In this study the changes occurring in the endo- and exo-metabolome of yeast cells that allow them to grow exponentially in the presence of inhibitory concentrations of ethanol were characterized by high resolution 1
H NMR spectroscopy. Previously, Ding et al.
and Li et al.
used GC-MS-based metabolomic approaches to study the yeast response to ethanol-induced stress at the endo-metabolome level. Based on the metabolites that were reported to be, simultaneously, detected and identified in those studies, the work herein have significantly increased the metabolic coverage at the endo-metabolome level (30% increase), emphasizing the importance of complementary strategies in metabolomics. The simultaneous analysis of the exo-metabolome further increased our understanding, allowing the quantification of metabolites whose presence was only detected in the extracellular environment, and providing clues on whether the changes observed in the endo-metabolome may be due to different catabolic/anabolic rates or to altered partition across the plasma membrane.
The changes induced by ethanol stress at endo- and exo-metabolome level identified in this work are summarized in . A number of metabolic effects from alcohol appear to be directly linked to the production of an excess of both NADH and acetaldehyde. Since ethanol exposure leads to this sort of reductive stress, the latter might trigger the induction of pathways aimed at the elimination of NADH excess (e.g. the conversion of pyruvic acid to lactic acid, the synthesis of glycerol or fatty acids, or the mitochondrial electron transport chain) and thus in the down-regulation of other metabolic pathways needed for optimal growth. NAD(P)+
and NAD(P)H, besides their role in determining the cell redox status 
, are critical determinants of in vivo
reaction kinetics, being involved in over 200 reactions throughout yeast metabolism 
. It is therefore reasonable to hypothesize that the huge differences in the yeast metabolome in the presence of increasing ethanol concentrations are, at least partially, linked with the perturbation of the cell redox status. In fermentative or respiro-fermentative growth conditions, the NADH formed during glycolysis is reoxidized through the reduction of acetaldehyde to ethanol, maintaining in this way the intracellular redox balance 
. However, Martini et al.
showed that the metabolic yield of ethanol production in S. cerevisiae
decreases with increasing concentrations of exogenously added ethanol, in agreement with the known toxic effects of ethanol, namely, decreased fermentation productivity and ethanol yield 
. Therefore, and since the formation of glycolytic NADH beyond the cellular capacity for its reoxidation will lead to reduced conditions, an inhibition in the yeast fermentative capability, particularly at the level of the final alcohol dehydrogenase reaction, may be causing an imbalance in the cell redox status. Another possible cause for such an apparent decreased ability to regenerate NAD+
may be the inhibitory effect of ethanol at the level of the mitochondrial respiratory chain, whose function depends deeply on membrane integrity. Strongly linked with the intracellular redox status is the type and amount of by-products formed during yeast metabolism 
. Indeed, the exo-metabolome analysis carried out herein showed a higher accumulation in the broth medium of acetate, pyruvate, acetoin and acetaldehyde when ethanol is exogenously added, in a dose dependent way. The results obtained are also consistent with the previous observation, based on transcriptomic analysis, that ethanol challenge leads to a strong induction of the expression of TDH1
, encoding a glyceraldehyde-3-phosphate dehydrogenase that utilises NAD+
as a cofactor, suggesting that ethanol stressed-cells may experience reductive stress 
. Also in agreement with this notion, the ratio of reduced to oxidized glutathione was observed, in this study, to increase in a dose-dependent manner in yeast cells growing exponentially in the presence of inhibitory ethanol concentrations.
Integrative overview of the metabolic changes occurring under ethanol stress or upon FPS1 deletion.
Li et al.
suggested that in the presence of ethanol stress there is an inhibition of glycolysis. The increased amount of intracellular and/or excreted glycolytic and TCA cycle intermediate metabolites, including pyruvate, citrate, malate, fumarate and succinate suggests an increase in overflow metabolism in turn of the pyruvate branch-point. The higher accumulation of pyruvate in the broth medium in the presence of ethanol stress is probably a consequence of an increase in difference between the fluxes upstream and downstream of pyruvate, which may be linked with the demand of energy supply for stress responsive processes. The referred overflow metabolism in turn of the pyruvate branch-point may underlie the changes registered in the intracellular content of some amino acids 
. Indeed, the current endo-metabolome analysis identified an overall increase in the intracellular content of most amino acids, which corroborates the observations made in previous metabolomic studies focused on the ethanol stress response 
. One general explanation that could account to an increase in the intracellular concentration of each amino acid could be an inhibition in protein synthesis in the presence of ethanol stress. However, this explanation apparently cannot account for the fact that not all the amino acids that were monitored increased their content with increasing ethanol concentrations as it is the case for L-arginine, L-lysine and L-ornithine. Moreover, the increase fold changes were found to vary significantly between the different amino acids monitored. In particular, the intracellular content of L-alanine and L-valine, two pyruvate-related amino acids, showed a dramatic accumulation in the exponential growth phase with increasing concentrations of ethanol, reinforcing the idea of overflow metabolism in turn of the pyruvate branch-point, as previously suggested to occur under other stress challenges, as was reported for very high gravity fermentation and in propionic acid stressed cells 
. The increased intracellular content of L-aspartate, L-asparagine and L-threonine was also shown to occur in yeast cells grown in the presence of increasing ethanol concentrations. These aminoacids are derived from oxaloacetate, an intermediate of tricarboxylic acid (TCA) pathway that occurs in the matrix of mitochondria. Similar trends were observed in the contents of the TCA pathway intermediates succinate, malate, citrate and fumarate. These observations are consistent with previous indications, suggesting that, even in the presence of glucose-repressing conditions, mitochondrial function is important for yeast tolerance against ethanol stress 
. During alcoholic fermentation, the TCA pathway operates as two branches, a reductive and an oxidative branch 
, aiming the supply of C4 and C5 compounds (oxaloacetate and 2-oxoglutarate), the precursors of L-aspartate and L-glutamate, respectively 
. Interestingly, the interconversion of L-glutamate and L-glutamine, whose concentrations were found to increase in yeast cells thriving in the presence of ethanol stress, has been implicated in the balance of the cell redox status 
. Since L-glutamate is formed from the condensation of ammonium and 2-oxoglutarate, the accumulation of lower amounts of 2-oxoglutarate in the broth medium in the presence of ethanol stress is consistent with the changes registered in the intracellular content of L-glutamate. Aromatic amino acid concentrations were also found to increase in the presence of increasing ethanol concentrations, which may correlate with the identification of aromatic amino acids biosynthetic enzymes as being required for maximal ethanol resistance 
. Although with different magnitudes, we also observed the intracellular accumulation of the amino acids that were supplemented in the growth medium due to the yeast strain auxotrophies (L-leucine, L-methionine and L-histidine) in a dose dependent way. The accumulation of L-leucine and L-methionine may rely on the fact that they may be degraded into fusel alcohols, such as isoamyl alcohol or methionol, in a NAD+
regenerating reactions 
. Interestingly, changes observed in the L-leucine content are consistent with a recent study suggesting that an enhanced uptake and/or utilization of L-leucine may be responsible for improved growth in the presence of ethanol 
. Furthermore, these observations may also indicate the diffusion of externally supplied hydrophobic amino acids across the plasma membrane, which may occur as a consequence of membrane permeabilization by ethanol.
The increased accumulation of several metabolites known for their function as cellular stress protectants was also identified in yeast cells growing under ethanol stress. This was the case of L-proline and trehalose, whose accumulation in yeast cells 
, together with the increased expression of the corresponding biosynthetic genes 
had been previously implicated in cell protection against ethanol stress. Both L-proline and trehalose have been shown to enhance the stability of membranes, and to contribute to protect proteins from denaturation and aggregation (reviewed in 
), two important features for cells growing in the presence of ethanol stress, which induces low water activity stress. In this work we show that L-proline and trehalose accumulation occurs in an ethanol-dose dependent manner in yeast cells growing exponentially in the presence of this stress.
This study also focused on the effect of FPS1
expression in the yeast metabolome, as summarized in . Consistent with the decreased exponential growth rate registered upon FPS1
deletion under control conditions, it was interesting to observe that, even in the absence of ethanol stress, there are significant differences at the metabolome level between the fps1Δ
deletion mutant and the corresponding parental strain. The major change registered was the 13-fold increase in the intracellular accumulation of glycerol in the absence of Fps1, accompanied by a decrease in extracellular glycerol concentration, which is in agreement with the proposed role for this aquaglyceroporin in facilitating the diffusion of glycerol across the plasma membrane 
. Simultaneously, a decrease in NAD+
content was also observed in yeast cells devoid of FPS1
, which may imply that glycerol-3-phosphate dehydrogenase activity, that catalyses a step required for NAD+
regeneration and leads to glycerol formation, is inhibited by the over-accumulation of glycerol. Indeed, FPS1
deletion was previously shown to decrease not only glycerol transport, but also total glycerol production 
. This result suggests that the fps1Δ
deletion mutant may experience a permanent state of redox imbalance, similar to the one observed in this work to affect wild-type cells growing under ethanol stress. Consistent with the hypothesis raised herein that fps1Δ
cells may experience reductive stress, Fps1 was previously identified as conferring resistance to the reductive stress inducers dithiothreitol and mercaptoethanol 
. Given the obtained results, fps1Δ
cells may exhibit an increased susceptibility to ethanol stress due to their intrinsic redox imbalance, which may be further exacerbated under ethanol stress. The higher ethanol tolerance exhibited by cells expressing FPS1
is hypothesized to be due to their increased ability to regenerate NAD+
, through the production and release of glycerol 
, and to the increased trehalose levels produced, assuming that a link between trehalose and glycerol synthesis may exist to fine-tune the concentration of these two molecules with partially overlapping functions as osmoprotectants. Consistent with this hypothesis is the increased glycerol content found in wild-type cells growing exponentially in the presence of ethanol stress, except for those exposed to 6% ethanol, which instead exhibit a very high increase in trehalose levels.
The intra-cellular concentration of a number of amino acids, including L-tyrosine, L-phenylalanine, L-alanine, and L-glutamine, and TCA-related metabolites, including succinate, fumarate and citrate was found to be increased in the fps1Δ
deletion mutant, when compared to the parental strain, mimicking ethanol stress repercussions exerted upon the parental strain. Particularly interesting is the observation that fps1Δ
cells display a decreased concentration of trehalose both in control conditions and under ethanol stress. Given the importance of this metabolite for ethanol tolerance 
, the decreased ability of the fps1Δ
deletion mutant to produce trehalose may also underlie its increased susceptibility towards ethanol stress.
Altogether, this study provides evidence that support the notion that ethanol stress induces reductive stress in yeast cells, which appears to be dealt with by the increase in the rate of NAD+ regenerating bioreactions. The action of Fps1 in mediating the passive diffusion of glycerol is highlighted as a key factor in the maintenance of redox balance, an important feature for ethanol stress resistance. The obtained results further reinforce the idea that metabolomic approaches may be crucial tools to understand the function of membrane transporters/porins, such as Fps1, particularly by providing clues on their physiological substrates and/or the effect of their expression at the global metabolism level. By providing a systems level view of the metabolome changes contributing to ethanol stress tolerance in yeast, this metabolic analysis appears as an important approach to guide the design of process conditions and of more robust yeast strains optimized to improve industrial fermentation performance.