Molecular mechanisms of the yeast adaptive response and tolerance to stresses encountered during ethanol fermentation

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 osmolarity, increased ethanol concentration, oxygen metabolism-derived reactive oxygen species (ROS), and elevated temperature. 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 simultaneously 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 production (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 conditions, 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: [email protected].

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 fermentation in high-sugar-containing media that mimics the composition of grape must (6). At an industrial scale, the fermentation temperature, which is usually maintained by using water-cooling systems, can increase beyond an optimal range due to high environmental temperature, especially during summer or in tropical countries. High temperature is therefore another stress that yeast cells potentially encounter during fermentation. To cope with these fermentation-associated stresses, appropriate 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 molecular 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

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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

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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, glycerol 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 autophosphorylates a conserved histidine residue, which in turn transfers the phosphate to Ypd1 and eventually to Ssk1 (11). The phosphorylated 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 autophosphorylation and self-activation. Active Ssk2 and Ssk22 then phosphorylate and activate the MAPK kinase (MAPKK) Pbs2, which in turn phosphorylates and activates the MAPK Hog1 (11,12). On the other

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 regulate 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 activators 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 isoenzymes 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 osmolyte, 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 chaperones (e.g., HSP12, HSP26, HSP42, HSP104) (21e23). In agreement

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

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with these findings, the general stress response transcription factors 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 hypersensitive 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

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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 reduction 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 membrane 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 hydrophilic 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 functions 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 ethanolinduced cytosolic acidification was recovered by vacuolar acidification 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

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

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genes encoding the plasma membrane Hþ-ATPases was moderately upregulated when grown in the presence of ethanol (38). Nevertheless, 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 (D9cisC18:1) 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). Between 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 hexokinase (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 specific 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 ethanolinduced membrane permeabilization (56). Similar to the regulation 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

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 highaffinity 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 membranes, 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 accumulation 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 molecules containing one or more unpaired electrons (5). The majority of intracellular ROS are generated as by-products of the mitochondrial 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 boxidation, 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 therefore 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 optimum conditions for effective fermentation, ethanol fermentation is commonly performed under semi-anaerobic conditions (2).

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

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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 osmotic stress have been shown to lead to increased generation of ROS (27,74). Consistent with these findings, several genes associated with oxidative stress were induced during wine fermentation (75). Furthermore, SOD1 gene encoding Cu/Zn-superoxide dismutase 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 antioxidants can be divided into two categories, ROS scavengers and regulators of intracellular redox balance (5). The major ROSscavenging 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 intermembrane 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

plays an important role in detoxifying mitochondrial matrixaccumulated 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 hydroperoxides into the corresponding alcohols and water by reacting with cysteine thiol groups. Yeast cells have three glutathione peroxidases, 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 residues 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 redoxactive cysteines that are required for the thiol reduction of other proteins by catalyzing cysteine thiol-disulfide exchange reactions

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

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(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, mitochondrial Grx5, and cis-Golgi Grx6/7 (81). In addition to the enzymatic defense system, some nonenzymatic molecules such as glutathione also play an important role in ROS detoxification. A tripeptide glutathione (g-glutamylcysteinyl-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 enzymatic 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 glutathione 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 transcription factors Yap1 and Skn7, and the general stress response transcription 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,

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 glutathione reductase (83). Under physiological conditions, Yap1 localizes 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 nuclear 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 productivity. 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

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

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, carbohydrate 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 transcription of genes in response to various stresses including heat, osmotic, oxidative, and ethanol stresses, via binding to the STRE element (52). Under stress conditions, Msn2/Msn4 are hyperphosphorylated, translocated into the nucleus, and shuttled in and out of the nucleus periodically under the control of the cAMPdependent protein kinase (PKA) pathway (52). Most HSPs function as molecular chaperones to assist the folding of newly synthesized proteins, refolding of misfolded proteins, 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 regulated by various co-chaperones, including Sti1, Cns1, Cdc37, Sba1, Aha1, Cpr6, Cpr7, and Ppt1, mainly through their activities on inhibition 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 stressinduced protein aggregates prior to delivery to the Hsp70mediated 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).

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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 thermotolerance 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 thermotolerant 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 transcription factors, leading to an induction of specific stressresponsive 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 thermotolerance (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 thermotolerant 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 increase in the ubiquitination of the damaged proteins for degradation 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/Skn7mediated pathways play major roles in responses to osmotic and oxidative stresses, respectively. On the other hand, the primary

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

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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 fermentation. Recently, we found that, in response to multiple simultaneous 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 homeostasis are the primary mechanisms required for responding to multiple simultaneous stresses. For practical ethanol fermentation, yeast strains with multistress 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 engineering 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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