D9 fatty acid
desaturase encoded by OLE1 gene through the desaturation of
palmitic (C16:1) and stearic (C18:1) acids, respectively (41,42). Be-
tween these two UFAs, You et al. (43) reported that oleic acid was
more effective in reducing ethanol toxicity. In addition to UFAs, the
membrane ergosterol levels also contribute to ethanol tolerance,
possibly due to its effect on increasing membrane rigidity (44).
Consistent with this idea, the mutants lacking genes involved in
ergosterol biosynthesis were hypersensitive to ethanol stress
(24,37,45,46).
High concentrations of ethanol have been shown to disrupt
protein structure, leading to denaturation of cellular proteins
including the key glycolytic enzymes pyruvate kinase and hexoki-
nase (Fig. 2) (47). In agreement, the expression of genes associated
with the stabilization or refolding of denatured proteins such as
those encoding heat shock proteins (HSPs) and trehalose metabolic
enzymes were rapidly upregulated upon ethanol exposure (48,49).
These ethanol-responsive genes include a wide range of HSP genes
(e.g., HSP12, HSP26, HSP30, HSP78, HSP82, HSP104, SSA3, and SSA4),
TPS1 encoding trehalose-6-phosphate synthase, TPS2 encoding
trehalose-6-phosphate phosphatase, and NTH1 encoding neutral
trehalase (48,49). Mutants lacking HSP12, HSP26, HSP30, HSP104,
SSA4, TPS1, or TPS2 genes were hypersensitive to ethanol
(24,26,45,46,50), suggesting their role in the acquisition of ethanol
tolerance. Consistent with these findings, it has been shown that
the HSP12, TPS1, and TPS2 genes were highly expressed in the
ethanol-tolerant mutant of sake yeast but not in the parent strain
(51).
HSPs are generally required for protecting yeast cells from
protein denaturation by assisting the folding and maintenance of
newly translated proteins, the refolding of misfolded proteins, and
the disassembly of protein aggregates (52). It is likely that ethanol
may not only affect the conformation of existing proteins but also
the folding of newly synthesized polypeptide chains. In addition to
their common role in protein folding, the specific roles of some
HSPs in response to ethanol stress have been reported. For instance,
the membrane-associated Hsp12 has been reported to be involved
in maintaining membrane integrity during ethanol stress (53). The
expression of HSP genes is mainly controlled by the heat shock
transcription factor Hsf1 and partially by the general stress
response transcription factors Msn2/Msn4 via binding to the spe-
cific sequences called the heat shock element (HSE) and the stress
response element (STRE), respectively (52). To prevent protein
denaturation, trehalose is also involved in the close interplay with
HSPs in stabilizing protein structure through its functions in protein
binding and reduction of water activity (54,55). In addition,
trehalose has been shown to play a role in alleviating ethanol-
induced membrane permeabilization (56). Similar to the regula-
tion of HSP genes, the expression of trehalose metabolism-related
genes such as TPS1, TPS2, and NTH1 is also under the control of
Msn2/Msn4 transcription factors and the STRE element (57,58). The
high accumulation of trehalose has been shown to be important for
improved growth under ethanol stress condition (59). In some
tropical S. cerevisiae strains, the amount of trehalose accumulated
Please cite this article in press as: Auesukaree, C., Molecular mechanisms of the yeast adaptive response and tolerance to stresses encountered
during ethanol fermentation, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.009
J. BIOSCI. BIOENG.,
before exposure to ethanol stress seems to play an important role in
their survival (60).
In addition to the aforementioned protectants, some amino
acids, i.e., proline, tryptophan, and arginine, also have a protective
effect against ethanol stress. Intracellular accumulation of proline
has been shown to confer tolerance to ethanol stress (Fig. 2). The
yeast strain with increased proline synthesis was shown to be more
tolerant to ethanol than the wild-type strain, while the mutants
lacking proline synthesis enzymes, such as the Dpro1 and Dpro2
mutants lacking g-glutamyl kinase and g-glutamyl phosphate
reductase, respectively, were hypersensitive to ethanol stress
(26,61,62). The expression of the PUT4 gene encoding a high-
affinity proline transporter, but not the genes involved in proline
synthesis and degradation, was strongly induced upon ethanol
exposure. These gene expression profiles suggest that the elevated
intracellular proline levels were not caused by an increased proline
synthesis but rather due to an increased proline uptake (63).
Consistent with this finding, the intracellular proline levels did not
increase immediately after ethanol challenge (63). Proline has been
shown to play an important role in stabilizing proteins and mem-
branes, and in inhibiting protein aggregation during protein
refolding (64e66), suggesting its role in protecting yeast cells
against the adverse effects of ethanol on proteins and membranes.
In addition, proline also has an ability to scavenge several reactive
oxygen species (ROS) including hydroxy radicals and superoxide
anions (67). In fact, Takagi et al. (68) showed that proline accu-
mulation reduced the ROS levels and increased the survival rate of
yeast cells grown under ethanol stress conditions. For tryptophan,
the deletion of genes encoding tryptophan biosynthesis enzymes,
i.e., TRP1, TRP2, TRP3, TRP4, and TRP5, resulted in hypersensitivity to
ethanol stress, whereas the overexpression of these tryptophan
biosynthesis genes or tryptophan permease gene TAT2, especially
TRP1 and TRP5, and tryptophan supplementation increased ethanol
tolerance (26,37,69). Recently, arginine has been reported to have a
protective effect against ethanol-induced damages on cell wall,
plasma membrane and intracellular organelles, possibly due to its
role in inhibiting ROS generation (70).
OXIDATIVE STRESS: CELLULAR RESPONSES AND TOLERANCE
MECHANISMS
Oxidative stress is the state that results from an imbalance of
intracellular pro-oxidant/antioxidant, in favor of the pro-oxidants
(Fig. 3). The most abundant intracellular pro-oxidants are reactive
oxygen species (ROS), which are a variety of oxygen-derived mol-
ecules containing one or more unpaired electrons (5). The majority
of intracellular ROS are generated as by-products of the mito-
chondrial electron transport chain during aerobic respiration,
especially when the oxygen reduction reaction is incomplete (5). In
addition to the mitochondria, the endoplasmic reticulum (ER) and
the peroxisomes are other significant sources of ROS via the
metabolic processes in these organelles that also use oxygen as an
electron acceptor, i.e., oxidative protein folding and the fatty acid b-
oxidation, respectively (71,72). The major species of intracellular
ROS consist of superoxide anion ðO2 $
Þ, hydrogen peroxide (H2O2),
and hydroxyl radical (OH) (73). ROS are toxic and can cause
damage to several cellular components including proteins, lipids,
and DNA, resulting in protein oxidation, lipid peroxidation, and
DNA oxidation (5).
In the presence of oxygen, all organisms including yeast there-
fore potentially experience oxidative stress induced by aerobic
metabolism. Since oxygen is required for promoting yeast growth
at early stages of fermentation and for maintaining yeast at opti-
mum conditions for effective fermentation, ethanol fermentation is
commonly performed under semi-anaerobic conditions (2).
VOL. xx, 2017
FIG. 3. Cellular effects of oxidative stress induced by fermentation-associated stresses and molecular mechanisms of oxidative stress response in yeast.
Landolfo et al. (6) showed that, during hypoxic fermentation in
high-sugar-containing medium, S. cerevisiae wine strains exhibited
increased levels of intracellular ROS and oxidative damage to cell
structures. In addition, some stresses present during ethanol
fermentation such as ethanol stress and high glucose-induced os-
motic stress have been shown to lead to increased generation of
ROS (27,74). Consistent with these findings, several genes associ-
ated with oxidative stress were induced during wine fermentation
(75). Furthermore, SOD1 gene encoding Cu/Zn-superoxide dis-
mutase was required for tolerances to not only oxidative stress but
also heat, ethanol, and osmotic stresses (24).
To prevent endogenous oxidative stress, yeast cells contain both
enzymatic and non-enzymatic antioxidant defense systems to
scavenge excessively produced ROS (Fig. 3). The enzymatic anti-
oxidants can be divided into two categories, ROS scavengers and
regulators of intracellular redox balance (5). The major ROS-
scavenging enzymes are superoxide dismutase (SOD), catalase,
and peroxidase, while the redox regulators include thioredoxin and
glutaredoxin. On the other hand, the non-enzymatic antioxidants
are typically small molecules that function as ROS scavengers such
as glutathione (5).
SODs function in the conversion of superoxide anion into
hydrogen peroxide, which is then reduced to water and oxygen by
catalase, glutathione peroxidase and thioredoxin peroxidase (76).
In S. cerevisiae, SODs are classified into two groups according to
their subcellular localization and metal cofactor, i.e., cytosolic Cu/
Zn-SOD (Sod1p) and mitochondrial Mn-SOD (Sod2p) (76). The
Cu/Zn-SOD enzyme, which localizes to the mitochondrial inter-
membrane space, is thought to function in scavenging not only
superoxide anions that leak from the mitochondrial electron
transport chain into the intermembrane space but also externally
and cytosolically-generated superoxide anions. While the Mn-SOD
Please cite this article in press as: Auesukaree, C., Molecular mechanisms of the yeast adaptive response and tolerance to stresses encountered
during ethanol fermentation, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.009
YEAST RESPONSE AND TOLERANCE TO FERMENTATION STRESS 5
plays an important role in detoxifying mitochondrial matrix-
accumulated superoxide anions (76). Hydrogen peroxide is still
harmful because it can further react to produce the highly reactive
hydroxyl radical via the Fenton reaction. Yeast cells therefore
contain catalases that catalyze the dismutation of hydrogen
peroxide into water and oxygen (5). S. cerevisiae has two catalases,
i.e., peroxisomal catalase Cta1 and cytosolic catalase Ctt1. Cta1 is
thought to function in the detoxification of hydrogen peroxide
generated from peroxisomal fatty acid b-oxidation while Ctt1
seems to have a more general role to cope with cytosolic hydrogen
peroxide (77). In addition to catalases, glutathione peroxidase and
thioredoxin peroxidase, which use glutathione and thioredoxin as
reducing reagents, are also involved in the reduction of hydroper-
oxides into the corresponding alcohols and water by reacting with
cysteine thiol groups. Yeast cells have three glutathione peroxi-
dases, i.e., Gpx1, Gpx2, and Gpx3, which have been shown to reduce
phospholipid hydroperoxides, suggesting their role in protecting
membrane lipids from peroxidation (78,79). In S. cerevisiae, five
thioredoxin peroxidases (or so-called peroxiredoxins) have been
reported (i.e., cytoplasmic peroxiredoxins Tsa1, Tsa2, and Ahp1;
nuclear peroxiredoxin Dot5; and mitochondrial peroxiredoxin
Prx1) (80). Prx1 is the 1-Cys peroxiredoxin while the others are the
2-Cys peroxiredoxins, based on the number of cysteine residues
required for the catalysis. The typical 2-Cys peroxiredoxins are
active as a homodimer and require two-redox active cysteine res-
idues to form a disulfide bond during the catalysis, while the 1-Cys
peroxiredoxin is monomeric and active as a peroxidase (80).
The thioredoxin and glutaredoxin systems are involved in
regulating the redox state of protein thiol groups. Thioredoxins and
glutaredoxins are small thiol oxidoreductases containing redox-
active cysteines that are required for the thiol reduction of other
proteins by catalyzing cysteine thiol-disulfide exchange reactions
6 AUESUKAREE
(81). Thioredoxins and glutaredoxins control protein redox states
by donating electrons to disulfide bridges of target proteins,
thereby resulting in oxidized forms. These two proteins are then
reduced back to their redox-active forms by different mechanisms:
thioredoxins are enzymatically reduced by thioredoxin reductase
and NADPH, whereas glutaredoxins are nonenzymatically reduced
by reduced glutathione (81). S. cerevisiae contains both cytoplasmic
and mitochondrial thioredoxin systems, which are composed of
thioredoxins (cytoplasmic Trx1 and Trx2, and mitochondrial Trx3)
and thioredoxin reductases (cytoplasmic Trr1 and mitochondrial
Trr2). On the other hand, yeast glutaredoxins have been reported to
localize to various compartments, i.e., cytoplasmic Grx1/8,
cytoplasm/mitochondria-colocalized Grx2, nuclear Grx3/4, mito-
chondrial Grx5, and cis-Golgi Grx6/7 (81).
In addition to the enzymatic defense system, some non-
enzymatic molecules such as glutathione also play an important
role in ROS detoxification. A tripeptide glutathione (g-glutamyl-
cysteinyl-glycine) is the most abundant small sulfhydryl compound
that functions as an endogenous antioxidant (82). In S. cerevisiae,
the synthesis of glutathione involves two ATP-dependent enzy-
matic steps: formation of g-glutamylcysteine from glutamate and
cysteine via the catalysis of g-glutamylcysteine synthetase Gsh1
and formation of glutathione from g-glutamylcysteine and glycine
via the catalysis of glutathione synthetase Gsh2 (82). In the gluta-
thione cycle, reduced glutathione (GSH) is oxidized to the disulfide
form (GSSG), which is reduced back to GSH by NADPH-dependent
glutathione reductase Glr1 (82).
In response to oxidative stress, transcriptional reprogramming
is mainly regulated by two oxidative stress-responsive transcrip-
tion factors Yap1 and Skn7, and the general stress response tran-
scription factors Msn2/Msn4 (Fig. 3) (83). Yap1 is a basic leucine
zipper (bZip) transcription factor while Skn7 is a transcription
factor that contains a DNA-binding domain homologous to that of
Hsf1. Yap1 and Skn7 coordinately control the transcription of a
number of oxidative stress-responsive genes including SOD1 and
SOD2 encoding superoxide dismutases, TRX2 encoding thioredoxin,
FIG. 4. Deleterious effects of elevated temperature on yeast cells and molecular mechanisms underlying heat stress response and thermotolerance.
Please cite this article in press as: Auesukaree, C., Molecular mechanisms of the yeast adaptive response and tolerance to stresses encountered
during ethanol fermentation, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.009
J. BIOSCI. BIOENG.,
TRR1 encoding thioredoxin reductase, TSA1 and AHP1 encoding
peroxiredoxins, as well as GPX2 encoding glutathione peroxidase
(83). Nevertheless, some genes have been reported to be regulated
by Yap1 in a Skn7-independent manner, especially those involved
in the glutathione system, e.g., GSH1 and GSH2 encoding enzymes
involved in glutathione biosynthesis, and GLR1 encoding gluta-
thione reductase (83). Under physiological conditions, Yap1 local-
izes to the cytoplasm due to an active Crm1-mediated nuclear
export. Upon exposure to oxidative stress, the thiol peroxidase
Hyr1 catalyzes the formation of disulfide bonds in Yap1, which
inhibits the nuclear export of Yap1 and allows its accumulation in
the nucleus (84,85). On the other hand, Skn7 is a constitutive nu-
clear protein, which seems to participate only in the peroxide
response. It has been shown that Skn7, in cooperation with Hsf1, is
involved in the upregulation of HSP genes during oxidative stress,
possibly due to the similarity of their DNA-binding domains (86).
Furthermore, its role in regulating the cell wall synthesis, cell cycle,
and osmotic stress response has also been reported (83). Although
the main function of Msn2/Msn4 is to control the general stress
response, the expression of some oxidative stress-responsive genes,
such as CTT1 encoding catalase, is also regulated by Msn2/Msn4
(87).
HEAT STRESS: CELLULAR RESPONSES AND TOLERANCE
MECHANISMS
During industrial ethanol fermentation, elevated fermentation
temperature beyond an optimal range can affect yeast metabolism
and viability, thereby resulting in a decrease in ethanol produc-
tivity. Heat stress significantly disturbs the stability of proteins,
enzymes, membranes, and cytoskeleton structures, leading to
protein dysfunction, metabolic imbalances, and cellular collapse
(Fig. 4) (52). Adaptive responses against heat stress include the
induction of HSPs to prevent protein aggregation, the synthesis of
some compatible solutes such as trehalose, cell wall remodeling,
and a transient interruption of the cell cycle. Among these, the
VOL. xx, 2017
induction of HSPs under the control of Hsf1 and Msn2/Msn4
transcription factors plays a major role in the heat stress response
(52).
Hsf1 is the heat shock transcription factor that regulates the
transcription of a number of target genes, including those involved
in protein folding and degradation, energy generation, carbohy-
drate metabolism, and maintenance of the cell wall integrity
(88,89). During activation, the homotrimeric Hsf1 specifically binds
to the conserved heat shock element (HSE) motif in the promoters
of its targets (52). Although Hsf1 is constitutively phosphorylated, it
is hyperphosphorylated upon heat shock under positive regulation
by its C-terminal regulatory domain (CTM) (90). The general stress
response transcription factors Msn2/Msn4 regulate the transcrip-
tion of genes in response to various stresses including heat, os-
motic, oxidative, and ethanol stresses, via binding to the STRE
element (52). Under stress conditions, Msn2/Msn4 are hyper-
phosphorylated, translocated into the nucleus, and shuttled in and
out of the nucleus periodically under the control of the cAMP-
dependent protein kinase (PKA) pathway (52).
Most HSPs function as molecular chaperones to assist the
folding of newly synthesized proteins, refolding of misfolded pro-
teins, and the disaggregation of protein aggregates (Fig. 4). HSPs
therefore play a key role in the biogenesis and quality control of
proteins under both non-stress and stress conditions (52). HSPs are
classified into Hsp100, Hsp90, Hsp70, Hsp 60, and the small Hsp
families according to their molecular weight and homology.
Hsp70s assist in the proper folding of nascent polypeptides,
protein translocation to the mitochondria and ER, refolding of
damaged proteins, and degradation of aberrant proteins. In
S. cerevisiae, there are nine cytosolic Hsp70s (Ssa1, Ssa2, Ssa3, Ssa4,
Ssb1, Ssb2, Ssz1, Sse1, and Sse2), two ER Hsp70s (Kar2, and Lhs1),
and three mitochondrial Hsp70s (Ssc1, Ssq1, and Ecm10) (52). The
Ssa family and Kar2 are involved in general protein folding,
whereas the other components are required for specific processes
or specific substrates. For instance, the Ssb family participates in the
folding of nascent polypeptides that emerge from the ribosome,
while the Sse family functions as nucleotide exchange factors for
Hsp70 chaperones during protein refolding.
In contrast to the Hsp70 chaperones, which unselectively
recognize unfolded or misfolded proteins, the Hsp90 chaperone
activity is required for the folding of specific target proteins
including many kinases and transcription factors such as Swe1,
Gcn2, and Hap1. Hsp90 functions in the final steps of maturation of
target proteins into active conformations and also in the refolding
of denatured target proteins back into their native forms (52).
S. cerevisiae has two Hsp90 isoforms, i.e., Hsp82 and Hsc82, whose
activity and target specificity are positively and negatively regu-
lated by various co-chaperones, including Sti1, Cns1, Cdc37, Sba1,
Aha1, Cpr6, Cpr7, and Ppt1, mainly through their activities on in-
hibition or enhancement of ATP binding to Hsp90. Sti1 and Cns37
facilitate Hsp70/Hsp90 bridging, Cdc37 assists Hsp90 in the folding
of specific protein kinases, Sba1 and Aha1 control ATPase activity of
Hsp90, while Cpr6, Cpr7, and Ppt1 relieve the Hsp90 ATPase
inhibition.
Unlike the Hsp70 and Hsp90 systems, Hsp104, a member of the
Hsp100 family, specifically function in the disassembly of stress-
induced protein aggregates prior to delivery to the Hsp70-
mediated refolding pathways (52). Small HSPs (sHSPs) bind to
unfolded proteins to prevent the irreversible formation of protein
aggregates. In yeast, Hsp26 and Hsp42 are two sHSPs that have
been well characterized. Hsp26 and Hsp42, like the other sHSPs,
exist in a large homo-oligomeric complex, which is dissociated into
dimeric from prior to interaction with the unfolded substrates.
Although both Hsp26 and Hsp42 share similar function, Hsp42 is
involved under both normal and stress conditions while Hsp26
activity is required only under stress conditions (52).
Please cite this article in press as: Auesukaree, C., Molecular mechanisms of the yeast adaptive response and tolerance to stresses encountered
during ethanol fermentation, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.03.009
YEAST RESPONSE AND TOLERANCE TO FERMENTATION STRESS 7
In addition to HSPs, a disaccharide trehalose also plays an
important role in preventing protein denaturation and aggregation
through its function as a protein stabilizer. Trehalose binds to the
unfolded proteins to maintain them in a partially-folded state until
being refolded by molecular chaperones (54). Upon exposures to
heat stress and also other protein-damaging stresses such as
oxidative, osmotic, and ethanol stresses, the expression of trehalose
metabolism-related genes including TPS1, TPS2, and NTH1 is rapidly
upregulated, resulting in an increase of intracellular trehalose
levels (27,54). In agreement, the TPS2 gene has been shown to be
required for tolerances to heat, oxidative, osmotic, and ethanol
stresses (24).
Although the molecular mechanisms rendering thermotol-
erance seem to be complicated and are not yet fully understood,
several cellular mechanisms involved in the protein quality control
machinery have been shown to play a crucial role in the acquisition
of thermotolerance (Fig. 4). Our studies in the natural thermoto-
lerant strains revealed that continuous high-level expression of
heat stress-responsive genes, such as HSP genes and trehalose
metabolism-related genes, seems to contribute to thermotolerance
of these strains (91). Similarly, Satomura et al. (92) found that the
YK60-1 strain acquired thermotolerance by an upregulation of the
MSN2 and MSN4 genes encoding general stress response tran-
scription factors, leading to an induction of specific stress-
responsive genes, especially those encoding HSPs and trehalose
synthetic enzymes, and an increase of intracellular trehalose levels.
Recently, it has been shown that the point mutations in CDC25 gene
encoding a guanine nucleotide-exchange factor of the cAMP-PKA
pathway led to the inactivation of this pathway. Importantly, this
inactivation was shown to be required for an acquisition of ther-
motolerance (93). The importance of this cAMP-PKA pathway
inactivation may be due to the fact that the low intracellular cAMP
levels in these mutants may induce the activation of the general
stress response transcription factors Msn2/Msn4, which in turn
enhance the transcription of stress-responsive genes including HSP
genes and trehalose metabolism-related genes. These findings
therefore suggest the important role of increased levels of HSPs and
trehalose in conferring thermotolerance. Furthermore, we also
found that the thermotolerant phenotype of one of our thermoto-
lerant isolates, i.e., the C3723 strain, is under the control of six
genes, two of which have been identified to be CDC19 encoding
pyruvate kinase (involved in ATP production) and RSP5 encoding
ubiquitin ligase (involved in protein degradation) (94,95). This
strain was found to have high pyruvate kinase activity, possibly due
to two silent mutations in the coding region of the CDC19 allele,
which may then cause constitutively active energy metabolism to
maintain cellular homeostasis during heat stress (94). In addition,
the base changes in the promoter region of the RSP5 allele of this
strain, which caused an increase of RSP5 transcription, were found
to confer the thermotolerant phenotype, possibly through an in-
crease in the ubiquitination of the damaged proteins for degrada-
tion by the ubiquitin-proteasome pathway (95).
CONCLUSION AND FUTURE PROSPECTS
Yeast cells encounter osmotic, ethanol, heat, and oxidative
stresses during fermentation (2). These stresses can cause both
specific and general effects on yeast cells. For instance, the osmotic
stress can specifically induce the loss of cellular turgor pressure,
while all these stresses can induce broad protein denaturation.
Therefore, both specific and general stress responses are required
for protecting yeast cells against multiple stresses present during
ethanol fermentation. The HOG pathway and the Yap1/Skn7-
mediated pathways play major roles in responses to osmotic and
oxidative stresses, respectively. On the other hand, the primary
8 AUESUKAREE
general stress response is the upregulation of genes responsible for
reducing and preventing protein denaturation, such as HSP genes
and trehalose metabolism-related genes. Although the expression
of these genes is largely under the control of the general stress
response transcription factors Msn2/Msn4 and the heat shock
transcription factor Hsf1, some stress-specific transcription factors
including Hog1 and Skn7 also participate in the induction of these
genes.
Yeast cells are simultaneously and continuously exposed to
multiple fermentation-associated stresses. Cellular mechanisms
responsible for protecting yeast cells against multiple simultaneous
stresses therefore may play a critical role during ethanol fermen-
tation. Recently, we found that, in response to multiple simulta-
neous stresses mimicking fermentation stress, the specific multiple
stress-tolerant strains (C3253, C3751, and C4377) exhibited
remodeled cell walls with greater robustness and relatively low
intracellular ROS levels compared to the control strains. However,
neither the continuous expression of HSP genes nor the induction of
trehalose biosynthesis was observed under this simultaneous
multi-stress condition (27). These findings suggest that the general
mechanisms including cellular protection afforded by the cell wall,
which is the first line of defense against external stresses, and the
capability for maintaining redox balance to control metabolic ho-
meostasis are the primary mechanisms required for responding to
multiple simultaneous stresses.
For practical ethanol fermentation, yeast strains with multi-
stress tolerance are ideal for efficient ethanol production. The
findings from these studies regarding the molecular basis of cellular
mechanisms required for the acquisition of tolerances against
stresses present during fermentation have provided an important
clue for improving stress tolerances in yeast by using genetic en-
gineering approaches. Nevertheless, the mechanisms of stress
adaptive responses are complicated networks and have not been
clearly understood. Further studies on stress tolerance mechanisms
are needed for the successful genetic manipulation of multiple
stress-tolerant yeast strains, which is indispensable for efficient
ethanol fermentation.
ACKNOWLEDGMENTS
Choowong Auesukaree would like to express his appreciation to
the Society for Biotechnology, Japan, which awarded him the Young
Asian Biotechnologist Prize in 2016, and to Adam Charles Kaplan for
editing the manuscript. This work was partially supported by the
National Research Council of Thailand, the Thailand Research Fund,
Mahidol University, and the Center of Excellence on Environmental
Health and Toxicology.
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