The Proteomic Response of Saccharomyces cerevisiae in Very High

Sep 23, 2008 - Most glycolysis and pentose phosphate pathway proteins were upregulated. Aminoacyl-tRNA biosynthesis and heat-shock protein abundances ...
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The Proteomic Response of Saccharomyces cerevisiae in Very High Glucose Conditions with Amino Acid Supplementation Trong Khoa Pham and Phillip C. Wright* Biological and Environmental Systems Group, Department of Chemical and Process Engineering, The University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom Received April 30, 2008

Ethanol yield by Saccharomyces cerevisiae in very high glucose (VHG) media with an amino acid supplement was investigated. Amino acid supplementation led to positive cell responses, including reduced lag time and increased cell viability in VHG media. A quantitative shotgun proteomic analysis was used to understand how amino acid supplemented S. cerevisiae responds to high osmotic conditions. iTRAQ data revealed that most proteins involved in glycolysis and pentose phosphate pathways were up-regulated under high glucose shock. Reactivation of amino acid metabolism was also observed at the end of the lag phase. The relative abundance of most identified proteins, including aminoacyl-tRNA biosynthesis proteins, and heat-shock proteins, remained unchanged in the hours immediately following application of glucose shock. However, the expression of these proteins increased significantly at the end of the lag phase. Furthermore, the up-regulation of trehalose and glycogen biosynthesis proteins, first maintaining then latterly increasing glycolysis pathway activity was also observed. This was verified by enhanced ethanol yields at 10 and 12 h (0.43 and 0.45 g ethanol/g glucose) compared to 2 h (0.32 g ethanol/g glucose). These data combined with relevant metabolite measurements demonstrates that enhanced ethanol fermentation under VHG conditions can be achieved with the aid of amino acid supplementation. Keywords: S. cerevisiae • iTRAQ • amino acid supplementation • enhanced ethanol fermentation • VHG conditions

Introduction Many studies have reported that, under high osmotic stress conditions, Saccharomyces cerevisiae reduces its nutrient uptake, protein synthesis, and many protein activities including ribosomal proteins. 1-3 The effects of many factors relating to ethanol fermentation under VHG conditions have been comprehensively reviewed.4 It has been shown that cells under VHG conditions demonstrate increased lag time and reduced ethanol yield.5 Many studies have investigated the effects of various media supplements, including yeast extract,6 ammonium,7 calcium and magnesium8 which influenced growth and cell viability as well as fermentation ability, and as a result led to a stimulation of both fermentation rate and ethanol production. The response of yeast to osmotic conditions is closely linked to the physiological state of the cells in culture. Moreover, cells in the exponential phase might be more sensitive to stress conditions than cells in the stationary phase.1 Recently, global gene expression measurements have been used to investigate fermentation under VHG conditions with maltose as the main substrate,9,10 and the profile of intracellular metabolites was also used to investigate the influence of VHG conditions.11 * To whom correspondence should be addressed: Prof. Phillip C. Wright, Biological and Environmental Systems Group, ChELSI, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, U.K. Tel: +44(0)114 2227577. Fax: +44(0)114 2227501. E-mail: [email protected].

4766 Journal of Proteome Research 2008, 7, 4766–4774 Published on Web 09/23/2008

However, a large-scale proteomic analysis of S. cerevisiae to characterize the response of this microorganism during the lagand early exponential phases, under osmotic stress conditions with an improvement of media quality, has not yet been clarified in detail yet. Thus, in this paper, cells grown under standard conditions in the mid-exponential phase were stressed by applying VHG conditions (with and without amino acid supplementation), and comparisons with cells under this stress conditions (with an improvement of media) to the control sample were made. The work presented here builds on our previously published work,12 where immobilization and media improvements were shown to aid in improving ethanol yield. Although, as we reported previously, the immobilization and media improvements led to this improved ethanol yield, a deeper understanding of the temporal proteomic response of S. cerevisiae under VHG conditions has not yet been clarified. In particular, how the S. cerevisiae proteome and intracellular amino acid concentrations changed during the early stages of the applied high glucose shock, as well as the contribution of the relationship between intracellular amino acids fluctuations and protein expressions. The initial response of S. cerevisiae to shock should be understood clearly, since this period plays a key role in enabling yeast to overcome the shock conditions. Building on this previous work,3,12 where a proteomic analysis on the relative protein expressions between free cells 10.1021/pr800331s CCC: $40.75

 2008 American Chemical Society

Proteomic Response to VHG Conditions and immobilized cells (with or without amino acid supplementation) at the late exponential phase was perfomed, this current study focuses on the response of free cells during the initial hours of fermentation (lag and early exponential phases). New proteomic and metabolite data is generated to augment the understanding of S. cerevisiae in responding to VHG conditions from different states (in this study) to different media conditions discussed previously.3,12 This information is to contribute to an understanding of the systems-wide response of cells under VHG conditions with the aim of improving fermentative ethanol yield. We seek here to provide more evidence on the benefits of amino acid supplementation for improved ethanol generation under VHG conditions, that was initially described elsewhere.12 The shotgun proteomics technique employing isobaric tags for relative and absolute quantitation (iTRAQ) was used, as this has been demonstrated to be reliable for assessing the S. cerevisiae proteome (see refs 3, 12-15 for details). Suggestions to further enhance ethanol generation under VHG conditions are also reported.

Materials and Methods Growth Conditions. Preculturing of S. cerevisiae was carried out as described elsewhere.3 Precultured cells were then centrifuged at 3000g at room temperature for 5 min, and then washed twice with deionized water before culturing in 250 mL Erlenmeyer flasks containing 100 mL of media with the same recipe detailed in the literature3 consisting of (g/L) yeast extract, 5; peptone, 3; KH2PO4, 5; NH4Cl, 1.5; MgSO4, 0.7; KCl, 1.7; casamino acids, 5.8; fresh yeast autolysate, 7.2, except the glucose concentration was 20 g/L (the standard condition). The fermentation conditions are as described in Pham et al.; briefly, S. cerevisiae was grown in flasks at 30 °C with shaking at 120 rpm.3 These samples were cultured for 8 h before adding additional glucose to induce stress. Since it was reported that cells in the exponential phase might be more sensitive to stress than cells in the stationary phase,1 at this time (8 h, with a corresponding residual glucose concentration of 15 g/L), 50 mL of media containing 900 g/L of glucose without or with an amino acid supplement was added to the cultures to achieve a final residual glucose concentration of 300 g/L (to induce stress). The mixture of amino acids used was a Complete Supplement Mixture (Sunrise Science Products).12 Because of the 50 mL of extra media, a reduction of OD650 values in these cultures occurred. Thus, to estimate the OD650 reduction, 50 mL of media containing glucose was also added to the control samples. All experiments were performed in biological triplicate. Measurements of Fermentation Parameters. Ethanol concentrations in the broths were quantified using a Finnigan Trace DSQ single Quadrupole GC-MS system coupled with an AS3000 autosampler (Thermo Electron Corporation) fitted with a 30 m × 0.25 mm i.d. × 0.25 µm df Stabilwax fused silica column (Thames Restek, Bucks, U.K.). See Pham et al.,3 for more details. The results were referenced to ethanol calibration curves to obtain the ethanol concentration. Residual glucose and glycerol concentrations were measured using a glucose (GO) assay kit (Sigma, U.K.), and a glycerol assay kit K-GCROL (Megazyme, Ireland). The S. cerevisiae growth rate was monitored by absorbance of the culture at 650 nm (OD650) using an Ultraspec 2100 Pro Spectrophotometer

research articles (Biochrom, England). The numbers of cells were also counted using a Thoma chamber (Marienfeld, Germany). Determination of Cell Viability, Dry Weight, Glycogen, and Trehalose Concentration. The methylene blue technique was used to determine yeast cell viability as described in detail elsewhere.12 The determinations of dry weight using a gravimetric method, glycogen, and trehalose concentrations using enzymes kits were done as also described in Pham and Wright.12 Intracellular Amino Acids Determination. Ten milliliters of culture was harvested and centrifuged at 3000g for 5 min, and cells were then quenched with 50 mL of -40 °C methanol. Suspended cells were centrifuged at 3000g for 5 min, and the pellet was washed twice with 50% methanol before resuspension in 500 µL of deionized water. Cells were then lysed by heating the suspension in boiling water.16 Intracellular metabolites were collected by filtering this suspension with cellulose nitrate membrane filters (pore size 0.45 µm; Whatman, England). Subsequently, amino acids were derivatized with N-methyl-N-t-butyldimethylsilyl-triflouroacetamide (MBDSTFA) as detailed elsewhere,17 with some modifications. Briefly, 20 µL of the samples was dried in a vacuum concentrator prior to dissolution in 16 µL of dimethyl formamide containing 0.1% pyridine, followed by addition of 16 µL of MBDSTFA. Samples were then incubated at 80 °C for 60 min. The identification and quantitation of intracellular amino acids were performed using the GC-MS system described above. One microliter was used for injection. A temperature gradient was performed with a hold at 120 °C for 5 min, followed by ramping at 5 °C/min to 300 °C, and holding for 5 min at 300 °C. The analysis was performed with the MS detector, with a 1 mL/min He carrier gas. The identification of each amino acid was achieved by comparing the MS spectra (corresponding to retention time) of the samples to the MS spectra of a standard amino acids solution. The quantitation values were determined using calibration curves based on the comparison between the peak areas (of the spectrum corresponding to each individual amino acid) of samples and a standard solution. A standard solution containing 1 mM for each amino acid was purchased from Sigma-Aldrich (U.K.). Labeling, Mass Spectrometry, and Data Analysis. Protein extraction, labeling, mass spectrometry, and data manipulation for proteomics analysis were performed as described elsewhere.3 In brief, cells harvested were washed twice with deionized water and twice with yeast extract buffer (50 mM 2-morpholinoethanesulfonic acid (MES), 10 mM EDTA, and 10 mM MgCl2 at pH 4.5). Proteins were extracted using glass beads (425-600 µm) coupled with vigorous vortexing for 9 min (45 s vortex and 45 s in an ice bath) in the yeast extract buffer and 5% (v/v) of a protease inhibitor cocktail (Sigma, U.K.). The yeast genome database (6298 proteins) downloaded from NCBI (June 2006) was used. The total protein concentration from all cell extracts was measured using the RC DC Protein Quantification Assay (Bio-Rad, Hertfordshire, U.K.) according to the manufacturer’s protocol. Biological triplicate samples were pooled for further proteomic analysis. For iTRAQ experiments, 100 µg of protein in 20 µL of 500 mM TEAB from each phenotype was reduced, alkylated, digested, and labeled with iTRAQ reagents according to the manufacturer’s (Applied Biosystems) protocol with some modifications. These included a 2-day tryptic digestion and 2 vol of ethanol used during labeling. The sampling times for the iTRAQ analysis were at 0 h (unstressed condition), 2 h (following application of stress), 10 h (end of Journal of Proteome Research • Vol. 7, No. 11, 2008 4767

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Pham and Wright The two most abundant charged peptides above a 5 count threshold were selected for MS/MS and dynamically excluded for 60 s with (50 mmu mass tolerance. Protein identification and quantification was carried out using ProQuant software v1.1 (Applied Biosystems, MDS-Sciex). The tolerance search parameters up to 0.15 and 0.1 Da were used for peptide and MS/MS, respectively; one missed cleavage of trypsin. The identification and quantification were performed based on only peptides with above 70% confidence. ProGroup Viewer software v1.0.6 was used to identify proteins with at least 95% confidence. The results obtained from ProGroup Viewer were exported to Microsoft Excel for further analysis (for more details see ref 3). To obtain the confidence ratios, the EF (Error Factor) of the ratio was considered (see refs 3 and 12 for detail). Information on proteins was referenced from the Saccharomyces Genome Database (http://www.yeastgenome.org/), as well as from KEGG (http://www.genome.jp/kegg/pathway.html) for major metabolic pathway reconstructions.

Results and Discussion

Figure 1. The growth of S. cerevisiae (A) and ethanol concentration (B) under standard conditions (20 g/L of glucose) (control samples) without (9) or with (0) a dilution of OD650 (see the text for details), and VHG conditions (300 g/L of glucose) without (O) with (b) amino acid supplementation. All experiments were performed in triplicate. The numbers in the dashed boxes indicate sampling times for iTRAQ proteomic analysis.

lag phase), and 12 h (exponential phase). See Figure 1A for a summary. After a 2-h incubation period, labeled samples were pooled and dried in a vacuum concentrator. Samples were fractionated using a SCX technique on a BioLC HPLC unit (Dionex, Surrey, U.K.), and each fraction was then dried in a vacuum concentrator. For LC/MS analysis, each dried SCX peptide fraction was redissolved in 100 µL of Switchos buffer containing 0.1% formic acid and 3% acetonitrile and then 20 µL of sample was introduced to the nano-LC-QStar ESI-qQTOF-MS/MS (Applied Biosystems, Framingham, MA; MDSSciex, Concord, Ontario, Canada). The LC gradient started with 3% Buffer B (0.1% formic acid in 97% acetonitrile) and 97% Buffer A (0.1% formic acid in 3% acetonitrile) for 3 min, followed by 3-25% or 30% Buffer B for either 60, 90, 120, or 127 min, then 90% Buffer B for 7 min, and finally 3% Buffer B for 8 min. The mass spectrometer was operated in the positive ion mode, with a selected mass range of 300-2000 m/z. Peptides with +2 to +4 charge states were selected for survey. 4768

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Cell Growth during the Lag Phase under Stress Conditions. Since 50 mL of media was added to the broths, dilutions in OD were observed (see Figure 1A) for the control (20 g/L) and VHG sample sets (without and with amino acid supplementation). There were two control sample sets used here, one was grown continuously without the addition of 50 mL of media, and another was grown with the addition of 50 mL media. Because a dilution occurred in one of two sets of control samples, a lower OD in one of the control samples sets was observed. However, the congruent growths of S. cerevisiae between two sets of control samples were also observed (Figure 1A). It was also noticed that the cell growth occurring between -2 and +2 h seems to be linear (instead of exponential phase); this might be caused by the oxygen limitation in the 3 biological replicate flasks. Growth of S. cerevisiae paused after VHG conditions were initiated, as a result of new lag phases under these conditions, while the growth in control samples (both sets) continued to reach the stationary phase, as seen in Figure 1A. There was a clear difference in S. cerevisiae growth under VHG conditions without and with amino acid supplementation. The S. cerevisiae lag phase was shorter under VHG conditions with amino acid supplementation, compared to cultures without such a supplement (4 h compared to 8 h). There was also an OD decrease under VHG conditions without amino acid supplementation (during the first 4 h), with the data here in agreement with previous observations.3 The faster growth recovery of S. cerevisiae under VHG conditions with amino acid supplementation indicates that the supplement helps cells tolerate VHG conditions. To investigate how the cells respond to VHG conditions with and without an amino acid supplement, compared to standard conditions, an analysis of the intracellular amino acid concentrations during the lag phase and early exponential phase was carried out, with results summarized in Figure 2. The Fluctuations in Intracellular Amino Acid Concentrations. A significant decrease of most amino acids during VHG conditions was found, especially for histidine, leucine, lysine, arginine, alanine, aspatate, and tryptophan as seen in Figure 2. However, the recovery of intracellular amino acid levels under VHG conditions with an amino acid supplement was faster at ca. 6 h in comparison to cultures without such a supplement, where the amino acid concentrations recovered after 10 h (see Figure 2). Interestingly for cells grown in VHG

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Figure 2. The concentrations of intracellular amino acids in cells grown in standard (0) and VHG conditions with (b) or without (O) amino acid supplementation. All experiments were performed in triplicate. Journal of Proteome Research • Vol. 7, No. 11, 2008 4769

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Figure 3. The expression of proteins at 2, 10, and 12 h after initiation of VHG conditions with amino acid supplementation compared to control conditions (at 0 h). Red, green and yellow colors represent numbers of down-regulated, up-regulated, and unchanged proteins, respectively.

conditions with amino acid supplementation, where 10 h after being shocked by high osmotic stress (VHG), the intracellular amino acids levels had recovered and surpassed the concentration of the nonstressed controls. The faster recovery of intracellular amino acids may have helped cells increase their tolerance, and as a result decreased the duration of the growth lag phase under VHG conditions (with amino acid supplementation), as shown in Figure 1A. The data depicted here are in agreement with observations published recently, when the addition of amino acids was used to study the effect of osmotic stress (NaCl) on yeast.16 When cells reached the stationary phase, there was a decrease of intracellular amino acids (because of nutrition limitations) in the cells as illustrated in Figure 2 for the control sample at 10 and 12 h, while a significant increase in the intracellular amino acid concentrations under VHG conditions (both with and without amino acid supplementation) were observed in these times. The concentrations of histidine, leucine, alanine, tyrosine, phenylalanine, and methionine were significantly increased after 6 h following the VHG shock for cells growing with amino acid supplementation, while these amino acids concentrations in cultures without an exogenous amino acid supplement increased after 10 h. The increase of intracellular amino acids was in agreement with the OD650 increase after 10 h for cells grown under VHG conditions. It can be seen that, after the supplementation of amino acids in VHG media, the OD was similar to the OD of the control samples at 12 h following application of stress (Figure 1A). As detailed previously,3 many proteins related to amino acids metabolism were down-regulated in response to VHG conditions, due to high osmotic stress arising from high concentrations of both glucose and ethanol. To date, many transcriptionallevel analyses for relative comparisons between normal and stress conditions have been performed.18-21 These studies show that the osmotic stress tolerance in S. cerevisiae was influenced by the up-regulation of genes involved in the biosynthesis and accumulation of glycerol, trehalose, and glycogen, with the roles of many other up-regulated genes yet to be determined.20,21 However, the response of yeast to such stress conditions cannot be fully characterized, since up- or down-regulations of individual genes cannot directly decide and exert biological functions.16 4770

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The intracellular amino acid depletion led to decreased transport through the membrane.22 Furthermore, the delayed up-regulation of many amino acid biosynthesis proteins suggests that there is amino acid limitation under VHG conditions due to high osmotic stress, and that amino acid supplementation can increase cellular tolerance under these conditions. The lack of amino acids might result from their increased demand in the cells to redistribute the intracellular concentration of many different enzymes to improve stress tolerance.16 Therefore, a strain with increased transport processes and amino acid biosynthesis might be a suitable for fermentation under VHG conditions. The metabolites were obtained from different sources (forms), such as ethanol and glucose in the broths, while amino acid and proteins were from the cells. Therefore, to facilitate the understanding (linking) of these data, some relationships between these metabolites (sources) were made and presented as follows: The relationship between OD and number of cells: OD ) 0.21 ∼ 106 cells (per 1 mL). To unify the calculation and evaluation of each amino acid concentration, a linear relation between cell dry weight and OD650 was made, dry weight ) OD650 × 0.46 (g/L of the broths). For iTRAQ analysis, 20 mL of the broths was collected and extracted as described in the Materials and Methods. From these relationships, the reader may link data for comparison, for example based on 106 cells. The Identification and Classification of Detected Proteins. To estimate how long proteome expression distributions took to recover, samples were taken at 0, 2, 10, and 12 h after application of the VHG conditions. These times were compared to a control sample harvested before initiation of VHG conditions (at 0 h) and amino acid supplementation. The important quantitative proteomic observations are summarized in Table SI (in the Supporting Information). We showed previously that most amino acids related and heat-shock proteins were decreased in relative abundance in response to VHG conditions compared to controls containing lower glucose concentrations. The expressions of important proteins are summarized in Table SI in the Supporting Information by function, and as a function of time in Figure 3. From Figure 3, we can see that 22%, 39%, and 33% of detected glycolysis proteins were up-regulated at 2, 10, and 12 h, respectively, compared to the control, while the remaining

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Figure 4. The relationship of most amino acid metabolic systems in relationship with proteins detected here (modified from ref 35). Most proteins were up-regulated, leading to increased amino acid metabolism activity in cells.

detected glycolysis proteins were unchanged in abundance. Correspondingly, the relative expressions of pentose phosphate pathway associated proteins were also increased. These data suggest that glycolysis, and by implication glycolysis protein activities, was not repressed by this stress condition, and the enhanced protein abundance led to the increased ethanol yield seen at 10 and 12 h (see below for details). A similar phenomenon was observed for starch, sucrose, and energy metabolic proteins. Another surprise here was the differential expression of amino acid related proteins, since there was a recovery in the expression of these proteins observed at 10 and 12 h post stress induction. A total of 38% of the detected amino acid metabolism related proteins were down-regulated at 2 h post glucose shock, with this ratio reducing to 27% by 10 h, and then to only 8% at 12 h postshock. Up-regulation of several amino acid metabolism related proteins was also observed. In this case, only 12% of up-regulated proteins were observed at 2 h post shock, with this ratio increasing to 31% at 10 h, and then to 54% at 12 h post glucose shock (Figure 3). Additionally, 29% of detected proteins with putative stress-response functions were down-regulated at 2 h postshock. However, the abundances of these proteins recovered, and even increased (up to 14%) by 10 h, with further increases to 43% at 12 h postshock. Unsurprisingly, most detected proteins involved with aminoacyl-tRNA biosynthesis were unchanged in expression at 2 h, but had subsequently dramatically increased abundances by up to 100% at 10 and 12 h postshock. The expressions of many of proteins are discussed in the following sections in the context of metabolite measurements, to supply

corroborative evidence of the benefit of amino acid supplementation to VHG cultures aimed at enhanced ethanol production. The Expression of Proteins in Response to VHG Conditions. 1. Expressions of Amino Acids Biosynthesis Related Proteins and t-RNA Synthetases. Under VHG conditions with amino acid supplementation, up-regulation of most proteins (compared to control samples) involved with important processes such as glycolysis, trehalose and glycogen biosynthesis, biosynthesis of amino acids, translation and growth process, cell division, cycle, and homeostasis was observed. Indeed, proteins relating to amino acids biosynthesis (e.g., Ilv3p, Ilv5p, Leu4p, Met6p, Din7p, Bat1p, Ura1p, and Ura2p) were significantly up-regulated as a function of time (see Figure 4). In brief, their expression was decreased (or unchanged) at 2 h, but was increased significantly at 10 and 12 h. The functions of these proteins (Ilv3p, Ilv5p, Leu4p, Met6p, Din7p, Bat1p, Ura1p, and Ura2p) can be found not only in generating amino acids as precursors, but also other processes such as maintaining mitochondrial DNA (Ilv3p, Ilv5p),23 regulation of mitochondrial genome stability (Din7p),24 or in iron homeostasis (Bat1p). The observed expression changes for Ilv3p, Ilv5p, Leu4p, Met6p and Din7p are in agreement with yeast growth observations, since high expressions of these proteins at 12 h were congruent with increased growth from 12-24 h (data not shown). These proteomic data are also in agreement with the fluctuations in intracellular leucine and valine concentrations depicted in Figure 2. In contrast to those proteins that were down-regulated at the onset of high glucose shock, expressions of Ura1p and Ura2p were significantly increased at both 10 and 12 h, suggesting Journal of Proteome Research • Vol. 7, No. 11, 2008 4771

research articles that the de novo synthesis of pyrimidine ribonucleotides might be also activated at this time. Most detected tRNA (tRNA) synthetases were significantly increased in relative abundance at 10 h post glucose shock, possibly as a result of the stimulation of high intracellular amino acid concentrations observed at this time. Table SI in the Supporting Information summarizes the expression changes for the detected aminoacyl-tRNA synthetases. Briefly, these synthetases are essential, as well as being related to the regulation of amino acids biosynthesis and amino acids transport.25 Moreover, the expression of most detected ribosomal proteins here increased significantly at 12 h, especially Rpl19ap and Rpl19bp. Obviously, the high expression of these proteins at 10 and 12 h resulted in increased growth, especially at 12 h. Additionally, ribosomal protein expression was up-regulated during the early stage glucose shock. We detected components of the large 60S ribosomal subunits, and components of the small 40S ribosomal subunits. In yeast, the biogenesis of the ribosome is an evolutionarily conserved process that starts in the nucleolus with transcription of rRNA precursors, and that ends in the cytoplasm with the formation of the mature 40S and 60S ribosomal subunits.26 Ribosomes are known as the core of the translation machinery, and are large ribonucleoprotein particles comprised of two unequally sized subunits (large subunit, 60S and small subunit, 40S). Therefore, the upregulation of ribosomal proteins at 10 h (with amino acid supplemented cultures) may have led to an increase in overall protein synthesis rates, resulting in the increased growth. 2. The Expression of Proteins Related to Stress Conditions. Cell viability in amino acid supplemented cultures was higher than in nonsupplemented cultures, and approached the value as the control sample (90%) 12 h post glucose shock. To date, it is known that yeast responds to osmotic stress by regulation of the uptake of amino acids in protein synthesis.27 Previously, we showed3 that Hsp70p, Hsp60p and Hsp12p expression decreased in response to VHG conditions, and this observation agrees with reported transcriptomic data.27 As seen in Table SI in the Supporting Information, the relative expression of Hsp12p, Hsp24p and Hsp26p decreased at 2 and 10 h post glucose shock. Subsequently, however, their expression recovered, and surpassed those of the nonshocked control samples. Furthermore, as seen in Figure 2, high concentrations of most intracellular amino acids were also observed at this time, suggesting that amino acid addition induces protein synthesis in S. cerevisiae in response to high glucose stress. Observations here are in agreement with data we observed previously for immobilized cells under VHG conditions with amino acid supplementation (compared to free cells or immobilized cells under VHG without amino acid supplementation).12 Furthermore, the expression of other heat-shock proteins (e.g., Ssa1p, Ssa2p, Ssa4p, Ssb1p, Ssb2p, Ssc1p and Sse1p) showed a slight decrease after 2 h of shock conditions; however, the abundance of these proteins recovered at 10 and 12 h (compared to the control). These data suggest that heat-shock proteins may play a role in protecting protein biosynthesis machinery. Together with the differential regulation of these heat-shock proteins, the expression of Gpd1p (glycerol-3-phosphate dehydrogenase) was also increased. This finding is in agreement with previous work,3 since high levels of glycerol formed at the beginning of fermentation under VHG conditions.3 Most observed stress response-related proteins, including Hsp104p, Tps1p, and Tps2p (trehalose-6-phosphate synthase) were up-regulated. These proteins relate to trehalose and 4772

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Figure 5. Concentrations of trehalose (O) and glycogen (0) before (at 0 h) and after (2, 10, and 12 h) stress conditions were applied.

glycogen synthesis. The expression of the heat-shock proteins was increased at 10 and 12 h in response to high osmotic stress. Further, the expression patterns of Hsp104p, Tps1p, and Tps2p are consistent with previous observations of synergism between protective reagents (trehalose, and glycogen) during heat-shock and ethanol stress.19,28 The expression of Tps1p, Tsp2p and other glycolysis-related proteins (Pgm2p, Ugp1p) were also increased at 2 h. This suggests that cells accelerated trehalose and glycogen biosynthesis during the initial shock in parallel with glycerol formation to potentially aid in the tolerance of high osmotic stress. Indeed, >2 h post glucose shock, increases of both trehalose and glycogen concentrations from 9.9 and 36.8 g/L at 0 h to 21.2 and 41.3 g/L at 2 h, respectively, were observed. However, the accumulation rates were different for each energy storage compound, since trehalose concentrations increased significantly between 2 and 12 h (compared to the control), while glycogen increased more slowly (see Figure 5). These data are in agreement with data reported by Devantier et al.11 The accumulation of these compounds may aid in stress tolerance,12 since there are suggestions that trehalose has a potential membrane protective effect.29 However, increased expressions of Gph1p and Nth1p were observed at 12 h, where it is hypothesized that this might aid in the release of accumulated trehalose and glycogen generated during the initial stages of glucose shock. Subsequently, when cells became acclimated to the high osmolarity, fermentation ability accelerated. This was confirmed by higher glycolysis protein expression at 10 and 12 h, with a concomitant (12 h) increase in glucose uptake and higher ethanol concentration/yield compared to 10 or 2 h (see Figure 1B). Furthermore, obviously, these values were also higher than the control, since nutrient limitation in the control sample occurred at these times. 3. The Expression of Glycolysis Proteins for Ethanol Production and Energy Metabolism Related Proteins. Most glycolysis proteins (e.g., Hxk1p, Glk1p, Adh1p, Adh4p, Ald3p, Ald6p and Pdc1p) significantly increased in abundance between 2 and 12 h. While Hxk1p and Glk1p concentrations increased significantly between 2 and 12 h, Hxp2p expression remained unchanged. It is thus likely that phosphorylation of glucose under VHG conditions in the early fermentation stages was mainly performed by Hxk1p and Glk1p. However, increased expression of these proteins did not lead to significant increases in ethanol concentration during this time (see Figure 1B). This may be because most glucose-6-phosphate derived from hex-

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Proteomic Response to VHG Conditions okinases and glucose kinase action was used for trehalose and glycogen biosynthesis. However, elevated expression of these proteins was maintained at 10 and 12 h, together with very high expression of alcohol dehydrogenases Adh1p and Adh4p (and Pcd1p). This suggests that fermentation was enhanced at 10 and 12 h (see Figure 1B). This is validated by the corresponding enhanced ethanol yields at these times (0.43, and 0.45 at 10 and 12 h, respectively, compared to 0.32 at 2 h). As seen in Table SI in the Supporting Information, most energy metabolism related proteins were also increased in abundance in response to glucose shock. These results are in agreement with Piper,28 since higher ATPase activity was found in response to stress conditions here, leading to higher energy consumption. Under anaerobic conditions, ATP is only gained by ethanol generation. Moreover, this is similar to the glycolytic flux in Escherichia coli reported by Koebmann et al.,30 where this flux is controlled by the ATP demand. Most detected proteins involved in nucleotide metabolism and TCA cycle were unchanged in abundance during stress, suggesting tight control in order to maintain cell activity. Slightly increased expression of Idp1p (mitochondrial form of NADP-specific isocitrate dehydrogenase), and Cit1p (citrate synthase) in the TCA cycle was observed at 10 and 12 h. Only one protein involved in nucleotide metabolism (Adk1p, adenylate kinase), increased in expression at 12 h. It is reported that Adk1p is required for purine metabolism, as well as for normal cell proliferation, but is not essential for viability.31 This data is in agreement with the up-regulation of Act1p, which is implicated in cell polarization, endocytosis, and other cytoskeletal functions.32 The actin cytoskeleton is a main factor that helps cells mediate responses to signals,33 with it being essential for polarized cell growth, and its important role in cell death.33 Thus, the Act1p upregulation observed at 10 h (in amino acid supplemented cultures) is indicative of cell well-being, since actin acts as a sensor in this regard (see Gourlay and Ayscough33 for reviews). The actin- and formin-interacting protein Bud6p, which relates to actin cable nucleation and polarized cell growth, was also up-regulated. Concluding Remarks. We show that the supplementation of cultures containing very high levels of glucose (VHG) with amino acids for aiding enhanced ethanol yield is successful. The combination of intracellular amino acid concentration and proteomic response measurements provides clear evidence for improvements in both the cell growth and tolerance of VHG conditions. Although there were a large number of intracellular amino acids as well as proteins that decreased in concentration in the early stages of VHG conditions, the levels of these intracellular compounds recovered faster in amino acid supplemented cultures. Together with this recovery, increased expression of proteins with cell viability and proliferation functions, as well as a strongly activated glycolysis pathway, was observed after 10 h. As a result, cells rapidly entered exponential growth, and subsequently, ethanol production was enhanced. Therefore, a metabolically engineered strain with an increased expression of transport processes and amino acids biosynthesis might be suitable for VHG fermentation. Data here provides an insight into intracellular processes for the effects of exogenous amino acid addition on the proteome, and the physiological changes of the cells, since a higher growth rate was found as well as the enhanced ethanol yields was also observed at 10 and 2 h compared to 2 h (0.43 and 0.45 (g ethanol/g glucose) at 10 and 12 h compared to 0.32 (g ethanol/g glucose) at 2 h, respectively). The application of VHG conditions, as well

as the addition of amino acids should be simple for the fermentation industry to apply. Tests with industrial and mutant strains, as well as the performance of amino acids addition and cost estimation, are the subject of future work. More detailed transcriptomics and metabolomics studies, as well as protein interactions are required to reveal an integrated understanding of the “biological system” of S. cerevisiae under VHG conditions. Forster et al.34 were successful in generating a genome-scale metabolic reconstruction of S. cerevisiae, where the whole cell functions were quantitatively computed from an in silico model. The combination of genome-scale metabolic reconstruction and quantitative constraints-based analysis allows analysis, interpretation, and prediction of phenotypic behavior of S. cerevisiae. Therefore, at present, the authors believe that proteomic data together with recent genomic data10 and metabolomic data11 will be useful in reconstructing and generating an integrated understanding of S. cerevisiae for VHG fermentation.

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