Proteomic Analysis of Saccharomyces cerevisiae under High Gravity

Jul 27, 2006 - Saccharomyces cerevisiae KAY446 was utilized for ethanol production, with glucose concentrations ranging from 120 g/L (normal) to 300 g...
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Proteomic Analysis of Saccharomyces cerevisiae under High Gravity Fermentation Conditions Trong Khoa Pham, Poh Kuan Chong, Chee Sian Gan, and Phillip C. Wright* Biological and Environmental Systems Group, Department of Chemical and Process Engineering, The University of Sheffield, Mappin Street, Sheffield, S1 3JD, UK Received July 27, 2006

Saccharomyces cerevisiae KAY446 was utilized for ethanol production, with glucose concentrations ranging from 120 g/L (normal) to 300 g/L (high). Although grown in a high glucose environment, S. cerevisiae still retained the ability to produce ethanol with a high degree of glucose utilization. iTRAQmediated shotgun proteomics was applied to identify relative expression change of proteins under the different glucose conditions. A total of 413 proteins were identified from three replicate, independent LC-MS/MS runs. Unsurprisingly, many proteins in the glycolysis/gluconeogenesis pathway showed significant changes in expression level. Twenty five proteins involved in amino acid metabolism decreased their expression, while the expressions of 12 heat-shock related proteins were also identified. Under high glucose conditions, ethanol was produced as a major product. However, the assimilation of glucose as well as a number of byproducts was also enhanced. Therefore, to optimize the ethanol production under very high gravity conditions, a number of pathways will need to be deactivated, while still maintaining the correct cellular redox or osmotic state. Proteomics is demonstrated here as a tool to aid in this forward metabolic engineering. Keywords: • S. cerevisiae • proteomics • ethanol production • high glucose concentration • iTRAQ • injection replicates

Introduction Saccharomyces cerevisiae is widely used as a biotechnological production organism, as well as a eukaryotic model system. It is attractive to work with since it is nonpathogenic, and due to its long history of application in the production of consumable products (such as ethanol and bakers’ yeast/breads), it has been classified as a GRAS (generally regarded as safe) organism. Currently, fermentative ethanol production by S. cerevisiae is still the most economical method, with many attempts being made to achieve higher ethanol concentrations.1-4 As an example, very high gravity (VHG, very high dissolved solids content in the fermentation medium5) fermentation technologies have been used to increase the ethanol productivity,6 with media containing sugar in excess of 250 g/L being used in order to achieve over 15% (v/v) ethanol production.4,7,8 However, a high glucose feed environment is likely to impose serious stress on S. cerevisiae and result in slow cell proliferation and a decline in cell viability.9 The reagent-based iTRAQ method has been proven to be a useful means for determining the relative expression level of individual proteins of up to four phenotypes simultaneously.10,11 This technique has been applied to microorganisms (S. cerevisiae12 and E. coli13,14) and for animal and human research (cancer,15 human saliva,16 pancreatic acinar cells,17 and a stemlike cell line18,19). In a previous study, 1217 unique proteins were * To whom correspondence should be addressed. Tel: +44(0)114 2227577. Fax: +44(0)114 2227501. Email: [email protected]. 10.1021/pr060377p CCC: $33.50

 2006 American Chemical Society

identified in S. cerevisiae using iTRAQ.12 Several studies have also sought to compare iTRAQ to other methods (2-DE;20 iCAT;15 DIGE, and cICAT21) to evaluate their relative performance, where iTRAQ performed well in comparison. It was not until recently that a deeper analysis of technical replicates within iTRAQ has been made.22 Using three MS replicate injections, an increment in the total number of proteins identified was found in S. cerevisiae (6%), Sulfolobus solfataricus P2 (33%), and Synechocystis sp. PCC6803 (50%), respectively.22 Moreover, the quantification reliability of iTRAQ experiments was obtained with high reproducibility across the injections, with an average CV of 0.09 for all three microorganisms.22 Many S. cerevisiae proteomic and/or transcriptomic studies provide us with an increasingly rich understanding of this organism’s response to various environmental perturbations. Examples include 2-DE and mRNA measurements of glucose and ethanol limitation;23 2-DE for responses to physiological fermentation stresses;24 and mRNA and 2-DE for an molecular understanding of the adaptation of S. cerevisiae for wine fermentation.25 Although Devatier et al.26 investigated intra- and extracellular S. cerevisiae metabolism, relative protein expressions under different glucose concentrations (from normal to VHG) during anaerobic (or microaerobic) fermentation have not been determined. In addition, the proteomes of brewing yeast are still largely unknown.24 For these reasons, we sought to monitor and understand how the wild-type S. cerevisiae KAY446 strain adapts to different glucose conditions, to gain a deeper knowledge of yeast physiology during a VHG fermentaJournal of Proteome Research 2006, 5, 3411-3419

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research articles tion process, facilitated by classical measurements of metabolites, and aided by iTRAQ proteomics. We focus on the influence of high glucose feed and growth rate on ethanol yield and corresponding physiological response. Modifications and suggestions to increase ethanol productivity will be also reported in the context of the literature and our results. The proteomic data is based on three injection replicates and a technical replicate. We have previously demonstrated the reliability of this data set.22

Materials and Methods Fermentation Conditions. Saccharomyces cerevisiae KAY446 (courtesy of Dr. Kathryn Ayscough, The University of Sheffield) was initially grown in 30 mL Erlenmeyer flasks containing 10 mL of liquid medium (1% yeast extract, 2% glucose, 2% peptone, 4 µg adenine/mL) in a shaken water bath (FALC, Germany) at 120 rpm at 30 °C for 12 h. Subsequently, the yeast cells were collected by centrifugation at 500 × g at room temperature for 5 min, followed by washing with deionized water prior to fermentation. An initial cell concentration of 2 × 106 cells/ mL (OD650 ∼ 0.25) was used for fermentation, and this process was performed in 250 mL Erlenmeyer flasks containing 100 mL of media 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; and glucose of differing amounts, 120, 210, 300. The cell suspension was incubated at 30 °C, with shaking at 120 rpm for 12 h, and then without shaking to allow for microaerobic growth until fermentation stopped. Samples were collected at time 0, and every 2 h during the first few days, and after that every 12 h. For every sample, the OD650, pH, ethanol concentration and residual glucose and glycerol concentrations were monitored, as described below. Cells were harvested at the 68th hour (late exponential phase) for protein extraction. All chemicals used in this study were supplied from Sigma (Sigma-Aldrich, UK) and Fisher (Fisher Scientific, UK), unless otherwise specified. Measurement of Fermentation Parameters. The concentration of ethanol in the broths was determined using a Finnigan Trace DSQ single Quadrupole GC-MS system coupled with an auto-sampler model AS3000 (Thermo Electron Corporation, USA) fitted with a 30 m × 0.25 mm i.d. × 0.25 µm df Stabilwax fused silica column (Thames Restek, Bucks, UK). Approximately 1 mL of the broths was collected and centrifuged at 9600 × g for 1 min at room temperature, and 100 µL of supernatant was withdrawn for GC-MS analysis. The total analysis run-time was 7 min with the MS detector, and a helium carrier gas at a flow rate of 1 mL/min. The temperature gradient was performed with a hold at 40 °C for 0.5 min, followed by a ramp at a rate of 10 °C/min to 110 °C and finally a hold for 1 min at 110 °C. 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, UK) and a glycerol assay kit K-GCROL (Megazyme, Ireland). The pH was tested in every sample using a pH meter. The S. cerevisiae growth rate was monitored by measuring the absorbance of the culture at 650 nm, OD650, using an Ultrospec 2100 Pro Spectrophotometer (Biochrom, England). Protein Extraction, Labeling, Mass Spectrometry and Data Analysis. The preparation of yeast cell extracts, labeling of samples with different phenotypes, as well as the identification of proteins from peptide fractions were done as described by Chong et al.22 Briefly, cells harvested at the 68th hour were 3412

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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).24 Protein was 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 yeast extract buffer and 5% (v/v) of a protease inhibitor cocktail. An amount of 100 µg of protein of each phenotype was precipitated with ice-cold acetone overnight at -20 °C before being resuspended in 20 µL of 500 mM TEAB pH 8.0. Protein from each sample was reduced, alkylated, digested, and labeled with iTRAQ regents (114, 115, 116, and 117 for protein extracted from 120 g/L, 120 g/L, 210 g/L, and 300 g/L glucose, respectively). The peptide fractionation was carried out using a strongcation exchange (SCX) column (PolySULFOETHYL A, PolyLC, Columbia, MD). These peptide fractions were then dried in vacuum concentrator before resuspension in 100 µL of Swichos buffer (0.1% formic acid and 3% acetonitrile) for the nano-LCESI-MS/MS (Dionex/LC Packings Ultimate coupled to a QStarXL, Applied Biosystems/MDS-Sciex) analysis. Data acquisition in the positive ion mode was performed with a selected mass range of 300-2000 m/z. Peptides with +2 to +4 charge were selected for tandem mass spectrometry, and the summation time for MS/MS events was set at 3 s. 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. Mass spectra generated were searched against yeast single genome database downloaded from NCBI (June 2005) using ProQuant software version 1.1 (Applied Biosystems, MDS-Sciex). The complete list of identified proteins and the ratios of protein expressions was collated and analyzed using ProGroup Viewer software version 1.0.6 (Applied Biosystems, MDS-Sciex) with at least 95% confidence. All data from ProGroup Viewer were exported to Microsoft Excel for statistical analysis and manipulation purposes. To obtain the confidence ratios in this study, the EF (Error Factor) of the ratio was considered.

Results and Discussion Ethanol Fermentation as a Function of Glucose Concentration. The glucose uptake and the growth behavior for S. cerevisiae are shown in Figure 1A, with the ethanol and glycerol production characteristics shown in Figure 1B. For the 120 g/L glucose culture, S. cerevisiae entered the exponential growth phase after 4 h and finished this phase at 68 h (Figure 1A). As the glucose concentration increased to 210 g/L and 300 g/L, the onset of exponential growth of S. cerevisiae began later compared to cultures supplemented with lower glucose concentrations. For example, the exponential phase began after 6 h and finished this phase after 88 h for the 210 g/L glucose culture, with a correspondingly lower growth rate. The growth rate (OD650) reduced slightly when S. cerevisiae was grown on 300 g/L glucose between 2 and 6 h. We can hypothesize that, under high glucose environments, S. cerevisiae was inhibited by a high osmotic stress environment, potentially leading to some cell death. However, after acclimation, its viability recovered and it commenced exponential growth. This may explain the 8 h lag phase observed for this condition in the 300 g/L glucose culture (Figure 1A). The maximum ethanol concentrations were (Figure 1B) 54.3 g/L at 68 h for 120 g/L glucose, and 72.4 g/L after 86 h for 210 g/L glucose, and finally 89.3 g/L after 96 h for 300 g/L glucose. Another observation within the cultures was with regard to solution pH. Higher glucose concentrations in the feed led to

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Figure 2. The number of protein showing either up- or downregulation with respect to their functional groups involved in primary and secondary metabolic pathways.

Figure 1. Residual glucose, ethanol, and glycerol concentrations, as well as the growth of S. cerevisiae in the broths under different glucose concentrations: 120 g/L (4), 210 g/L (0), and 300 g/L (O). Filled symbols are representative for (A) OD650, or (B) glycerol; open symbols are representative for (A) glucose, or (B) ethanol. Experiments were performed in triplicate.

lower final pH. At the completion of fermentations, the pH was 3.1, 2.8, and 2.3 for the 120 g/L, 210 g/L, and 300 g/L glucose cultures respectively. We can hypothesize that S. cerevisiae might produce acidic products, as well as glycerol, under high glucose concentrations. Further evidence was observed when Ald6p (cytosolic aldehyde dehydrogenase) and Gpd1p (NADdependent glycerol-3-phosphate dehydrogenase) were upregulated, since these proteins are directly related to the formation of acetic acid and glycerol, respectively. After 8 h fermentation, the glycerol concentration in the 300 g/L glucose culture was higher than the other cultures. At the same sampling time (68 h), the concentration of glycerol in the 120 g/L, 210 g/L, and 300 g/L glucose cultures was 3.8, 5.2, and 8.6 g/L, respectively (see Figure 1B). Therefore, under VHG conditions, besides ethanol as a major product, glycerol is also formed as a possible counterbalancing product to maintain the redox or osmotic balance of the cells. The problem of combined high osmotic conditions generated by a high glucose and ethanol concentration imposed more stress and further enhanced inhibition of the ethanol production process. It is proposed that S. cerevisiae accelerated the production of acidic compounds and glycerol rather than synthesizing ethanol after 88 h to minimize this phenomenon. This explanation will be examined in future experiments by applying metabolic flux analysis (MFA) to track intracellular metabolites. Here we only aim to examine aspects of proteomics under VGH conditions for fermentation. Relative Protein Expression under Different Glucose Concentrations. In this study, 413 proteins were found from three injection replicates. Only those proteins identified and quantified with at least 2 MS/MS experiments are used for subsequent results and discussion here. Proteins which showed >(1.5-fold

change (EF < 3) were recorded as significant (in either 210 g/L glucose or 300 g/L glucose compared to 120 g/L glucose) (refer to Table 1 in Supporting Information). From these criteria, 74 proteins were down-regulated and 35 were up-regulated more than 1.5-fold in the 210 g/L glucose condition compared to the 120 g/L glucose condition, while 87 down-regulated and 41 upregulated proteins were recorded for 300 g/L glucose compared to 120 g/L glucose. Among these proteins, 50 down-regulated and 22 up-regulated proteins were found in both 210 g/L and 300 g/ L glucose cultures. Greater changes in protein expression were observed in the culture supplemented with the higher initial glucose concentration (i.e., more protein regulation was found in the 310 g/L culture than in the 210 g/L culture when compared to the control at 120 g/L). Many proteins related to carbohydrate and amino acid metabolism were identified. Of these proteins, proteins involved in carbohydrate metabolism increased their expression more than decreased under VHG conditions, and this phenomenon was opposite for proteins involved in amino acid metabolism (Figure 2). Since most of the regulated-proteins identified play important roles in metabolic pathways, these were classified by reference to the KEGG database (http://www.genome.jp/kegg/). The results are shown in Table 1 in the Supporting Information, and on our website at http://wrightlab.group.shef.ac.uk/projects/. The Glycolysis/Gluconeogenesis Pathway. From Figure 3, the number of up-regulated carbon metabolism proteins is greater than that of down-regulated proteins. Most proteins that play major roles in glycolysis were identified here. From a practical point of view, we will focus first on Pdc1p (pyruvate decarboxylase) and Adh1p (alcohol dehydrogenase), since these proteins have a major role in ethanol production. The PDC1 gene encodes for the pyruvate decarboxylase that converts pyruvate into acetaldehyde and CO2. The up-regulated expression of PDC1 has received much attention, because this gene normally accounts for about 80% of the pyruvate decarboxylase activity.27 Here, protein Pdc1p increased its expression up to 1.5 fold alone in 300 g/L glucose compared to the 120 g/L glucose condition. Protein Pdc5p was up-regulated around 2-fold in both 210 g/L and 300 g/L glucose cultures compared to the 120 g/L glucose culture. This protein is also regarded as Journal of Proteome Research • Vol. 5, No. 12, 2006 3413

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Figure 3. Relative protein expression levels of glycolysis/gluconeogenesis related proteins in the 210 g/L and 300 g/L glucose conditions compared to the 120 g/L glucose concentration.

a key enzyme in alcohol fermentation, as its regulation depends on the glucose and ethanol environment.28 It is worth noting this protein can be repressed by thiamine and is involved in amino acid catabolism.29 Protein Pdc6p was slightly upregulated (+1.5; +1.7) in 210 g/L and 300 g/L glucose cultures compared to the 120 g/L glucose culture. The gene PDC6 only shows expression in media containing ethanol and galactose and not in media containing glucose for fermentation purposes.30 Since, in some cases, gene and protein expression do not correlate well (potentially due to different turnover rates, post-translational modifications etc), for this reason, we can hypothesize the up-regulation of this protein may be as an adaptation to glucose stress. There is a similar phenomenon seen for alcohol dehydrogenase Adh1p (+1.5; +1.6), whose function is to convert acetaldehyde to ethanol. From Figure 1B, the ethanol concentrations at 68 h for 120, 210 and 300 g/L are 54.3 ( 0.2, 55.1 ( 0.3, and 65.7 ( 0.6 g/L, respectively. After taking the error into consideration, the ethanol concentration at 210 g/L was marginally higher than the control 120 g/L culture. These results show that the increase in expression levels of Pdc1p, Pdc6p, and Adh1p are related to high glucose environment and may provide the additional activity during ethanol production. As mentioned previously, S. cerevisiae might produce acidic products in the high glucose environment, for instance, acetic acid (as we noticed a lowered pH with increased levels of glucose). This compound is produced from an intermediate metabolite of acetyl-CoA synthesis from acetate generated from acetaldehyde (see Figure 4), under the catalysis of enzymes Alds and Acss.31 We could see that the up-regulation of Pdc1p, Pdc5p, and Pdc6p led to a likely increase in the amount of acetaldehyde, and as a consequence, this leads to an increase in the expression of Ald6p (+1.8; +2.1). It is also means that acetate formation was accelerated, since it has been previously demonstrated that the deletion of the ALD6 gene (which encodes for Ald6p) results in a decrease in acetate production.32 Moreover, among genes encoding ALD, the constitutive expression of ALD6 gene was suggested to benefit the greatest contribution to acetic acid production.31 Conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate is an important step in glycolysis, since the NADH formed in this step has to be reoxidized for glycolysis to proceed (see Figure 6 in the Supporting Information).33 This process is carried out by glyceraldehyde-3-phosphate dehydrogenase encoded by three genes, TDH1, TDH2, and TDH3, in which 3414

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TDH3 accounts for 50-60% of the enzymes found in the cell.34 The up-regulated expression of Tdh1p (+1.7; +1.9) is found in 210 g/L, and 300 g/L glucose, compared to the 120 g/L glucose condition. In contrast, levels of both Tdh2p (+1.2; +1.0) and Tdh3p (+1.1; +1.2) remained unchanged in elevated glucose conditions. Unlike the regulations of most glycolysis proteins, the expression levels of phosphofructokinase (Pfk1p and Pfk2p) were down-regulated (-3.8; -4.6) in 300 g/L glucose compared to the 120 g/L glucose environment. No change in expression of Pfk1p and Pfk2p was seen in the 210 g/L glucose environment. Phosphofructokinase is responsible for phosphorylation of fructose-6-phosphate to fructose-1,6-biphosphate, and its activity is affected by a variety of effectors and has been the paradigm for “multimodulated enzymes”.35 Some studies suggest that over-expression of the genes encoding phosphofructokinase did not increase fermentation efficiency in S. cerevisiae,36,37 and the control of a pathway may be rarely attributed to a single step.38 In this study, the down-regulation of Pfk1p and Pfk2p here may be related to the accumulation of fructose1,6-bisphosphate, since the accumulation of this compound despoils the phosphate pool in the cell and may cause cell death,39 thereby restricting the glucose flux in glycolysis.40 This data suggests the down-regulation of Pfk1p and Pfk2p leads to a delay in growth rate for S. cerevisiae to complete exponential growth (see Figure 1A). Pgi1p (glucose-6-phosphate isomerase) showed slight upregulation (+1.5 fold) in both 210 g/L and 300 g/L compared to 120 g/L glucose. This is a phosphoglucose isomerase, which catalyzes the interconversion of glucose-6-phosphate and fructose-6-phosphate (also a precursor of the cell wall components chitin and manno-protein), and it is also required for cell cycle furtherance and finishing the gluconeogenic occurrences of sporulation.41,42 The regulation of this protein relates to the nutrition content of S. cerevisiae, since cells lacking Pgi1p are unable to grow on either fructose or glucose as a sole carbon source.41 Fructose-6-phosphate is formed from sedoheptulose-7-phosphate and glyceraldehydes-3-phosphate catalyzed by transaldolase Tal1p in the nonoxidative pentose phosphate pathway.43 This protein (Tal1p) was also upregulated (+1.5; +1.8) in both elevated glucose conditions, and it is known to constitutively express under most conditions.43 Furthermore, the over-production of Tal1p has previously been shown to improve the growth of recombinant S. cerevisiae strains during ethanol fermentation from xylose.44 Gpd1p (NAD-dependent glycerol-3-phosphate dehydrogenase), a key enzyme of glycerol synthesis, essential for growth under osmotic stress,45 showed evidence of up-regulation (+1.9 fold and +1.8 fold in 210 g/L and 300 g/L glucose compared to the 120 g/L glucose) in this study. Its expression has been shown to be regulated by a high-osmolarity glycerol response pathway.46 The up-regulation of Gpd1p, and hexokinase Hxk1p (+1.5; +1.7) might lead to an increase in glycerol formation (which we did see in these conditions here). In brief, the expression of glycolysis proteins was upregulated in the 300 g/L glucose culture, but the highest ethanol yield was only 89.3 g/L. Why did S. cerevisiae not produce more ethanol? The answer might be a combined inhibition of products that generated high osmotic stress, as well as a redox balance requirement for cells. Indeed, there was a reduction in growth rate at the beginning of the fermentation under VHG conditions. It can be hypothesized that S. cerevisiae spent a lot of time repairing damaged cells, synthesizing necessary

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Figure 4. The glycolysis/gluconeogenesis pathway shown in relationship to other metabolic pathways, such as the citrate cycle, alanine and aspartate metabolism, glutamate metabolism, lysine biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis, and starch and sucrose metabolism (with reference to KEGG). Proteins shown in bold type with blue background and ratio in red being representative for up (+) regulation; proteins in bold type with a green background and ratio in blue is representative for down (-) regulation in this study. The expression ratios of these proteins are also shown in Table 1 in the Supporting Information.

compounds to help resist this stress, in addition to assimilating glucose for biomass, over synthesis for ethanol production. Once cells became adapted to the VHG environment, they switched toward fermentation to produce ethanol as a primary product. This can be seen by the enhanced rate of ethanol generation in the 300 g/L glucose culture compared to 210 g/L and 120 g/L glucose after 48 h. Ethanol production continued until the accumulation of this product became toxic. The importance of the redox balance in the cells will be discussed later. Storage Carbohydrates. The glucose-6-phosphate, produced from the glycolysis/gluconeogenesis pathway (see Figure 4) can be converted to produce storage carbohydrates, i.e., glycogen and trehalose. Glycogen and trehalose are the main storage carbohydrates involved in thermal, osmotic, oxidative, and

ethanol stress in S. cerevisiae,47 since they provide energy during non-proliferation periods. During fermentation with the high level of glucose at 300 g/L, proteins related to glycogen and trehalose synthesis were down-regulated. Examples include glycogen synthase/glycogen phosphorylase, Gph1p (-3.7), Tps1p (-4.3), and Glc3p (-1.6) (see Table 1 in the Supporting Information). Trehalose biosynthesis is performed by a two step process via the catalytic activity of two proteins, Tps1p and Tps2p (trehalose-6-phosphate phosphatase). Tps1p converts glucose6-phosphate and UDP-glucose into R,R-trehalose-6-phosphate, which is subsequently converted with water into trehalose and phosphate by Tps2p.48 The down-regulations of Tps1p and Tsp2p may result in a change in trehalose biosynthesis, since the deletion of TPS1 results in a loss of not only Tps activity, Journal of Proteome Research • Vol. 5, No. 12, 2006 3415

research articles but also trehalose biosynthesis.49 Moreover, at our sampling time (68h), S. cerevisiae grown in 120 g/L glucose had just reached the stationary phase (see Figure 1B); thus the accumulation of trehalose was higher than for the other samples. Glc3p (1,4-glucan-6-(1,4-glucano)-transferase) is a glycogen branching enzyme involved in glycogen accumulation.50 Gph1p is a glycogen phosphorylase, and its activity and expression is regulated by AMP-mediated phosphorylation, as well as by both stress-response elements and the HOG MAP kinase pathway, respectively.51,52 The regulation of these proteins is related to glycogen accumulation in S. cerevisiae, and their expression increase correlatively with glycogen accumulation. This happens when cells reach the stationary phase, or when nutrients are exhausted. At our sampling time (68 h), S. cerevisiae grown on 120 g/L glucose entered the stationary phase, whereas cells grown on other glucose concentrations just reached the late or nearly late exponential phase (Figure 1B). These details provide a possible explanation for the expressions of proteins Glc3p, and Gph1p being down regulated under 300 g/L glucose condition alone. In contrast to the down-regulation of Tps1p, Glc3p, and Gph1p, protein Pgm2p (phosphoglucomutase) increased its expression (+2.4; +2.6). This protein converts glucose-1-phosphate to glucose-6-phosphate. Its role is important for carbohydrate metabolism such as glycolysis, the pentose phosphate shunt, and glycogen, and trehalose and galactose metabolism, and its up-regulation here is in response to osmotic stress. The Requirement for Redox Balance Led To Secondary Products Generation. The highest glucose concentration in this study was not high enough to completely inhibit the growth of S. cerevisiae, but was high enough to force it to adapt to maintain its growth. Here, S. cerevisiae was grown in flasks with high glucose and restricted O2 (without O2 supplementation, as well as CO2 accumulation leading to decreased O2). This led to cells fermenting glucose as an efficient means of generating and consuming the energy from this substrate. As mentioned previously, under high initial glucose conditions, S. cerevisiae growth was retarded, since cells were synthesizing other compounds to aid survival under these shock conditions. Glycerol is one of the choices, since its formation and accumulation helps to protect the cell against lysis under osmotic stress conditions.53 During ethanol fermentation, NAD+ and NADH are generated via glycolysis and other processes (see Figure 6 in the Supporting Information). NADH is formed both in the cytosol and in the mitochondria.54 In the cytosol, NADH is generated by catalysis of glyceraldehyde-3-phosphate dehydrogenase (Tdhs) (via glycolysis) and also in assimilatory reactions;54 in the mitochondria NADH is formed via the TCA cycle and pyruvate dehydrogenase.55 Other processes, including the synthesis of organic acids (succinic acid, acetic acid, and pyruvic acid) as bypass products also generate NADH. The resultant formation of NADH leads to an imbalance in the cellular NAD+/NADH ratio. Glycerol was formed in the early stages of our batch cultivations, and this is in agreement with other studies.56 The up-regulation of Tdh1p (see Figure 3) here provides evidence for the increase of NADH via the glycolysis pathway in S. cerevisiae under VHG conditions. Why can S. cerevisiae not maintain its redox balance via ethanol fermentation to produce ethanol only? The redox balance of NADH is reinstated via a mitochondrial internal and external NADH dehydrogenase, alcohol dehydrogenase, cytosolic glycerol-3-phosphate dehydrogenase, and mitochondrial 3416

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redox shuttles.57 During fermentation, ethanol is formed from glucose, which produces 2 mol of ATP from 2 mol of ADP. The energy rebalance of the ethanol fermentation is the conversion of pyruvate to ethanol, performed as a final step to reoxidize the NADH generated from the oxidation of glyceraldehyde-3phosphate in the glycolysis pathway. In other words, 1 mol of NADH generated from the oxidation of glyceraldehyde-3phosphate to 1,3-diphosphoglycerate is used in the conversion of pyruvate to ethanol. Theoretically, 2 mol of NADH generated in the reaction converting glucose to pyruvate can be completely used to form ethanol to maintain redox balance. To successfully perform this process requires the synchronization of protein activity through this system. However, some of the requisite protein activities such as Adhs and Pdcs are also involved in other processes such as tyrosine, fatty acid, and glycerolipids metabolism, or in the biosynthesis of bile acid. The Pdc activity is also necessary for the production of cytosolic acetyl-CoA. For this reason, the up-regulation of proteins Adhs and Pdcs did not present for the conversion of pyruvate to ethanol only. Pyruvate generated from the glycolysis pathway was used by other products which generated more NADH, thus driving the cell further away from redox balance. The redox reactions are carried out mainly in the cytosol, where most of cellular metabolism happens, and the mitochondria. Since the membranes of the organelles and the inner membrane of the mitochondria are not permeable to NADH; therefore, the redox balance is regulated in each compartment.58 Cytosolic NADH is mainly reoxidized by Adh1p to produce ethanol, but this reaction is a redox-neutral process.59 Therefore, the reoxidation of the excess NADH is performed by Gpd1p, as a result, glycerol also formed to maintain the redox balance under VHG conditions (see Figure 6 in the Supporting Information). In summary, more bypass products were formed under high glucose conditions compared to normal conditions. Tricarboxylic Acid Cycle (TCA cycle). The TCA cycle is a major metabolic pathway, since via the oxidative decarboxylation of acetyl-CoA in this cycle, the reducing equivalents to respiratory complexes are provided. Further, its function also mainly involves the synthesis of amino acids, heme, and glucose.60 The Idh1p protein was down-regulated (-1.6; -2.6) in both 210 g/L and 300 g/L glucose compared to 120 g/L glucose. Idh1p is a subunit of mitochondrial NAD+-dependent isocitrate dehydrogenase and is involved in the oxidation of isocitrate to R-ketoglutarate (2-oxoglutarate). The down-regulation of this protein potentially leads to a reduction in the conversion of isocitrate to R-ketoglutarate, with R-ketoglutarate being known as an initial substrate for both lysine biosynthesis and glutamate metabolism. Therefore, the down regulation of Idh1p, together with the decrease of R-ketoglutarate under high glucose conditions (210 g/L and 300 g/L), might lead to a decrease in expression of Gdh1p (glutamate metabolism). The detailed functions of these proteins are discussed later in the section on amino acid metabolism. Fum1p (fumarase) decreased its expression level (-1.7; -2.8) in both the 210 g/L and 300 g/L glucose conditions, and Mdh1p (mitochondrial malate dehydrogenase) was down-regulated (-2.0) in the 300 g/L glucose concentration alone. Two of these proteins (Fum1p and Mdh1p) are neighbors in the TCA cycle. Thus, the down-regulation of Fm1p and Mdh1p might lead to a decrease of fumarate in the TCA cycle and correctively lead to a decrease in expression of Arg4p (-1.6; -5.9), an argino-

Response of Yeast to High Glucose Concentration

succinate lyase, regulated by the general amino acid control mechanism.61 Amino Acid Metabolism. S. cerevisiae entered the exponential phase later in higher glucose concentrations than for the lower glucose concentrations. In osmotic stress environments (high glucose concentrations), a significant decrease in protein expression levels for de novo biosynthesis metabolism has been reported,62 and our data supports this. Most proteins in amino acid metabolism and metabolism of other amino acids decreased their expression when under high osmotic stress. The decrease in these pathways can be affected by a decrease in ATP consumption for biosynthesis.39 Glutamate plays a main role in nitrogen catabolism and anabolism and is also known as a major source of cellular nitrogen.63 Therefore, the down-regulation of NADP+ dependent glutamate dehydrogenase, Gdh1p (-1.6; -2.1) was a noticeable feature here, since, as reported by Nissen et al.,64 the deletion of GDH1 leads to an increase in ethanol formation as well as decreased glycerol production. But here, at our sampling time (68 h), both the ethanol and glycerol concentrations in the high glucose environments (210 and 300 g/L glucose) were higher than the control sample (120 g/L glucose), in spite of the down-regulation of Gdh1p. Therefore, we hypothesize that this protein might show a decrease in its function in other metabolic pathways, for example in glutamate synthesis, as its function is also to synthesize glutamate from ammonia and R-ketoglutarate, and its expression is regulated by carbon sources and nitrogen.65 Under fermentative conditions, the utilization of R-ketoglutarate is regulated by a mechanism generated from glutamate biosynthesis and catabolism, without decreasing the integrity of the connected energy-providing systems.66 Like the regulation of Gdh1p, the regulation of proteins histidinol dehydrogenase, His4p (-1.9; -3.1); dihydroxyacid dehydratase, Ilv3p (-2.6; -4.7); tryptophan synthase, Trp5p (-1.8; -2.1); mitochondrial and cytoplasmic valyl-tRNA synthesis, Vas1p (-5.6; -7.3); and asparaginyl-tRNA synthetase, Ded81p (-1.7; -2.5) was also down-regulated in both 210 g/L and 300 g/L glucose conditions compared to 120 g/L glucose. These proteins relate to the biosynthesis of histidine (His4p), valine, leucine, and isoleucine (Vas1p, Ilv3p), tryptophan (Trp5p), and metabolism of alanine and aspartate (Ded81p). Among these proteins, the regulation of His4p was noticeable. This protein catalyzes four steps (the second, third, ninth, and tenth) in histidine biosynthesis, since it carries phosphoribosylATP pyrophosphatase, phosphoribosyl-AMP cyclohydrolase, and histidinol dehydrogenase activities.67,68 Therefore, the down-regulation of this protein led to a significant decrease in histidine biosynthesis. The biosynthesis of branched-chain amino acids was also decreased because of a down-regulation of Ilv3p and Ilv5p (-1.7 in 300 g/L glucose alone). The downregulation of Trp5p was regulated by the general control system of amino acid biosynthesis.69 Proteins related to tRNA synthetase such as Ded81p, Cdc60p (cytosolic leucyl-tRNA synthetase), and Dps1p (cytoplasmic aspartyl-tRNA synthetase) were also down-regulated. While the down-regulation of Ded81p was found in both 210 g/L and 300 g/L glucose conditions, that of Cdc60p (-2.1) and Dps1p (-3.3) were found only in 300 g/L glucose. Another protein, Aro4p (3-deoxy-Darabino-heptulosonate-7-phosphate), also showed downregulation (-3.1) in 300 g/L glucose alone. The function of this protein is important, since it catalyzes the first step in aromatic amino acid biosynthesis, and the regulation of Aro4p might

research articles

Figure 5. Relative protein expression levels of heat shock proteins in the 210 g/L and 300 g/L glucose conditions compared to the 120 g/L glucose concentration.

relate to the accumulation of tyrosine in S. cerevisiae, since Aro4p is feedback-inhibited by tyrosine.70 Once again, the regulation of proteins in amino acid pathways showed that in high glucose environments, S. cerevisiae had reduced viability (but did not lead to widespread cell death) and led to a delay in the time to complete exponential growth. Proteins showing up-regulation seem to be those that are necessary for maintaining the viability and activity of cells. S. cerevisiae, under the conditions here, was able to survive due to these responses and maintain ethanol production. Briefly, most proteins involved in amino acid metabolism decreased their expression under high glucose concentrations; therefore, the addition of these amino acids in the media for highly efficient VHG fermentation may be necessary. Proteins Involved in eIF (eukaryotic initiation factor) and Heat-Shock Proteins. Most of the proteins involved in eIF decreased their expression when S. cerevisiae was grown at higher glucose concentrations. Tif1p is known as a translation initiation factor 4A (eIF4A).71 This protein decreased its expression in the high glucose environments (-3.5; -9.4). The downregulation of this protein might affect the initiation of polypeptide synthesis, due to its activities being related to promote the “melting” of secondary structure in mRNAs, which may otherwise slow down translation initiation.72 This target is achieved by interaction between eIF4A and other initiation factors such as eIF4E and eIF4G.73 The down-regulation of both proteins, Anb1p (-2.9; -8.1) and Hyp2p (-3.0; -8.1), involved in anaerobic conditions during fermentation with high glucose conditions was reported previously.74 These proteins are the translation initiation factor eIF-5A. They facilitate formation of the first peptide bond and suffer an essential hypusination modification,75 and they are also essential for S. cerevisiae cell viability.74,76 In general, proteins involved in eIF were downregulated in the high glucose environments. This eIF downregulation correlated with amino acid protein pathway downregulation, and the reducing viability of S. cerevisiae at elevated glucose concentrations. Of the 22 putative heat-shock proteins annotated in the genome, 18 of them were identified with more than 2 distinct peptides, and of these 12 were down-regulated greater than 1.5 fold when S. cerevisiae was grown at higher glucose concentrations (see Figure 5). These heat-shock proteins are known as response-factors to stress environments, and some of them are associated directly to the de- or reactivation of Journal of Proteome Research • Vol. 5, No. 12, 2006 3417

research articles damaged proteins. The expression levels of the six proteins of the Hsp70 family (Ssa3p, Ssa2p, Ssb1p, Ssb2p, Sse1p, and Sse2p) decreased significantly (from -1.5 to -4.0 fold) when S. cerevisiae was grown in the higher glucose conditions. These proteins bind to unfolded (or partially unfolded conformation) proteins to assist appropriately in the folding of proteins and aggregation prevention.63 While the down-regulation of Ssa2p is related to SRP-dependent co-translational protein-membrane targeting and translocation, that of Ssb1p and Ssb2p may be involved in the folding of newly synthesized polypeptide chains.77 At higher glucose concentrations studied here, the regulation of Sse1p (mitochondrial matrix ATPase), Sse1p, and Sse2p decreased significantly (from -1.5 to -4.0 fold). The function of Ssc1p is related to translocation of protein into the matrix and protein folding.78 In brief, the main function of the Hps70 family is to expedite the protein translation across membranes and prevent the cell from protein denaturation under stress conditions, as well as assist in folding of nascent polypeptides.79 The down-regulation of proteins in the Ssa group is directly related to the down regulation of Sti1p (Hsp90 co-chaperone), since this protein interacts with the Ssa group of cytosolic Hsp chaperones.80 Hsp82p, the cytoplasmic chaperone (Hsp90 family), was also down-regulated, and this protein interacts with the cochaperone Sti1p.81 The down-regulated Hsp12p protein is involved in the prevention of cell membrane desiccation,82 and it is affected by many stress factors, including high alcohol conditions, glucose depletion, etc.82,83 Hsp26p was also down-regulated here. Since this protein is involved in cell growth, the reduction in the expression level is a consequence of cells entering the stationary phase and during sporulation.84 The expression of the heat-shock proteins identified in this study is directly related to the glucose stress condition, with most proteins in the Hsp70 family, Hsp12p, Hsp26p, and Hsp82p decreasing in expression. So far, the direct interaction of these heat-shock proteins under elevated glucose conditions has not been reported yet at the proteomics level. However, Rossignol et al. reported that the HSP26 and SSE2 genes were down-regulated during rehydration and after inoculation under the 200 g/L initial glucose condition.85 Another study at 200 g/L of an equimolecular mixture of glucose and fructose resulted in the HSP26 gene being differentially expressed in different strains (ICV 16 and ICV 17).86 Moreover, the SSB1 gene was down-regulated by glucose exhaustion in a previous study.24 Therefore, we can conclude that the down-regulation of these proteins might lead to a deactivation of damaged proteins more than a reactivation of damaged proteins, and the down-regulation of these proteins was unexpected since more cells might be damaged under VHG conditions.

Conclusions Under a high glucose environment (high osmotic pressure), S. cerevisiae KAY446 still showed an ability to produce ethanol. Our data provides an understanding of the response of S. cerevisiae KAY446 in the relationship between the shifting of physiological parameters and the global expression of proteins under VHG conditions for ethanol fermentation. Under VHG conditions (300 g/L glucose), the rapid change of expression of proteins resulted in a high energy-yield, which led to the formation of a primary product, i.e., ethanol and secondary byproducts (such as glycerol and acetic acid). As a counterbalance to achieve redox balance, as well as to minimize the combined high osmotic conditions, glycerol formation was 3418

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Pham et al.

favored over ethanol. Surprisingly, the expression of heat-shock proteins did not increase to aid in adaptation to high osmotic conditions, since they decreased in expression. This phenomenon was similar for proteins relating to amino acid metabolism. To optimize the ethanol fermentation under VHG conditions, the elimination of secondary metabolites should be considered, while still maintaining the balance in the NAD+/NADH ratio. Moreover, the down-regulation of proteins relating to amino acid metabolism also led to a decreased amount of some amino acids in the cell (such as alanine and histidine). Therefore, the addition of these amino acids in the media for VHG fermentation may be necessary. A good yeast strain must have the capability to surmount this stress. This study can be further extended to other strains by comparing the protein expression of laboratory and industrial strains of S. cerevisiae to obtain an optimal strain for ethanol fermentation under VHG conditions.

Acknowledgment. We gratefully acknowledge the Ministry of Education and Training of Vietnam for financial support. P.C.W. thanks the EPSRC for provision of an Advanced Research Fellowship (GR/A11311/01) and for funding (GR/ S84347/01). Supporting Information Available: List of proteins that changed regulation in the 210 and 300 g/L glucose compared to the 120 g/L glucose condition; list of proteins found in three injections with >2 peptides; figure showing the central mode of redox balance and bioenegetics in glycolysis/ gluconeogenesis. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Kegami, T.; Yanagishita, H.; Kitamoto, D.; Negishi, H.; Haraya, K.; Sano, T. Desalination 2002, 149, 49-54. (2) Ikegami, T.; Yanagishita, H.; Kitamoto, D.; Haraya, K.; Nakane, T.; Matsuda, H.; Koura, N. S. T. Biotechnol. Technol. 1997, 11, 921-924. (3) O’Brien, D.; Craig, J. C., Jr. Appl. Microbiol. Biotechnol. 1996, 44, 699-704. (4) Kelsall, D.; Lyons, T. P. Management of fermentations in the production of alcohol: moving toward 23% ethanol. In The alcohol textbook: a reference for the beverage, fuel and industrial alcohol industries; Jacques K. A.; Lyons, T. P.; Kelsall D. R., Eds.; Nottingham University Press: UK, 1999; pp 49-87. (5) Thomas, K. C.; Hynes, S. H.; Ingledew, W. M. Appl. Environ. Microbiol. 1994, 60, 1519-1524. (6) Yupeng, Z.; Yen-Han, L. Biotechnol. Lett. 2003, 25, 1151-1154. (7) Bayrock, D. P.; Michael, Ingledew, W. J. Ind. Microbiol. Biotechnol. 2001, 27, 87-93. (8) Bafrncova´, P.; Mogroviova´, D.; Sla´vikova´, I.; Pa´tkova´, J. D. Z. Biotechnol. Lett. 1999, 21, 337-341. (9) Thomas, K.; Ingledew, W. M. J. Ind. Microbiol. 1992, 10, 61-68. (10) Schneider, L. V.; Hall, M. P. Drug Discovery Today: Targets 2005, 10, 353-363. (11) Zhang, Y.; Wolf-Yadlin, A.; Ross, P. L.; Pappin, D. J.; Rush, J.; Lauffenburger, D. A.; White, F. M. Mol. Cell Proteomics 2005, 4, 1240-1250. (12) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Mol. Cell Proteomics 2004, 3, 11541169. (13) Aggarwal, K.; Choe, L. H.; Lee, K. H. Proteomics 2005, 5, 22972308. (14) Lee, J.; Cao, L.; Ow, S. Y.; Barrios-Llerena, M. E.; Chen, W.; Wood, T. K.; Wright, P. C. J. Proteome Res. 2006, 5, 1388-1397. (15) DeSouza, L.; Diehl, G.; Rodrigues, M. J.; Guo, J.; Romaschin, A. D.; Colgan, T. J.; Siu, K. W. M. J. Proteome Res. 2005, 4, 377-386. (16) Hardt, M.; Witkowska, H. E.; Webb, S.; Thomas, L. R.; Dixon, S. E.; Hall, S. C.; Fisher, S. J. Anal. Chem. 2005, 77, 4947-4954.

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