Yeast Cellular Stress: Impacts on Bioethanol Production

Bioethanol is the largest biotechnology product and the most dominant biofuel globally. Saccharomyces cerevisiae is the most favored microorganism employed for its industrial production. However, obtaining maximum yields from an ethanol fermentation remains a technical challenge, since cellular stresses detrimentally impact on the efficiency of yeast cell growth and metabolism. Ethanol fermentation stresses potentially include osmotic, chaotropic, oxidative, and heat stress, as well as shifts in pH. Well-developed stress responses and tolerance mechanisms make S. cerevisiae industrious, with bioprocessing techniques also being deployed at industrial scale for the optimization of fermentation parameters and the effective management of inhibition issues. Overlap exists between yeast responses to different forms of stress. This review outlines yeast fermentation stresses and known mechanisms conferring stress tolerance, with their further elucidation and improvement possessing the potential to improve fermentation efficiency.


Introduction
The biotechnological potential of Saccharomyces cerevisiae has been exploited traditionally for the purposes of baking, brewing, and wine making. This species has also been incredibly useful in basic biochemistry and genetics studies [1]. Yeasts can be found thriving in diverse sugar-rich habitats and can be considered to be microbial weed species due to their dominant, biocide producing nature [2]. Its fermentative nature evolved between 125 and 150 million years ago and is shared by many yeast species, but the acquisition of ethanol tolerance by S. cerevisiae is a more recent event occurring after the whole genome duplication event, which is believed to have happened 100 million years ago [3,4]. This~6000 gene-containing unicellular eukaryote was the first organism to have its genome fully sequenced [5,6]. Today it remains an indispensable model system for experimental studies and a mainstay of biotechnology. The curated Saccharomyces Genome Database (SGD; www.yeastgenome.org) contains vast amounts of information for this microorganism, obtained through "omics" technologies, traditional biochemistry and molecular biology techniques [7]. Yeast continues to play a pivotal role in the study of fundamental biological processes including aging, cell cycle and stress, as a model organism and in emerging biotechnological areas including drug screening, computational and systems biology-see, for example [8][9][10][11][12][13][14][15][16][17][18][19][20]. The high genetic tractability of this microorganism allows for the manipulation of metabolic pathways; for example biofuel researchers often minimize carbon fluxes to natural metabolic by-products to maximize ethanol productivity (see also Section 3.1) [21,22].
The displacement of conventional transport fuels with biofuels including high octane number bioethanol increases fuel security, whilst also being an effective strategy for reducing transport sector emissions [23]. First-generation bioethanol remains the most dominant biofuel globally, yet policy now YNL027W Transcription factor Involved in responding to alkaline stress [42] Elo1p YJL196C Medium-chain fatty acyl elongase Enables production of longer fatty acids in heat and chaotrope stress [43] Ena1p YDR040C Sodium ion pump Involved in responding to alkaline stress [44]

Hac1p
YFL031W Transcription factor Activates genes involved in the unfolded protein response [45] Hog1p

Yeast Stress Responses
Fluctuating environments affect all living organisms. Yeast's response to fermentation associated stress conditions includes the switching of growth and metabolic strategies, including the synthesis of stress proteins functioning to mitigate cell damage and stress. These responses are is coordinated by cell stress stimulus sensing and signal transduction, triggering downstream transcriptional and post-translational responses [64]. The stress response elements (STRE; consensus CCCCT) are activated during numerous stress conditions including oxidative, heat shock, nitrogen starvation, low external pH, weak lipophilic acids and ethanol [65][66][67][68][69][70]. Yeast when subjected to ethanol stress initially struggles to maintain energy production. This compromised energy production leads to the increased expression of genes associated with energy metabolism [71]. While stress is often perceived as detrimental, which it often is doing industrial fermentations, it should also be remembered that environmental challenges and stresses can improve evolutionary fitness and drive the selection of more robust traits, strains and species [72,73].
The genetics underlying stress tolerance have been extensively investigated in S. cerevisiae and genetic and metabolic improvements having been attempted with success [68][69][70][71][74][75][76][77][78][79][80][81]. It is common for genes found to be up-regulated in the presence of ethanol stress to be targets for genetic engineering approaches [82]. A common experimental approach for the eventual isolation of desirable industrial traits including high stress tolerance is directed evolution. Selective pressures imposed on microorganisms promote the eventual emergence of traits that provide a selective advantage [83][84][85]. Repetitive batch cultivations in the presence of lignocellulosic hydrolysates for example evolved strains displaying increased tolerance to all inhibitors present [86]. Evolutionary methods are essentially "blind", but they enable tolerance to develop through changes in many genes, including ones whose functions are not fully known. Alternatively, more rational methods might be employed. This would involve identifying systems which are known to be involved with stress tolerance and making specific changes which aim to enhance these functions. For example, ethanol tolerance improvement could be achieved through approaches including enhancing the unfolded protein response (UPR), or engineering the enzymes involved in the biosynthesis of cell membrane lipids [87][88][89][90]. Tools including genome shuffling and global transcription machinery engineering have successfully bred stress tolerant yeast [91][92][93][94][95]. The emergence of powerful technologies including CRISPR-Cas9 should also allow for the better engineering of industrially relevant phenotypic traits [96,97].

Compatible Solutes or Inert Osmolytes
Many microorganisms produce, or increase production of, small molecules in response to external stresses. These compatible solutes are typically hydrophilic and facilitate the protection of biological macromolecules and cells [98,99]. The main yeast compatible solutes are glycerol, proline and trehalose ( Figure 1). With the exception of glycerol, these compounds are kosmotropic and promote the ordering of biological macromolecules and assemblies, see Section 3.3 [100]. The physical biochemistry underpinning the mechanism of stabilization has not been fully elucidated [101]. It is likely to involve both direct and indirect effects [102]. Some compatible solutes bind to, and stabilize, biological macromolecules [103]. Kosmotropes generally act indirectly by increasing the ordering of water molecules, i.e., reducing the entropy of the system [104,105]. This enables them to "neutralize" the effects of chaotropes such as ethanol and to protect biological macromolecules from thermal, acid, or alkali denaturation. In the case of osmotic stress, they increase the osmolarity of the cytoplasm and thus help equalize the osmotic pressure on both sides of the plasma membrane [106].
Glycerol is a three carbon, trihydroxy alcohol. It is a by-product of ethanol fermentation in S. cerevisiae, having a role in maintaining cellular redox balance, with imbalances leading to oxidative stress [107]. Glycerol production also prevents osmotic-induced oxidative stress, caused by the intracellular accumulation of reactive oxygen species (ROS) [108]. Aquaporins and glycerol facilitators allow for the rapid export of glycerol for the conditioning of the external cellular environment. It is important in the adaptation to low osmolarity, due to its stabilizing effects on cellular components [106]. Glycerol is a three carbon, trihydroxy alcohol. It is a by-product of ethanol fermentation in S. cerevisiae, having a role in maintaining cellular redox balance, with imbalances leading to oxidative stress [107]. Glycerol production also prevents osmotic-induced oxidative stress, caused by the intracellular accumulation of reactive oxygen species (ROS) [108]. Aquaporins and glycerol facilitators allow for the rapid export of glycerol for the conditioning of the external cellular environment. It is important in the adaptation to low osmolarity, due to its stabilizing effects on cellular components [106].
Proline is an imino (secondary amino) acid which is synthesized in yeast from glutamate by the actions of the enzymes Pro1p, Pro2p and Pro3p [55]. Unlike glycerol, proline biosynthesis does not appear to be upregulated in response to stress. Natural levels may contribute to stress resistance and strains engineered to produce more, or consume less, proline, show greater tolerance to a range of stresses [109]. Trehelose is a disaccharide of two glucose subunits joined by an α (1→1) glycosidic bond. It is synthesized in yeast from UDP-glucose and glucose 6-phosphate by the enzymes Tps1p and Tps2p [110]. The genes encoding these enzymes have stress response elements upstream ensuring then they are coregulated [111]. The transcription factors responsible for activating the genes encoding these enzymes, along with others regulated by STREs, are Msn2p and Msn4p [50]. These proteins translocate to the nucleus under stress conditions [74,112]. Trehalose itself assists in the activation of the heat shock response via the transcription factor Hsf1p [113].

The Unfolded Protein Response (UPR)
The unfolded protein response facilitates generic, cell wide responses to stresses which results in disruptions to "proteostasis" or the accumulation of unfolded proteins in the endoplasmic reticulum [114]. The UPR also affects general processes related to secretory pathway homeostasis and therefore plays a maintenance role in yeast cell wall integrity [115]. Its rapid initiation is ensured by cells constitutively expressing mRNA encoding Hac1p [116]. In the non-stressed state this mRNA is maintained in the unspliced form which is not translationally active. Increased levels of unfolded protein are sensed by the chaperone Kar2p. This causes it to stop binding Ire1p, a protein with both nuclease and kinase activities [117][118][119][120]. Ire1p can also directly detect unfolded proteins and damaged phospholipids [121,122]. HAC1 mRNA is spliced by Ire1p and the RNA ligase Trl1p [116,120,123]. It is then translated to produce the transcription factor Hac1p which then translocates to the nucleus where it binds to the UPR response element. This sequence lies upstream from genes which are regulated by the UPR [124]. Hac1p then recruits RNA polymerase and other proteins required for transcriptional activation and increases the expression of a range of genes which code for proteins such as chaperones, and enzymes for phospholipid biosynthesis, cell wall biogenesis, Figure 1. Structures of the three main compatible solutes deployed by yeast. Note that there is little structural similarity between these molecules, except that they all relatively small and hydrophilic.
Proline is an imino (secondary amino) acid which is synthesized in yeast from glutamate by the actions of the enzymes Pro1p, Pro2p and Pro3p [55]. Unlike glycerol, proline biosynthesis does not appear to be upregulated in response to stress. Natural levels may contribute to stress resistance and strains engineered to produce more, or consume less, proline, show greater tolerance to a range of stresses [109]. Trehelose is a disaccharide of two glucose subunits joined by an α (1→1) glycosidic bond. It is synthesized in yeast from UDP-glucose and glucose 6-phosphate by the enzymes Tps1p and Tps2p [110]. The genes encoding these enzymes have stress response elements upstream ensuring then they are coregulated [111]. The transcription factors responsible for activating the genes encoding these enzymes, along with others regulated by STREs, are Msn2p and Msn4p [50]. These proteins translocate to the nucleus under stress conditions [74,112]. Trehalose itself assists in the activation of the heat shock response via the transcription factor Hsf1p [113].

The Unfolded Protein Response (UPR)
The unfolded protein response facilitates generic, cell wide responses to stresses which results in disruptions to "proteostasis" or the accumulation of unfolded proteins in the endoplasmic reticulum [114]. The UPR also affects general processes related to secretory pathway homeostasis and therefore plays a maintenance role in yeast cell wall integrity [115]. Its rapid initiation is ensured by cells constitutively expressing mRNA encoding Hac1p [116]. In the non-stressed state this mRNA is maintained in the unspliced form which is not translationally active. Increased levels of unfolded protein are sensed by the chaperone Kar2p. This causes it to stop binding Ire1p, a protein with both nuclease and kinase activities [117][118][119][120]. Ire1p can also directly detect unfolded proteins and damaged phospholipids [121,122]. HAC1 mRNA is spliced by Ire1p and the RNA ligase Trl1p [116,120,123]. It is then translated to produce the transcription factor Hac1p which then translocates to the nucleus where it binds to the UPR response element. This sequence lies upstream from genes which are regulated by the UPR [124]. Hac1p then recruits RNA polymerase and other proteins required for transcriptional activation and increases the expression of a range of genes which code for proteins such as chaperones, and enzymes for phospholipid biosynthesis, cell wall biogenesis, DNA repair and aerobic metabolism [117,122,[125][126][127]. They precise subset of genes upregulated depends partly on the conditions giving rise to stress [122,128,129]. Cell wall stress also activates the broad transcriptional response of the UPR through the mitogen-activated protein (MAP) kinase cascade, with ER stress also activating the CWI pathway, demonstrating that the UPR and CWI are co-ordinately regulated in Saccharomyces cerevisiae in order to protect cells against related stressors [130].

Osmotic Stress
High initial fermentable sugar concentrations is particularly pertinent to first-generation bioethanol production, with high initial external osmolarity in an alcoholic fermentation facilitating the passive diffusion of cellular water down the concentration gradient, leading to hyperosmotic stress [106]. Effects include rapid decrease of cell volume and turgor pressure, with the concurrent generation of reactive-oxygen species (ROS), leading to redox state imbalances and oxidative stress. Oxidative stress and osmotic stress differ significantly but display overlapping responses with the robust yeast continuing to proliferate over a range of external water activities (a W ) [108]. Intracellular volume and water balance are tightly regulated through cell osmoregulation, for the proper maintenance and functioning of biochemical and biological processes [66].
Interest in the molecular mechanisms of yeast osmoadaptation originated from the need to improve the performance of yeast strains under industrial conditions. Hyperosmotic stress disrupts cytoskeleton structure, causes the remodeling of chromatin and can even lead to cell cycle arrest and apoptosis [131][132][133]. An intracellular shift in metabolism results, mediated by the high-osmolarity-glycerol (HOG) pathway and cell wall integrity (CWI) pathways, producing glycerol as a compatible solute and providing cell surface stability respectively, with these processes being co-coordinatively linked [66,115,134]. This cross talk between the two pathways suggests the evolution of highly coordinated stress responses.
The CWI pathway responds to damage to the cell wall. Extracellular domains of transmembrane receptors sense chemicals which may damage the cell wall [135]. The pathway also responds to osmotic, ethanol and pH stress. How these stresses are sensed is currently unknown [136]. Following sensing of cell wall associated stress, a signaling pathway connects the cell membrane with the MAP kinase Slt2p [136,137]. This kinase then activates two transcription factors, Swi4p/Swi6p and Rlm1p [57,138]. The first of these normally regulates cell cycle progression, but in this case regulates some genes involved in cell wall biosynthesis [139,140]. Rlm1p controls the expression of at least 80 genes including those which code for enzymes involved in the synthesis of cell wall components [141,142]. The HOG pathway also responds to cell surface receptors activating a signaling pathway which culminates in the activation of the MAP kinase Hog1p [46,66,143]. Phosphorylated Hog1p acts both in the nucleus and the cytoplasm. In the nucleus it phosphorylates and activates transcription factors which control the expression of over 600 genes [144,145]. This includes genes responsible for controlling the production of compatible solutes and the pausing of the cell cycle in G2 phase [146]. In the cytoplasm Hog1p directly reduces the export of glycerol thus increasing the cellular concentration of this compatible solute [147,148].

Heat Stress
Stress due to high temperatures can be encountered in ethanol fermentations, particularly in the early stages or if the external temperature is higher than the optimum for the fermentation [149,150]. Excess temperatures also increase sensitivity to ethanol [151]. A brief discussion of the effects of, and remedies for, heat stress is included here due to their similarity with chaotrope stress, see Section 3.3 [65]. High temperatures represent a general threat to living systems. Proteins, nucleic acids and phospholipid bilayers are denatured by increased temperature. Therefore, cells respond by counteracting this. Chaperone protein expression is upregulated In the so called heat shock response [152]. These proteins assist folding of nascent proteins and those which become partially unfolded as a result of denaturation by temperature and other stresses. Membrane composition is altered. Shorter and unsaturated fatty acids in phospholipids are replaced by longer and less saturated ones [153]. Both longer and less saturated fatty acids have higher melting temperatures and thus increase membrane rigidity. This is achieved by the expression of enzymes which extend the number of carbon atoms in fatty acids, e.g., Elo1p [43]. The amount of ergosterol in the membranes also increases [154]. Ergosterol is the predominant sterol in yeast membranes; increasing the mole fraction of this sterol in the membrane causes it to become more rigid and promotes the formation of lipid rafts [155][156][157]. Some strains which have been artificially evolved to have enhanced long term thermotolerance produce fecosterol in place of ergosterol which further rigidifies the membrane (Figure 2) [158]. In industrial fermentations, heat stress can be mitigated by thermostatic control of the reaction vessels. The use of thermotolerant strains also reduces the negative effects of heat stress [159][160][161]. Given the similarities between heat and chaotrope stress these strains are also likely to have improved tolerance to high ethanol concentrations [65,162,163]. Alternatively, additives to the fermentation mix, such as magnesium sulphate, can improve thermotolerance [164]. a result of denaturation by temperature and other stresses. Membrane composition is altered. Shorter and unsaturated fatty acids in phospholipids are replaced by longer and less saturated ones [153]. Both longer and less saturated fatty acids have higher melting temperatures and thus increase membrane rigidity. This is achieved by the expression of enzymes which extend the number of carbon atoms in fatty acids, e.g., Elo1p [43]. The amount of ergosterol in the membranes also increases [154]. Ergosterol is the predominant sterol in yeast membranes; increasing the mole fraction of this sterol in the membrane causes it to become more rigid and promotes the formation of lipid rafts [155][156][157]. Some strains which have been artificially evolved to have enhanced long term thermotolerance produce fecosterol in place of ergosterol which further rigidifies the membrane (Figure 2) [158]. In industrial fermentations, heat stress can be mitigated by thermostatic control of the reaction vessels. The use of thermotolerant strains also reduces the negative effects of heat stress [159][160][161]. Given the similarities between heat and chaotrope stress these strains are also likely to have improved tolerance to high ethanol concentrations [65,162,163]. Alternatively, additives to the fermentation mix, such as magnesium sulphate, can improve thermotolerance [164].

Chaotrope Stress
Chaotropes are compounds which cause the disordering of biological macromolecules such as proteins and nucleic acids, as well as the disruption of biological assemblies held together by non-covalent interactions such as cellular membranes [100,165]. Many chaotropes do not to interact directly with the macromolecules and assemblies. Instead, they disrupt the hydrogen bonding networks in the solvent (water) thus increasing the overall entropy of the system [166,167]. However, the mechanism of chaotropicity is likely to vary between different compounds [165,168]. Since chaotropes disrupt critical cellular macromolecules and assemblies they exert a detrimental effect on biological systems. They affect key features of proteostasis and impose a dosage dependant "fitness cost" due to the "toxicity" of misfolded proteins [169,170]. The products of ethanol fermentations are chaotropic. Thus, the production of alcohol will ultimately be self-limiting as there will come a point where the concentration of the chaotropic compound exceeds the cell's ability to mitigate it [32]. These mitigations are similar to those seen in high temperatures stress [65]. The ER's quality control system, involving the unfolded protein response becomes activated [87]. This leads to increased expression of ER-located molecular chaperones whilst also aiding in the coordinated transition from fermentative metabolism to slower mitochondrial respiration (post-diauxic shift) during starvation [171]. The fatty acids in membrane phospholipids become longer and less saturated, catalyzed by Elo1p and Ole1p respectively. Compatible solutes such as glycerol and trehelose are produced [172].
Ethanol, a chaotropic solute, is utilized commercially as a protein denaturant, with chaotropic agents in general entropically disordering macromolecular systems, adversely affecting cell constituents including phospholipid bilayers, proteins, and other hydrated cell components [100]. The chaotropic activity of common fermentation products including ethanol (+5.93 kJ kg −1 mol −1 ) and some by-products of lignocellulose pre-treatment including vanillin (+174 kJ kg −1 mol −1 ), have been quantified using the agar gelation-temperature model system [100]. The growth rates susceptibility of S. cerevisiae to these substances has also been determined [173][174][175]. Chaotropes, including the aliphatic alcohols, cross and perturb cellular membranes in the high millimolar to molar range leading to a reduction in cell viability, slower growth/proliferation and lower obtainable ethanol yields [37,76,173].
Mitigation of chaotropicity in industrial settings is difficult to achieve. If a fermentation runs to completion, the alcohol poisons the cells. Furthermore, the production of glycerol, which is miscible with ethanol, presents an additional problem since the ethanol must be purified from this mixture. Kosmotropes are they opposite of chaotropes. They cause ordering and rigidity in biological macromolecules and phospholipid membranes. Typically, they reinforce the hydrogen bonding networks in the solvent, reducing the overall entropy of the system [176]. In theory, kosmotropes could be added to fermentation mixes to alleviate chaotrope stress. Indeed, the compatible solutes trehelose and proline are kosmotropic [100]. Glycerol presents an intriguing paradox. It is a compatible solute produced in response to stress by many microorganisms. However, in the agar gel setting assay it was shown to be mildly chaotropic [100]. Other studies suggest glycerol can enhance the hydrogen bonding network in water-a kosmotropic attribute [177,178].
There are a number of issues with using kosmotropes to neutralise chaotrope stress. First, some kosmotropes are themselves toxic to yeast cells and impair efficient growth and fermentation [175]. Second, there is currently no method of rapidly measuring or predicting the net solution chaotropicity. So, knowing the concentration of kosmotrope required to neutralize a given concentration of ethanol is currently impossible [179]. Third, the addition of chemicals to the fermentation mix will alter they osmolarity of the medium and may cause osmotic stress in the yeast. Fourth, the additional cost of purchasing kosmotropic compounds and, potentially, separating them from the ethanol at the end of the process may render this strategy nonviable.

Other Stresses
Stresses do not occur in isolation. Almost all instances of compounds which cause one form of stress also induce others [32,101]. For example, ethanol causes both osmotic and chaotropic stress [99]. In the context of ethanol fermentations, oxidative, and pH stress are less commonly encountered or occur as a consequence of other stresses.
Oxidative stress can be caused by chaotropic and osmotic stress during fermentations. Both can cause the buildup of reactive oxygen species. These free radicals cause non-specific damage to biomacromolecules and membrane lipids [180]. Uncorrected damage to DNA can cause hereditable mutations which are likely to be deleterious. Reactive oxygen species can also cause lipid peroxidation which damages cell membranes [181]. Yeast cells have a complex, integrated responses to free radical production [182]. This includes enzymes which repair double and single strand breaks in DNA, which reverse lipid peroxidation and which directly reduce reactive oxygen species [183][184][185][186].
Yeast cells normally condition the growth media to a slightly acidic pH. This requires the export of protons across the cell membrane by the plasma-membrane H + -ATPase, Pma1p [52]. Thus, alkaline conditions represent an external stress for yeast with growth being slow or undetectable above pH 8.2 [187,188]. Multiple systems respond to alkaline stress [188]. One key pathway involves the calcium activated phosphatase calcineurin complex which is comprised of one of two catalytic subunits, Cna1p or Cmp2p, and one regulatory subunit, Cnb1p [40,41,189]. This activates the transcription factor Crz1p which controls the expression of stress related genes [190]. In addition, signaling through protein kinase A is transiently inhibited leading to the nuclear localization of the transcription factors Msn2p and Msn4p which bind to stress response elements, see above [191]. One consequence of alkaline stress is a reduction in mRNA transcription rates and in the stability of mRNA molecules [192].
A key response is the upregulation of the ATP dependent sodium ion transporter Ena1p [193]. This is regulated by both the calcineurin and protein kinase A pathways [194]. It is not immediately obvious why the export of sodium ions into the alkaline medium would mitigate the stress caused by excess hydroxide irons. It is generally considered that, unlike many mammalian sodium transporters, Ena1p does not cotransport protons although some early evidence suggests that it may do so [195]. The export of sodium ions may also assist in maintaining the membrane potential under conditions where protons are likely to be consumed.
Excessively acidic conditions also cause stress in yeast. Weak acids such as acetic acid, citric acid and boric acid can inhibit yeast growth and induce apoptosis [196]. These compounds are transported out of the cell by multidrug resistance transporters [197,198]. Pma1p activity is increased in order to transport excess protons out of the cell [199]. Acidification of the external medium results in a corresponding decrease in the pH of the vacuole suggesting that the cell transports excess protons to this compartment in order to maintain cytoplasmic pH [199].
In fermentations, the pH can be monitored and controlled by inline sensors and the addition of appropriate amounts of acid or base. The amounts can be determined either by feedback control or estimation using the Henderson Hasselbalch equation.

Conclusions
S. cerevisiae is considered a robust and versatile microorganism and has become an outstanding model system for the elucidation of Eukaryotic cell stress biology. Responses to each form of stress typically involve complex interactions of signaling pathways and transcription factors. This can affect the expression how many different genes. Although genomic and proteomic studies have revealed many candidate genes and proteins for the mitigation of various forms of stress, unravelling and understanding these data is challenging. In particular, understanding which genes are critical for response compared to those whose expression changes consequentially (e.g., many stress responses require energy and so ATP generating metabolic pathway enzymes are likely to be upregulated as a consequence). Distinguishing between those changes which make a minor contribution to stress responses compared to more significant contributions is critical. Here is should be noted that fold changes in expression levels do not provide a reliable guide as to which changes are the most important. This can only be determined by further biochemical experiments, for example assessing the response of strains with key genes deleted. The major players are likely to be those which act in systems which have been identified by traditional molecular biology approaches to be involved in stress remediation. The heat shock response, for example, produces coordinated sets of actions which mitigate the effects of heat on cellular components. Unsurprisingly, elements of this response are also used in adaptations to other forms of stress.
Understanding these responses is critical to enabling rational efforts to engineer strains of yeast which are better adapted to stressful conditions. Inevitably, there must be limits to the amount and duration of stress which yeast can endure. These limits will ultimately impose maximum yields in ethanol fermentations and determine whether, or not, it will be economically viable to transition away from fossil fuels towards sustainable biofuels. Cellular stresses incurred by yeast throughout a bioethanol fermentation impact on cell growth and fermentative metabolism, reducing final obtainable ethanol titers. The plethora of general and stress-specific response and tolerance mechanisms makes yeast an industrially applicable microorganism. There are two main strategies for minimalizing the effects of stress on an ethanol fermentation. First, we can minimize the stress caused to the yeast. For example, temperatures and pH values of fermentations can be strictly control. To do this requires an understanding of the situations which cause the yeast cells to be stressed and thus reduce ethanol productivity. Second, we can select or engineer strains which are resistant to stresses. That many stress responses overlap assists us in this process. For example, yeast which are highly resistant to temperature stress are also likely to be able to resist high concentrations of ethanol. Again, this requires a knowledge of the biochemistry of stress sensing, stress responses and of unmitigated stresses. Therefore, it is clear that continued research efforts to understand the biochemistry of stress in yeast is vital for improving the economic and environmental sustainability of the biofuel industry.