Proteomic Analysis of Calcium Alginate-Immobilized Saccharomyces cerevisiae under High-Gravity Fermentation Conditions 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, U.K. Received June 23, 2007
Saccharomyces cerevisiae KAY446 cells immobilized in calcium alginate gel, and supplemented with additional amino acids, were successfully used in enhancing ethanol production. This combination succeeded in improving the ethanol yield and reducing the fermentation time. The ethanol yield under these conditions was 0.40 g of ethanol/g of glucose, with a final ethanol concentration of 118 g/L after 72 h. This is compared to yields with immobilized cells alone of 0.35 g of ethanol/g of glucose and freely suspended cells with no amino acid supplementation of 0.30 g of ethanol/g of glucose, under the same VHG conditions. The maximum specific ethanol production rates were 0.98, 0.73, and 0.61 g (g dry weight)-1 h-1 for immobilized cells under VHG conditions with and without amino acid supplementation and free cells, respectively. A proteomic analysis showed significant stimulation of many pathways during fermentation under these conditions, including the Ras/cAMP, glycolysis, starch, and sucrose pathways, amino acids biosynthesis, and aminoacyl-tRNA synthetases. The upregulation of ribosomal, heat-shock proteins and proteins involved in cell viability confirmed that protein biosynthesis was accelerated and revealed likely mechanisms for improving cellular viability. Keywords: Saccharomyces cerevisiae • proteomics • ethanol production • high glucose concentration • amino acid supplementation • immobilized cells • calcium alginate • iTRAQ
Introduction Ethanol is an important industrial solvent or chemical feedstock, with two broad production routes, these being chemical (the catalytic conversion of ethylene) or biological (fermentation using microorganisms). During the energy crisis of the 1970s, many attempts focused on the development of alternative energy resources, with ethanol being one of the foci. Consequently, a large number of cars use either Gasohol (76% gasoline and 24% ethanol) or pure ethanol as a fuel.1 The current refocus on alternative energy in light of global warming issues has focused attention on ethanol as one of the potential solutions. Until now, yeast-based fermentative ethanol production is still an economic method in many regards. However, most fermentation processes aimed at ethanol production are carried out with a low carbohydrate concentration (usually less than 200 g of glucose/L) to prevent either glucose or ethanol inhibition of yeast cells. As a result, final ethanol concentrations are low at 6–10% (v/v),2 resulting in high energy inputs for distillation of these broths. As an example of the gains in energy efficiency that could be achieved if ethanol yields could be increased, Rose3 showed that if the yield was increased to 12% (v/v) ethanol (from fermentation conditions consisting of * To whom correspondence should be addressed: Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, U.K. Telephone: +44(0)114 2227577. Fax: +44(0)114 2227501. E-mail: p.c.wright@ sheffield.ac.uk. 10.1021/pr070391h CCC: $40.75
2008 American Chemical Society
approximately 250 g of glucose/L over 72 h), this would reduce the energy requirement for distillation by 40% compared to that required for a 6% (v/v) ethanol process. The achievement of this goal in a routine industrial process requires that a number of challenges be addressed: (i) mitigation of cellular osmotic stresses imposed by a high initial glucose concentration, (ii) alleviation of inhibitory effects on the cell by higher concentrations of ethanol, (iii) the time to complete fermentation, and (iv) how to minimize the concentrations of residual substrates in the broths. Application of immobilized cells by the brewing industry broke out in the mid-1970s at both laboratory and pilot plant scales.4 Immobilization has many advantages for fermentation systems, including ease of handling and separation of cells from broths, and ease of increasing cell concentration, resulting in the possibility of continuous fermentation at high dilution rates without cell washout.5 Many different techniques are used for immobilizing cells, including entrapment, adsorption, and use of preformed carriers (see ref 6 for reviews). From the early 1980s to the present, calcium alginate has been used as a preferable carrier for entrapment at both laboratory and industrial scales,2,6,7 and the entrapment process is facile. The most important advantage for this carrier is the provision of a gentle environment for entrapping cells, and it is permitted for use in food and pharmaceutical products.8 Furthermore, using cells entrapped in calcium alginate carriers can help increase the cell concentration, subsequently helping to decrease ferThe Journal of Proteome Research 2008, 7, 515–525 515 Published on Web 01/03/2008
research articles mentation times, as well as increasing the tolerance of cells to both substrate and product inhibition.9 Although many studies have sought to elucidate the biochemical composition of immobilized yeast cells10,11 and characterize the activity of some key enzymes12 during fermentation, the global expressions of proteins under these conditions have not yet been quantitatively measured. It is known that immobilized cells modify their metabolism and growth, due to the effects of nutrient limitation, the cellular microenvironment, and the physical contact of cell carriers.13–15 However, specific changes and their functional impact compared to freely suspended cells remain to be determined, both under typical glucose levels and at very high glucose concentrations. In previous related work, Pham et al.16 reported that under VHG conditions, the relative abundance of most detected glycolysis proteins increased compared to the levels of those from cells grown at lower glucose concentrations. These effects led to enhanced ethanol production, while most detected proteins involved in the synthesis of amino acids, biosynthesis of vitamins, and synthesis of heat-shock proteins were downregulated, leading to a decrease in cell viability and the extent of proliferation. The release of the original iTRAQ reagents generated much attention, as this method is a useful means for determining the expression level of individual proteins of up four (and soon eight) phenotypes simultaneously.17 Recently, evaluations of the iTRAQ technique concentrating on reproducibility across multiple injections into an electrospray ionization mass spectrometer,18 as well as with regard to technical, experimental, and biological variations, have been reported.19 The results showed that high quantification reliability of an iTRAQ workflow can be obtained across multiple injections, with an average CV of 0.09 reported across the three microorganisms that were tested.18 Experimental (or iTRAQ) variations were shown to exhibit behavior similar to that of biological variations, with relatively low errors rates oberved.19 Against this background of good reliability, multiple injections of iTRAQ-labeled peptides into a LC-ESI-MS/MS instrument were used here to determine global differential relative expression patterns of proteins in yeast cells immobilized in calcium alginate beads versus freely suspended cells, both systems being subjected to high-gravity fermentation conditions. Moreover, we supplemented the VHG cultures with additional amino acids, as our previous proteomics investigation indicated these may be limiting.16
Materials and Methods Cell Growth Conditions and Immobilization. Saccharomyces cerevisiae KAY446 (courtesy of K. 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, and 4 µg of adenine/mL) in a shaken water bath (FALC) at 120 rpm and 30 °C for 12 h. To obtain a direct comparison between free and immobilized cells, all cells were harvested from the same inoculation culture and treated identically during the study. Subsequently, the yeast cells were collected by centrifugation at 3000g and room temperature for 5 min and then washed twice with 0.9% (w/v) NaCl. The number of yeast cells was calculated for immobilization, and these cells were then mixed with a calculated volume of 1.5% (w/v) sodium alginate to yield a final concentration of 25 × 106 cells/mL of this suspension. The suspension was transferred to a 50 mL sterilized syringe with a 2 mm internal diameter 516
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Pham and Wright needle. Yeast-gel bead formation was carried out by dropping this suspension drop by drop into 500 mL of a 0.1 M CaCl2 (Sigma) solution and incubating it at room temperature for 20 min.20 The diameters of the beads ranged from 3.5 to 4.5 mm, as determined using a caliper. For all calculations involving beads, the assumption was that they were spherical. The number of initial cells was calculated for immobilization in calcium alginate to reach around 25 × 106 cells/cm3 of beads. Subsequently, numbers of beads were also calculated before commencement of fermentation to reach a concentration of immobilized cells in broths at 2 × 106 cells/mL. Moreover, fermentation with free cells at the same cellular concentration was carried out as a control. Triplicate cultures were used. Before being subjected to VHG ethanol fermentation, both the immobilized cells and the free cells were precultured for 4 h in media with the same composition (listed below), but with a low concentration of glucose (20 g/L). The ethanol fermentations were carried out with an initial cell concentration of 2 × 106 cells/mL of broth, and these processes were performed in 250 mL Erlenmeyer flasks containing 100 mL of media consisting of 5 g/L yeast extract, 3 g/L peptone, 5 g/L KH2PO4, 1.5 g/L NH4Cl, 0.7 g/L MgSO4, 1.7 g/L KCl, 5.8 g/L casamino acids, 7.2 g/L fresh yeast autolysate, and 300 g/L glucose. In the case of immobilized cells with amino acid supplementation, the mixture of amino acids used consisted of a Complete Supplement Mixture (CSM)(Ade, His, Leu, Met, Trp, Ura) (Sunrise Science Products) added to a concentration of 550 mg/L of medium. The cell suspensions were 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. Measurement of Fermentation Parameters. The ethanol concentrations in the triplicate cultures were determined using a Finnigan Trace DSQ single quadrupole GC-MS system coupled with a model AS3000 autosampler (Thermo Electron Corporation) fitted with a 30 m × 0.25 mm (inside diameter) × 0.25 µm df Stabilwax fused silica column (Thames Restek, Bucks, U.K.). The preparation and analysis of samples were performed as described by Pham et al.16 Residual glucose and glycerol concentrations were measured using a glucose (GO) assay kit (Sigma) and a K-GCROL glycerol assay kit (Megazyme). The pH was tested in every sample using a pH meter (Fisherbrand Hydrus 300). Cell concentrations were monitored by measuring the absorbance of the culture at 650 nm (OD650) using an Ultrospec 2100 Pro spectrophotometer (Biochrom). Moreover, numbers of cells were counted using a Thoma chamber (Marienfeld). Immobilized Cell Extraction. To release yeast cells from beads for the determination of cell numbers, five beads were harvested from each culture and dissolved in 10 mL of a 5% EDTA solution shaken at 120 rpm for 15 min at room temperature. The result was calculated for 100 mL of medium. For protein extraction, approximately 100 beads were harvested from each of the triplicate cultures and dissolved in 100 mL of 5% EDTA. Subsequently, cells released from the beads were centrifuged at 3000g for 5 min, followed by being washed twice with deionized water and then twice with yeast extract buffer, including 50 mM 2-morpholinoethanesulfonic acid (MES), 10 mM EDTA, and 10 mM MgCl2 at pH 4.5.21 Finally, pellets were resuspended in yeast extract buffer with 5% (v/v) yeast protease inhibitor cocktail (Sigma), and 2 volumes of glass beads (425–600 µm) equivalent to the pellet volume was also
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Response of Immobilized Yeast Cells to High Glucose Concentrations added. Separate protein extraction for each culture was performed by vortexing samples in a Disruptor Genie (Scientific Industries) for 9 min (45 s vortex and 45 s in an ice bath). The protein concentration was determined in triplicate using the RC DC protein quantification assay (Bio-Rad, Herfordshire, U.K.). Biological replicates were pooled. Determination of Glycogen and Trehalose Levels. The determination of glycogen and trehalose levels was carried out mostly on the basis of the work by Thomson et al.22 with some modifications. Briefly, 2 mL of sample was centrifuged at 3000g for 5 min and washed twice with 0.9% cold NaCl. After the supernatant was discarded, pellets were resuspended in 2 mL of 0.2 M citrate buffer at pH 4.8, and then 1 g of glass beads (425–600 µm) was added for votexing for 50 min (votexing for 10 min alternating with 5 min on ice). Supernatants were subsequently collected by centrifugation at 3000g for 5 min at 4 °C and used for the analysis of trehalose and glycogen levels. This was done by incubation at 37 °C overnight with trehalase [0.1 unit/mL in 0.2 M citrate buffer (pH 5.7)] or amyloglucosidase [1.4 units/mL in 0.2 M citrate buffer (pH 4.8)], respectively; as a result, glucose was generated as a final product. Finally, these glucose concentrations were determined using a glucose assay kit (GO) (Sigma). Moreover, the glucose concentrations in the cases of non-enzyme-treated supernatants were also determined, with corrections then applied to calculation of the concentrations of trehalose and glycogen from glucose concentration results. Determination of Dry Weight. A gravimetric method using cellulose nitrate membrane filters (pore size of 0.45 µm; Whatman) was performed for the determination of cell dry weight. The filters were predried in a microwave oven (Sharp R-206) for 10 min. To determine dry weight, 10 yeast-gel beads were harvested; cellular release was carried out as described above, and the liberated cells were then centrifuged at 3000g for 5 min. Samples were subsequently washed twice with 20 mL of cold water before being dried with a microwave for 30 min. Determination of Cell Viability. The methylene blue technique was used to determine the viability of yeast cells, with all steps based on those of Alfenore et al.23 with some modifications. Briefly, 100 µL of a yeast suspension (diluted to reach an OD of 0.5–0.8) was mixed with 100 µL of a 0.3 mM sterilized methylene blue solution (in 68 mM Na3 citrate), votexed, and incubated for 5 min, followed by cell counting in a Thomas counting chamber. As a result, live cells were unstained while nonviable cells were stained. The viability of cells was represented by the percentage of “viable” cells calculated from the number of unstained cells divided by the total number of stained and unstained cells. Protein Extraction, Labeling, Mass Spectrometry, and Data Analysis. The preparation of yeast cell extracts, the labeling of different phenotypes, and the identification of proteins from peptide fractions were conducted as described by Chong et al.18 Briefly, cells harvested at the 60th hour, and the cell extraction was done as described in Immobilized Cell Extraction. Protein of each phenotype (100 µg) was precipitated with ice-cold acetone overnight at -20 °C before being resuspended in 20 µL of 500 mM TEAB at pH 8.0. Proteins from each sample were reduced, alkylated, digested, and labeled with iTRAQ reagents (114, 115, and 117) for proteins extracted from free cells, immobilized cells, and immobilized cells with amino acid supplementation, respectively. The remaining iTRAQ reagent
(116) was labeled by samples extracted from 300 g/L glucosegrown yeast from a previous study.18 This sample and the current sample (116 and 114, respectively) were used as a biological replicate. Peptide fractionation was carried out by strong-cation exchange (SCX) chromatography as described elsewhere.18 These peptide fractions were then dried by vacuum concentration before being resuspended in 100 µL of Switchos buffer (0.1% formic acid and 3% acetonitrile) for the nano-LC-ESI-MS/MS analysis. Data acquisition in the positive ion mode on a QStarXL tandem MS instrument (Applied Biosystems/MDS Sciex) was performed with a selected mass range of m/z 300–2000. Peptides with a charge of +2 to +4 were selected for tandem mass spectrometry, and the summation time for MS/MS events was set at 3 s. The two most abundantly charged peptides above a five count threshold were selected for MS/MS and dynamically excluded for 60 s with a (50 mmu mass tolerance. Mass spectra generated were searched against the yeast singlegenome database (6298 proteins) downloaded from NCBI (June 2005) using ProQuant, version 1.1 (Applied Biosystems/MDS Sciex). The search parameters allowed for peptide and MS/ MS tolerance up to 0.15 and 0.1 Da, respectively, one missed cleavage of trypsin, oxidation of methionine, and modification of MMTS. The complete list of identified proteins and the ratios of relative protein expressions was collated and analyzed using ProGroup Viewer 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 (see refs 16, 18, and 19 for more details). 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 reconstructing major metabolic pathways.
Results and Discussion Behavior of Immobilized Cells and Kinetic Fermentation Parameters. The populations of free and immobilized cells as functions of time are shown in Figure 1. The growth of S. cerevisiae free cells occurred slightly faster than for immobilized
Figure 1. Numbers of cells during fermentations: (white bars) nonviable cells, (black bars) free cells, (red bars) immobilized cells, and (green bars) immobilized cells in media with an amino acid supplement. All experiments were performed in triplicate. The Journal of Proteome Research • Vol. 7, No. 2, 2008 517
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Figure 2. Residual glucose concentrations in the broths with free cells (4) and immobilized cells without (O) or with (0) an amino acid supplement. Experiments were performed in triplicate. The inset illustrates the fluctuation of the glucose concentration in the broths during the early hours of fermentation.
cells; however, there was little difference between numbers of free cells and immobilized cells with amino acid supplementation at 60 h. Previously, a decrease in viability and proliferation of immobilized cells were reported,15 and our data here agree (Figure 1) but also show this problem can be mitigated by amino acid supplementation, where ca. 90% of these immobilized cells were still viable under VHG conditions. Glucose consumption, formation of ethanol, and formation of glycerol are shown in Figures 2 and 3, with some kinetic parameters summarized in Table 1. During the early stage of immobilized cell fermentation (with or without amino acid supplementation), glucose fluctuation occurred in the broths, while this phenomenon was not observed for the free suspension process (see the inset in Figure 2 for details). A decrease in glucose concentration in the broths appeared within 2 h. This phenomenon can be explained by the diffusion of external glucose from the broths into the gel matrix when the beads were first transferred to the medium. Cells need to acclimate to the new medium; therefore, the glucose consumption by immobilized cells during this period was insignificant. After this period (after the second hour), when cells began to increase their rate of consumption of glucose, we hypothesize that the CO2 generated during fermentation began to accumulate in the capillary gel matrix, resulting in significant gas in the beads,24 forcing medium (and glucose) back out. From Figure 2, we can see that this process occurred within the first six hours of fermentation; thus, we can divide the curves in Figure 2 into two states, the first stage being unstable (6 h) and the second state being the stable stage. The stable stage commenced when there was a balance among the diffusion of CO2, ethanol (from cells to gel beads and then to the broths), and glucose (from the broths to the gel beads and then to cells). Table 1 shows an increased rate of generation of ethanol from glucose for immobilized cells and for immobilized cells with amino acid supplementation of 0.35 and 0.40 g of ethanol/g of glucose, respectively, compared to a rate of 0.30 g of ethanol/g of glucose obtained with free cells. A significant change in the maximum specific ethanol production rate was observed in immobilized cells [0.73 g (g dry weight)-1 h-1] compared to free cells [0.61 g (g dry weight)-1 h-1]. This increase is even more dramatic for immobilized cells with amino acid supplementation, where a maximum specific ethanol production rate of 0.98 g (g dry weight)-1 h-1 was 518
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Figure 3. (A) Concentrations of ethanol (empty symbols) and (B) glycerol (filled symbols) in the broths with free cells (triangles) and immobilized cells without (circles) or with (squares) an amino acid supplement. Experiments were performed in triplicate.
observed. Moreover, in the case of immobilized cells with amino acid supplementation, a significantly enhanced maximum glucose turnover rate of 2.21 g g-1 h-1 was seen in comparison to that of free cells (1.76 g g-1 h-1). Concomitant with these kinetic data, a stimulation of glycolysis proteins was recorded. Compared with free cells, immobilized cells showed significant changes in proteome relative abundances (see the Supporting Information for the full list), as described below. This phenomenon was similar for immobilized cells grown under different conditions. As seen in Figure 3A, the detection of ethanol starts after 12 h and finishes at the 72nd hour for immobilized cells with amino acid supplementation, at the 84th hour for unsupplemented immobilized cells, and at the 96th hour for free cells. To estimate the effect of substrates on the reaction rates of immobilized cells and free cells, the data in the second stage (Figure 2) were used and are plotted in Figure 4. When the residual glucose concentration in the medium remained high, the bioconversion rate was independent of glucose concentration, and the reaction rates fluctuated slightly. In this period, the activity of immobilized yeast depended not only on the activity of enzymes in the cell but also on the gel matrix, since the diffusion of substrates and products relied on the size of the gel matrix. Even though fermentation is performed by a multienzyme system, the kinetics of the whole fermentation process are
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Table 1. Fermentation Kinetic Parameters Using Free and Immobilized Cells with Various Media free cells parameter
initial glucose concentration (g/L) fermentation time (h) residual glucose concentration (g/L) final biomass concentration (g/L) final ethanol concentration (g/L) ethanol yield (g/g) final glycerol concentration (g/L) glycerol yield (g/g) maximum specific glucose uptake rate [g (g dry weight)-1 h-1] maximum specific ethanol production rate [g (g dry weight)-1 h-1] maximum specific glycerol production rate [g (g dry weight)-1 h-1]
immobilized cells VHG with amino acids
VHG
VHG
300 ( 5.6
300 ( 6.3
300 ( 6.2
102
84
72
86.3 ( 9.45
63.7 ( 12.2
45.9 ( 10.1
4.7 ( 0.3
3.9 ( 0.5
4.8 ( 0.6
90.2 ( 4.6
104.6 ( 5.1
118.3 ( 5.8
0.30 ( 0.01
0.35 ( 0.01
0.40 ( 0.01
11.3 ( 0.6
12.0 ( 0.8
12.5 ( 0.9
0.038 ( 0.003 0.040 ( 0.004 0.042 ( 0.005 1.76 ( 0.13 2.04 ( 0.16 2.21 ( 0.15
0.61 ( 0.03
0.73 ( 0.04
0.98 ( 0.06
0.14 ( 0.08
0.12 ( 0.07
0.15 ( 0.04
limited by the activity of particular key enzymes in the reaction chain.24 Therefore, relatively simple enzyme kinetics can be used to illustrate the whole process.24 To describe the kinetics quantitatively, the slope of log[v/(V - v)] against log[glucose] (known as a Hill plot)25 was used. Subsequently, the slope, known as the Hill coefficient (h), was calculated using the linear regression function in Microsoft Excel (see Figure 5). From Figure 5, the Hill coefficients were 1.87, 1.96, and 2.03 for free cells, immobilized cells without amino acid supplementation, and immobilized cells with amino acid supplementation, respectively. The effect of immobilization as well as amino acid
Figure 4. Dependency of reaction rates on glucose concentration: (4) free cells and immobilized cells without (O) or with (0) amino acids supplement.
Figure 5. Hill plot representing the activity of a particular key enzyme in free cells and immobilized cells: (4) free cells and immobilized cells without (O) or with (0) an amino acid supplement.
supplementation on the kinetic behavior is clear. The consumption rate of glucose was enhanced by immobilization, and a further increase was realized after the addition of an amino acid supplement, where h increased from 1.96 to 2.03. These results show that ethanol fermentation was enhanced by using immobilized cells with amino acid supplementation. For immobilized cells with amino acid supplementation, the fermentation time was reduced by 12 h to 72 h, with a corresponding increase in the specific glucose uptake rate (see Table 1). Amino acid supplementation appears to aid cell viability, thus enhancing the productivity of ethanol production. The metabolism of immobilized cells was enhanced, since an upregulation of most glycolysis proteins compared to free cells was observed at the proteomic level (see the Supporting Information, and below). Identification and Quantitation of Protein Expression. The relative quantitation of proteins from immobilized cells with and without amino acid supplementation compared to free cells was carried out. Moreover, a biological replicate of the 300 g/L glucose condition was also performed. The comparison of these samples should result in an expression ratio of 1.0. The average expression ratio observed for this biological replicate was 1.15 (EF < 3), with a standard deviation of 0.39. From the replicate MS/MS injections applied here, a total of 523 proteins were identified and quantified, only considering those with more than two identified peptides per protein. The relative expressions and error factor (EF) values for these proteins are depicted in Figure 6 in log10 space. Figure 6 reveals that more proteins in immobilized cells with amino acid supplementation were upregulated than in unsupplemented immobilized cells (all ratios were compared to that of freely grown cells) while more downregulated proteins were seen in immobilized cells growing under VHG conditions alone. Our overall aim is to understand how S. cerevisiae produced more ethanol under VHG and immobilization conditions, especially with amino acid supplementation. Toward this goal, proteins which were differentially expressed by more than (1.5fold are used for discussion with regard to their EF value, as we showed previously the statistical basis for this cutoff with our S. cerevisiae strain.19 In the following discussion, the differential expression values for proteins are given in terms of two ratios [e.g., protein ((a; (b)]. The first value (a) The Journal of Proteome Research • Vol. 7, No. 2, 2008 519
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Figure 6. Expression of proteins in immobilized cells compared to free cells (O) and immobilized cells with an amino acid supplement compared to free cells (0). All data are presented in log10 space.
represents the differential expression of the proteins obtained from immobilized cells grown under VHG conditions compared to free cells under the same conditions, while the second value (b) represents the differential expression of proteins sourced from immobilized cells grown under VHG conditions with amino acid supplementation compared to free cells grown under VHG conditions alone. A positive value represents upregulation, while a negative value represents downregulation. For example, protein Hsc82p (-1.2; +2.2) was downregulated by 1.2-fold in immobilized cells when compared to free cells (although technically no change like this is within out 1.5-fold cutoff) and upregulated by 2.2-fold in immobilized cells with amino acid supplementation compared to free cells. Ras/cAMP Pathway. The Ras/cAMP pathway is essential for the control and incorporation among cell growth, cell cycle progression, and metabolic activities. The formation of cAMP is carried out by Cyr1p (+1.6; +1.9) (adenylate cyclase), which is activated directly by Ras2p (-1.1; +1.8) (a small GTP-binding protein), with this protein activated by Cdc25p (+1.4; +1.6) [guanine nucleotide exchange factor (GEF)] by stimulating Ras2p exchange of GDP to GTP26–28 (see Figure 7 for details). Under the immobilization condition with amino acid supplementation studied here, these proteins were all upregulated, meaning that this pathway was also activated in response to this stress condition. Moreover, together with this pathway activation, proteins involved in the synthesis of carbohydrate storage compounds such as glycogen and trehalose were also upregulated. Moreover, the upregulation of Cdc25p together with Ssa1p (+1.5; +2.2) (a member of the Hsp70p heat shock protein family) agreed with work done by Geymonat et al.,29 since the function of Ssa1p is to bind to Cdc25p to maintain activity of Cdc25p under stress conditions. The data here support this hypothesis, since both Cdc25p and Ssa1p were upregulated here. When Ras/cAMP is activated, this results in an upward expression of cAMP-dependent protein kinases.30 Another interesting observation is the upregulation of Tps2p (+1.8; +2.4), since this protein is known to be induced by stress and repressed by the Ras/cAMP pathway.31 From the proteomic data, as well as the measurements of trehalose accumulation (refer to Figure 9A), we can conclude that the response of Tps2p may be to play a role in aiding cells to adapt to stress more than to be repressed by the Ras/cAMP pathway. Under conditions tested here, differential expressions of proteins that both directly and indirectly generate cAMP were upregulated. The accumulation of CO2 in the matrix gel yielded 520
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Pham and Wright anaerobic conditions and led to beads floating to the culture surface. This resulted in CO2 release and the sinking of the beads again. This process accelerated the diffusion of medium into the beads, and then into yeast cells. Therefore, cells immobilized in the beads were subjected to a cyclic environment, and as result, the Ras/cAMP pathway was activated. This pathway was activated due to the nutrition demand of entrapped cells, due to cyclically reduced rates of diffusion from the liquid medium to the gel matrix, in comparison with free cells. The activation of this pathway together with the upregulation of glycolysis proteins shows that enhanced ethanol production can be achieved using immobilized cells under VHG conditions along with amino acid supplementation. Glycolysis Pathway. During fermentation with immobilized cells, with the same culture compositions, both ethanol and glycerol were formed faster than in freely suspended cultures. As seen in the Supporting Information, 16 of the identified glycolysis proteins were upregulated in immobilized cells compared to freely grown cells. More details about the functions of these proteins during fermentation are discussed elsewhere.16 There is an inverse correlation between the rate of glucose uptake and the concentration of glucose 6-phosphate. The greater the amount of glucose consumed, the lower the abundance of glucose 6-phosphate, due to feedback inhibition of glucose uptake by glucose 6-phosphate.32,33 Some studies suggest that the fermentation rate is limited by glucose uptake.34,35 In the immobilized cells, the demand for glucose induced the activity of transport proteins. This was evidenced by an upregulation of Hxk1p (+1.4; +1.5), Hxk2p (+1.6; +2.0), and Glk1p (+1.7; +2.2). Immobilization may damage the cell wall (nonlethal), but this helps increase the rate of transport of glucose into the cells.36,37 Others have demonstrated enhanced ethanol productivity using immobilized S. cerevisiae with polyethylene glycol and dextran supplementation,38 with the hypothesis being that a decrease in water activity directly affects ethanol metabolism, leading to enhanced generation of this product. The decrease in water activity can cause changes in intracellular pools of certain metabolites, leading to a rearrangement in an already operating metabolic pathway and/or initiation of dormant pathways. As a result, this effect helps to change yields of substances in maintenance metabolism, as well as effect new metabolic behavior.39 However, the mechanism for this observation remains unproven. Here, the upregulation of proteins relating to both the Ras/cAMP pathway and glucose transporters tends to support this hypothesis. Moreover, the upregulation of hexokinase may lead to immobilized cells diverting their metabolism toward glycogen and trehalose compounds, since the accumulation of these compounds may help cells protect against stress, as well as the regulation of these compounds playing a role in maintaining cellular redox balance.40 Glucose 6-phosphate is the branch point between catabolic and anabolic pathways. The role of hexokinase is to facilitate the supply of C-constituents for macrocellular components. Quantitative measurements have shown that immobilized cells contain larger amounts of carbon storage compounds, DNA, and RNA than free cells.10,41 The relationship between glucose 6-phosphate and the synthesis of DNA and RNA is shown in Figure 7. The formation of AMP and ATP may relate to the activity of Pfk1p and Pfk2p, since these compounds are the two main effectors of these proteins; however, the regulation of these effectors is complex and ill-understood.42 While AMP is
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Figure 7. Glycolysis pathway depicted in relationship to the Ras/cAMP pathway, starch and sucrose pathway, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis. Metabolites are shown with a blue background (except for energy compounds that have a red background), while upregulated proteins are depicted with a green background. The blue line shows the interaction of transmission of signals with activities of proteins.
a positive effector increasing the activity of an enzyme, ATP is an allosteric inhibitor leading to a decrease in the affinity of the enzyme. Moreover, the formation of fructose bis-phosphate is known as a stimulating factor of phosphofructokinase.12 The relationship between AMP and ATP and the synthesis of DNA and RNA is shown in Figure 7. During ethanol fermentation, both pyruvate decarboxylases and alcohol dehydrogenases play important roles in the conversion of pyruvate to ethanol as a terminal product of glycolysis. These proteins exhibited significant upregulation under immobilized conditions. Together with the upregulation of Pdc1p (+2.0; +2.5) and Pdc5p (+1.7; +2.1), the upregulation of Pdc6p (+1.2; +1.9) may help increase the activity of pyruvate decarboxylases during VHG fermentation with immobilized cells. While Adh1p (+1.7; +2.4) and Adh4p (+1.5; +2.0) are cytoplasmic enzymes, Adh3p (+1.5; +1.7) is located in the mitochondria.43 As mentioned before, VHG conditions have been widely used for ethanol fermentation. Many aspects of this technique have been characterized. Recently, global gene expression measurements have been used to investigate the fermentation under VHG conditions with maltose as a main substrate.44,45 As observed by Rautio et al., the levels of expression of hxk-1 and glk-1 increased after 9 h and the levels
of expression of adh-1, adh-3, and adh-4 increased after 4 h, leading to a large decrease in residual glucose concentration, and a large increase in ethanol concentration.45 Moreover, the expression levels of gpd-1, gpd-2, and tps-1 were also increased, leading to an increase in the glycerol concentration.45 The proteomics observations made here are highly consistent with these gene expression observations made previously. The upregulation of these proteins consolidates the enhanced conversion mechanism of glucose to ethanol for the immobilized cells supplemented with amino acids. Utilization of ATP and Synthesis of Trehalose and Glycogen. The intracellular pH in immobilized cells has been shown to be slightly lower than that in free cells.46 Due to this reduced pH, together with the high glucose uptake profile, it is thought that the permeabilization of an immobilized cell membrane may be accelerated.47–49 Since there is a leak of protons through the membrane, cells accelerate ATP consumption to maintain a constant intracellular pH.46 Here, the levels of expression of proteins involved in the formation and utilization of ATP were upregulated to respond to the energy balance of cells under immobilized conditions.50 While the expression of proteins in the upper part of the glycolysis pathway may increase levels of glucose 6-phosphate for The Journal of Proteome Research • Vol. 7, No. 2, 2008 521
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Figure 8. Model describing the activation of the Ras/cAMP pathway for the transition of signals in immobilized cells compared to freely suspended cells. Its activation indirectly accelerated alcohol fermentation under the conditions studied here.
synthesis of trehalose, the upregulation of proteins in the lower part of this pathway may accelerate ATP generation.50 As ATP levels decline, the pH falls, resulting in the mobilization of trehalose and glycogen through the stimulation of cAMP synthesis. The decrease in the level of storage carbohydrates stimulates fermentation via glycolysis, regenerating ATP levels in cells, as well as increasing proton efflux to maintain the intracellular pH51 (see Figure 8), increasing the extent of fermentation. The accumulation and utilization of trehalose and glycogen are important for understanding the physiological response of S. cerevisiae to environmental changes. For instance, while glycogen is a prerequisite for sporulation, trehalose is necessary for germination.40 In this study, the concentrations of glycogen and trehalose in entrapped cells were 1.5-2-fold higher in immobilized cells than free cells (see Figure 9A, B). At the 60 h sampling time, the concentrations of these compounds were higher than in free cells. From the trehalose and glycogen contents, as well as the expression of proteins related to the metabolism of these compounds, it can be seen that the trehalose and glycogen concentrations in immobilized cells were higher than in free cells (see Figure 9A, B). The levels of expression of proteins that synthesize [Gph1p (+1.6; +2.1) and Tps2p (+1.8; +2.5)] trehalose and glucogen were increased. The expression of Nth1p (+1.6; +2.0) (neutral trehalase), which converts trehalose and water into two molecules of glucose,52 was also upregulated. These integrated protein abundance and metabolite data confirm the hypothesis that the Ras/cAMP pathway is activated under immobilization conditions, resulting in enhanced ethanol production. A decrease in the cytoplasmic pH in immobilized cells may shift metabolism to produce more bypass products compared with free cells. Examples include acidic products (acetic acid and succinic acid),16 resulting in an increased level of glycerol 522
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Figure 9. Concentrations of trehalose (A) and glycogen (B) at the 48th and 60th hour sampling times for the iTRAQ experiment: (left bars) free cells, (center bars) immobilized cells, and (right bars) immobilized cells with an amino acid supplement. All experiments were performed in triplicate.
for maintenance of the redox balances in the cells. At the 60th hour sampling time, the concentration of glycerol was higher for immobilized cells (10.6 and 11.8 g/L without and with amino acid supplementation, respectively) than free cells (8.3 g/L), but there was little difference between entrapped cells with and without amino acid supplementation (Figure 3B). Differential Expression of Ribosomal Proteins. Of the 69 ribosomal proteins identified using tandem mass spectrometry, 52 were upregulated in immobilized cells with amino acid supplementation. Examples include four of the 40S subunit proteins [Rps24ap (+2.5; +4.1), Rps24bp (+2.5; +4.1), Rps15p (+1.8; +7.7), and Rps6bp (+1.4; +8.4)] and two of the 60S subunit proteins [Rpl11bp (-1.2; +2.4) and Rpp0p (+2.7; +3.8)]. The main function of ribosomal proteins is to organize protein synthesis.53 The upregulation of the Rps group might result in an increase in the rate of export of 20S rRNA to cytoplasm, since this protein is essential for rendering the pre-40S particles competent in translocation to the cytoplasm, and for the final steps in nucleoplasmic maturation.54 The differential regulation of proteins in the Rps group might also relate to the export machinery, and the export efficiency.55 The upregulation of these proteins shows that use of immobilized cells for ethanol fermentation leads to an increased level of protein in immobilized cells, with the addition of amino acids providing the greatest increase. The differential expression of these proteins po-
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Response of Immobilized Yeast Cells to High Glucose Concentrations tentially involves the Ras/cAMP pathway (which is related to environmental change56), as discussed above. Together with the upregulation of aminoacyl-tRNA synthetases discussed below, there is enough evidence to suggest that protein synthesis in immobilized cells under VHG conditions with amino acid supplementation was accelerated significantly. Proteins Essential for Viability. The growth of cells in the gel matrix occurred slightly slower than in free cells (see Figure 1), with this effect mitigated when the amino acid supplement was added. When immobilized, cells may have a reduced space in which to bud. This explains why the number of immobilized cells was smaller than the number of free cells. However, with addition of amino acids, the viability of immobilized cells was improved, leading to enhanced cell proliferation. From the measured proteome, an increase in the relative levels of expression of cytoplasmic proteins [e.g., Ipp1p (-1.2; +1.8), Rps3p (-1.2; +7.5), Rsm10p (+2.0; +3.8), and Spt15p (-1.2; +3.2)], relating to the synthesis of these compounds or processes, supports this observation. Immobilized cells showed a high viability at the sampling time, where 80 and 90% of immobilized cells without and with amino acid supplementation, respectively, were viable (see Figure 1). The upregulation of Ipp1p (-1.2; +1.8) (cytoplasmic inorganic pyrophosphatase) likely led to accelerated oxygen exchange in the immobilized cells, since its function is to catalyze the rapid exchange of oxygen from Pi with water. In yeast, inorganic pyrophosphate (PPi), generated as a byproduct of many metabolic pathways,57 is known as a central phosphorus metabolite.58 The hydrolysis of PPi is important for maintaining the forward direction of metabolites in these metabolic pathways.57 Moreover, together with the expression of Ipp1p, Pho88p (-1.2; +1.9) (a putative membrane protein), involved in phosphate transport, was also upregulated. Another interesting upregulated protein necessary for viability is Spt15p. The major function of Spt15p is to encode a TATA-binding protein, an essential general transcription factor.59 Recently, using global transcription machinery engineering for reprogramming gene transcription, Alper et al.60 succeeded in improving both glucose and ethanol tolerance using a spt-15 mutant formed from the combined effect of three separate mutations in the spt-15 gene (serine substituted for phenylalanine, Phe177Ser, and similarly Tyr195His and Lys218Arg). This mutant strain significantly enhanced the viability of cells. Moreover, by overexpression of leu-1 in S. cerevisiae in another study, ethanol production under VHG conditions was also enhanced.44 Therefore, we can assert that improving the viability of cells can result in enhancing both ethanol and glucose tolerance. Together with the upregulation of these proteins, the enhanced biosynthesis of amino acids, and aminoacyl-tRNA proteins, confirms that the synthesis of proteins in immobilized cells with amino acid supplementation was accelerated. The upregulation of Rps3p (protein component of the 40S ribosomal subunit) further supports this, since this protein is involved in the ribosomemRNA-aminoacyl-tRNA interaction during translation.61 Viability-related mitochondrial proteins,62 such as Rsm10p (mitochondrial ribosomal protein), were also upregulated. Proteins Related to de Novo Biosynthesis of Amino Acids and Aminoacyl-tRNA Biosynthesis. Aminoacyl-tRNA synthetases catalyze the esterifications of specific amino acids to one of their compatible cognate tRNAs to form aminoacyl-tRNA, required for protein biosynthesis. These synthetases are es-
sential, as well as being related to the regulation of amino acid biosynthesis and amino acid transport.63 In amino acidsupplemented media with immobilized cells alone, increased relative levels of expression of aminoacyl-tRNA synthetases was observed (see the Supporting Information and Figure 7 for their pathway roles). The supplementation of media with amino acids such as histidine, leucine, tryptophan, uracine, methionine, and adenine led to an increased level of expression of proteins related to the equivalent aminoacyl-tRNA of these amino acids. Surprisingly, not only were these proteins upregulated, but other proteins related to other aminoacyl-tRNA proteins (not present in the amino acids supplementation, such as cysteine, isoleucine, tyrosine, and phenylalanine) were also upregulated. Since amino acids are basic compounds for the synthesis of proteins, the synchronized upregulation of proteins involved in amino acids synthesis, aminoacyl-tRNA synthetases, and ribosomal proteins demonstrates that the synthesis of both functional and structural proteins was stimulated. This led to an increase in the relative abundance of proteins involved in many processes, including glycolysis, the starch and sucrose pathway, the cAMP pathway, heat shock proteins, and cell viability. Heat Shock Proteins. Of the 12 identified heat shock proteins (HSP), 11 of them were significantly upregulated in immobilized cells with amino acid supplementation. HSP from the Ssas group [Ssa1p (+1.5; +2.2), Ssa2p (-1.1; +2.0), and Ssa3p (+1.6; +4.4)], which encode chaperone proteins that combine the Ssa subfamily of cytosolic Hsp70p family,64 were generally upregulated. These proteins are necessary for binding newly translated proteins to respond to folding and to prevent aggregation. Moreover, Hsp70p family proteins also relate to the regulation of other HSP. Most Ssasp group proteins are cytosolic, except for Ssa1p and Ssa2p, which are also found in the cell wall.65 Ssa1p has a weak ATPase activity, with this function being enhanced by interacting with the DnaJ/Hsp40 cochaperones, for example, Sis1p and Sti1p. These latter two proteins [Sis1p (+1.5; +1.7) and Sti1p (+1.0; +1.8)] were also upregulated. The plasma membrane-localized Hsp12p protein (-1.2; +1.9) is also stimulated by osmotic stress.66 Together with upregulation of Ssa1p, the upregulation of Hsp104p (+1.0; +1.8) is also notable, as there is cooperation between this protein and Ssa1p to refold and reactivate denatured or aggregated proteins.67 Hsc82p (-1.2; +2.2) is necessary for activation of many key cellular proteins such as regulators and signalers, with this protein also known to reactivate denatured or aggregated proteins.68 The mitochondrial heat shock proteins, Hsp78p (+1.2; +2.0), known to fold and sort proteins in mitochondria were also differentially expressed here. Hsp78p is also known to cooperate with the mitochondrial heat shock protein Ssc1p to prevent the aggregation of misfolded matrix proteins.69,70
Conclusions The combination of two technical aspects here, immobilized cells and improved quality of VHG media using an amino acid supplement, succeeded in improving the specific ethanol yield. Proteomic data here provide a deeper understanding of the response of S. cerevisiae KAY446 to immobilization under VHG conditions with and without the supplementation of amino acids compared to free cells under the same VHG conditions. The data show there is a significant stimulation of immobilized cells under VHG conditions with additional amino acids, since The Journal of Proteome Research • Vol. 7, No. 2, 2008 523
research articles most key proteins related to the Ras/cAMP pathway, glycolysis pathway, starch and sucrose pathway, amino acid biosynthesis, as well as ribosomal proteins, and aminoacyl-tRNA synthetases were upregulated under this condition. Moreover, together with the upregulation of these proteins, the upregulation of heat shock proteins and proteins necessary for viability helped immobilized cells respond to VHG conditions, and the stress environment formed by the calcium alginate matrix. As a result, there was a significant improvement in ethanol yield during ethanol fermentation under VHG conditions using these immobilized cells. A cost-benefit analysis will still be carried out to consider this industrially, due to the extra costs of feedstock materials such as additional amino acids.
Acknowledgment. We gratefully acknowledge the Ministry of Education and Training of Vietnam for financial support. PCW thanks the EPSRC for provision of an Advanced Research Fellowship (GR/A11311/01) and for funding (GR/S84347/01 and EP/E036252/1). Supporting Information Available: Complete list of proteome relative abundances. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Ogbonna, J. C.; Mashima, H.; Tanaka, H. Bioresour. Technol. 2001, 76, 1–8. (2) Holcberg, I. B.; Margalith, P. Appl. Microbiol. Biotechnol. 1981, 13, 133–140. (3) Rose, D. Process Biochem. Int. 1976, 11, 10–12. (4) Corrieu, G.; Blachere, H.; Ramirez, A.; Navarro, J. M.; Durand, G.; Duteurtre, B.; Moll, M. An immobilized yeast fermentation pilot plant used for production of beer; 5th International Fermentation Symposium, Dijon, France, 1976. (5) de Alteriis, E.; Silvestro, G.; Poletto, M.; Romano, V.; Capitanio, D.; Compagno, C.; Parascandola, P. J. Biotechnol. 2004, 109, 83– 92. (6) Norton, S.; D’Amore, T. Enzyme Microb. Technol. 1994, 16, 365– 375. (7) Purwadi, R.; Taherzadeh, M. J. Bioresour. Technol. (in press). (8) Smidsrod, O.; Skjak-Braek, G. Trends Biotechnol. 1990, 8, 71– 78. (9) Talebnia, F.; Niklasson, C.; Taherzadeh, M. J. Biotechnol. Bioeng. 2005, 90, 345–353. (10) Doran, P. M.; Bailey, J. E. Biotechnol. Bioeng. 1986, 28, 73–87. (11) Doran, P. M.; Bailey, J. E. Biotechnol. Bioeng. 1987, 29, 892–897. (12) Hilge-Rothmann, B.; Rehm, H. J. Appl. Microbiol. Biotechnol. 1990, 33, 54–58. (13) Melzoch, K.; Rychtera, M.; Habova, V. J. Biotechnol. 1994, 32, 59– 65. (14) Lee, T. H.; Ahn, J. C.; Ryu, D. D. Y. Enzyme Microb. Technol. 1983, 5, 41–45. (15) Galazzo, J. L.; Bailey, J. E. Biotechnol. Bioeng. 1990, 36, 417– 426. (16) Pham, T. K.; Chong, P. K.; Gan, C. S.; Wright, P. C. J. Proteome Res. 2006, 5, 3411–3419. (17) 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. (18) Chong, P. K.; Gan, C. S.; Pham, T. K.; Wright, P. C. J. Proteome Res. 2006, 5, 1232–1240. (19) Gan, C. S.; Chong, P. K.; Pham, T. K.; Wright, P. C. J. Proteome Res. 2007, 6, 821–827. (20) Bucke, C. Cell immobilization in calcium alginate. In Methods in Enzymology; Mosbach, K. , Ed.; Academic Press: New York, 1987; pp 175–189. (21) Trabalzini, L.; Paffetti, A.; Scaloni, A.; Talamo, F.; Ferro, E.; Coratza, G.; Bovalini, L.; Lusini, P.; Martelli, P.; Santucci, A. Biochem. J. 2003, 370, 35–46. (22) Thomsson, E.; Larsson, C.; Albers, E.; Nilsson, A.; Franzen, C. J.; Gustafsson, L. Appl. Environ. Microbiol. 2003, 69, 3251–3257. (23) Alfenore, S.; Molina-Jouve, C.; Guillouet, S. E.; Uribelarrea, J. L.; Goma, G.; Benbadis, L. Appl. Microbiol. Biotechnol. 2002, 60, 67– 72.
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