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Proteomic Analysis of Erythritol-Producing Yarrowia lipolytica from Glycerol in Response to Osmotic Pressure
Proteomic Analysis of Erythritol-Producing Yarrowia lipolytica from Glycerol in Response to Osmotic Pressure
Journal of Microbiology and Biotechnology. 2015. Jul, 25(7): 1056-1069
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : December 10, 2014
  • Accepted : March 01, 2015
  • Published : July 28, 2015
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About the Authors
Li-Bo Yang
Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, P.R. China
Xiao-Meng Dai
Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, P.R. China
Zhi-Yong Zheng
Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, P.R. China
Li Zhu
Jiangsu Rayguang Biotechnology Co., Ltd., Wuxi, Jiangsu 214125, P.R. China
Xiao-Bei Zhan
Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, P.R. China
xbzhan@yahoo.com
Chi-Chung Lin
Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, P.R. China
xbzhan@yahoo.com

Abstract
Osmotic pressure is a critical factor for erythritol production with osmophilic yeast. Protein expression patterns of an erythritol-producing yeast, Yarrowia lipolytica , were analyzed to identify differentially-expressed proteins in response to osmotic pressure. In order to analyze intracellular protein levels quantitatively, two-dimensional gel electrophoresis was performed to separate and visualize the differential expression of the intracellular proteins extracted from Y. lipolytica cultured under low (3.17 osmol/kg) and high (4.21 osmol/kg) osmotic pressures. Proteomic analyses allowed identification of 54 differentially-expressed proteins among the proteins distributed in the range of pI 3-10 and 14.4-97.4 kDa molecular mass between the osmotic stress conditions. Remarkably, the main proteins were involved in the pathway of energy, metabolism, cell rescue, and stress response. The expression of such enzymes related to protein and nucleotide biosynthesis was inhibited drastically, reflecting the growth arrest of Y. lipolytica under hyperosmotic stress. The improvement of erythritol production under high osmotic stress was due to the significant induction of a range of crucial enzymes related to polyols biosynthesis, such as transketolase and triosephosphate isomerase, and the osmotic stress responsive proteins like pyridoxine-4-dehydrogenase and the AKRs family. The polyols biosynthesis was really related to an osmotic response and a protection mechanism against hyperosmotic stress in Y. lipolytica . Additionally, the high osmotic stress could also induce other cell stress responses as with heat shock and oxidation stress responses, and these responsive proteins, such as the HSPs family, catalase T, and superoxide dismutase, also had drastically increased expression levels under hyperosmotic pressure.
Keywords
Introduction
There are variable environmental conditions in which fermentations were conducted using microorganisms, such as osmotic, iron, oxidative, and heat shock stresses [50] . Osmotic pressure is known to exert profound influence on cell growth and its metabolism. When microorganisms are exposed to a high osmotic pressure environment, osmotic shock usually results in a temporary growth arrest to adapt to changes of cellular metabolism [43] . Under such condition, microorganisms induce an “osmotic such response” to resist damages due to high osmotic pressure, and alter their complex biological networks dynamically, involving genes, proteins, metabolites, physiology, etc. [22] ; for example, accumulation of compatible solutes, shrinking cell volume, decreasing growth of yeast, and changing the metabolic flux of nutrients [3] . In osmophilic yeast, generating intracellular or extracellular compatible solutes is the conventional strategy to compensate for variations of osmotic conditions.
There are a variety of compatible solutes such as polyols, amino acids, carbohydrates, and glycosides induced in different yeast species under high osmotic condition [57] . Polyols, such as glycerol, erythritol, arabitol, and mannitol, which are small molecules, are compatible solutes that can be safely accumulated at a high level in the yeast without impairing the stability of proteins and nucleic acids [59] . Furthermore, erythritol with its four carbon atoms per molecule has been widely used as a sweetener in the food and pharmaceutical industries [37] . The commercial production of erythritol is commonly conducted with glucose, sucrose, or enzyme-hydrolyzed corn starch as the carbon source. To maintain profitability during economic downturn, low-cost substrates are used to replace the traditional substrates. For example, raw or crude waste glycerol from the biodiesel industries have been proven to be an excellent substitutable substrate for erythritol production [47 , 48 , 52] .
High osmotic pressure (including high substrate concentration and high salinity) has been demonstrated to be beneficial for erythritol production in osmophilic microorganisms [60] . In this study, Yarrowia lipolytica , an unconventional osmophilic yeast, was used to produce erythritol from glycerol under various osmotic pressures values. To determine the changes in the expression level of key intracellular proteins and compare the patterns of the protein response to changing osmotic pressure, proteomic analysis of Y. lipolytica cultivated under high and low osmotic pressure was conducted. Although gene expression analysis systems such as proteomics, transcriptomics, and metabolomics have been successfully used to investigate the effect of osmotic pressure in Saccharomyces cerevisiae and Pichia pastoris [16 , 43] , there is no publication on the effect of osmotic pressure on the translational levels during erythritol fermentation from glycerol with Y. lipolytica . However, the two-dimensional electrophoresis (2-DE) approach has been successfully used to investigate the metabolic changes of Y. lipolytica during transition to hyphal growth [39] and amino acids catabolism [34] .
Y. lipolytica possesses some interesting physiological properties, such as the ability to grow in a hyperosmotic environment at very low pH (2.0-3.0). High osmotic pressure was beneficial for erythritol production from glycerol, which had been demonstrated. The activity of erythrose reductase was up-regulated under high osmotic pressure. Furthermore, the optimal initial osmotic pressure was determined to be 4.21 osmol/kg by us recently [60] . By analyzing the Y. lipolytica exhibiting particular growth and fermentation characteristics at the proteomics level, the crucial proteins playing pivotal roles in cell survival under a hyperosmotic environment can be identified. In addition, these crucial proteins responding to high osmotic pressure may also be involved in unprecedented functions. Consequently, our studies contribute to better understanding of Y. lipolytica in response to osmotic pressure, which conceivably plays a critical role in the elucidation of the adaptation of Y. lipolytica metabolism to the hyperosmotic environment [26 , 34] . In addition, erythritol production affected by osmotic pressure may be the results of enzyme expression changes in multiple metabolic pathways, such as energy, metabolism, cell rescue, and osmotic and redox stress response. Therefore, it is necessary to develop a global proteomic approach to explain the osmotic stress response mechanism at the protein expression level. Our objectives were to use two-dimensional electrophoresis coupled with mass spectrometry to compare the differences in expression levels of the responsive proteins and to identify the key enzymes in response to changes of osmotic pressure.
Materials and Methods
- Microorganism and Media
The Y. lipolytica strain CICC 1675 used in this work has been described previously [60] . The seed medium contained (g/l) glycerol 50; yeast extract 10; MgSO 4 ·7H 2 O 0.5; and KH 2 PO 4 0.2, pH 6.0. Erythritol fermentation medium contained (g/l) glycerol 200; urea 1.68; yeast extract 1.0; MgSO 4 ·7H 2 O 0.5; KH 2 PO 4 0.2; CaCl 2 ·2H 2 O 0.5; ZnSO 4 ·7H 2 O 0.1; MnSO 4 ·4H 2 O 0.01; and FeSO 4 ·7H 2 O 0.1. All media were sterilized at 115℃ for 20 min before filter-sterilized CaCl 2 ·2H 2 O and FeSO 4 ·7H 2 O were added under sterilized conditions.
- Culture Methods
For seed preparation, a loopful of Y. lipolytica from a fresh slant was inoculated into 50 ml of seed medium in 250 ml flasks and incubated on a rotary shaker at 250 rpm for 24 h. Batch fermentations were conducted in 3 L of erythritol fermentation medium using a 7 L stirred-tank reactor (BioFlo 115; New Brunswick Scientific Co., NJ, USA). All temperatures of the fermentations were maintained at 30℃. The pH was controlled at 3.0 with NaOH (2 mol/l) added dropwise automatically. The agitation speed and the aeration were m aintained at 800 rpm and 1.0 vvm , respectively. Batch fermentations were ended when glycerol was depleted. For the high osmotic pressure condition, 30 g/l NaCl was added into the erythritol fermentation medium to adjust the osmotic pressure to 4.21 osm ol/kg. Medium with no NaCl addition was the low osmotic pressure condition (3.17 osmol/kg). All fermentations were conducted with three replications.
- Analytical Methods
Cell density was detected optically at 600 nm by a UV spectrophotometer (model UV-2000; UNICO, Dayton, NJ, USA) and converted to dry cell weight (DCW) according to the equation: DCW = 1.12 (OD 600 ) – 0.19. Glycerol and erythritol were determined by HPLC (Agilent 1200 series, Santa Clara, CA, USA) with an Aminex HPX-87H ion-exclusion column (300 mm × 7.8 mm; Bio-Rad Laboratories Inc., Hercules, CA, USA) [60] .
- Determination of Osmotic Pressure
To determine the actual osmotic pressure of the fermentation broth, the supernatant samples obtained by centrifugation (10,000 × g , 10 min) were analyzed by freezing-point depression using an osmometer (Osmomat 030; Gonotec, Germany) [56] .
- Growth Tests Under Various Levels of Osmotic Pressure
Y. lipolytica was grown in the seed medium at 30℃ in a rotary shaker at 250 rpm for 24 h. Yeast cell suspension was diluted to a proper concentration (OD 600 ≈ 1) with sterile distilled water, and 1:10 serially diluted in sterile distilled water. Then 2 µl was spotted onto hyperosmotic agar plates containing 0, 10, 20, 30, 40, 50, 60, and 70 g/l NaCl. All plates were incubated at 30℃ for 4 to 6 days.
- Protein Sample Preparation
The yeast cells were harvested from the fermenter when the OD 600 was almost equal to 10 (at the end of exponential phase) under high and low osmotic pressure. For the extraction of the total intracellular protein, cells were centrifuged (10,000 × g , 5 min, 4℃) and washed three times with cold Tris buffer (50 mmol/l, pH 7.4). The washed cell pellet was resuspended in 1 ml of lytic buffer (50 mmol/l Tris HCl, 150 mmol/l NaCl, 5 mmol/l EDTA, and 1 mmol/l phenylmethanesulfonyl fluoride, pH 7.4) with protease inhibiter cocktail tablets (Roche, USA), and homogenized by grinding with glass beads (0.5 mm in diameter; Sigma) in a bead beater in a fast prep cell breaker (Bio 101; level of 5.5, 4 times for 30 sec). The glass beads and unbroken cells were removed by centrifugation (13,000 rpm for 15 min). Cell extracts in the supernatant were collected into a new Eppendorf tube. The protein concentration was determined using the Bradford assay (Bio-Rad) and protein samples were stored at -80℃ until analysis.
- Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)
Protein samples were dissolved in rehydration buffer containing 8 mol/l urea, 2% (w/v) CHAPS, 1% (v/v) IPG ampholytes (pH 3-10 NL; GE Healthcare, USA), 1% (w/v) dithiothreitol (DTT), and 0.002% (w/v) bromophenol blue. Samples were centrifuged twice at 12,000 × g for 15 m in at 4℃. Then 450 µl samples containing about 800-1,000 µg protein were added to 24 cm, pH 3-10 NL immobilized IPG dry strips (GE Healthcare Biosciences, Uppsala, Sweden) for rehydration for at least 12 h at 20℃ in a strip holder. Subsequently, the first dimension of isoelectric focusing (IEF) was carried out on an Ettan IPGphor (GE Healthcare Biosciences) with the following operating procedures: 100 V step for 2 h, 250 V step for 2 h, 500 V step for 2 h, 500-1,000 V gradient for 2 h, 1,000-5,000 V gradient for 2 h, 5,000-10,000 V gradient for 2 h, and 10,000 V step continuing until 120 kVh was reached. Thereafter, the IPG strips were equilibrated using 10 mg/ml DTT and 25 mg/ml iodoacetamide in 4 ml of equilibration buffer (50 mmol/l Tris-HCl, pH 8.8, 6 mol/l urea, 30% (w/v) glycerol, 2% (w/v) SDS, and 0.002% (w/v) bromophenol blue) for 15 min, respectively. The second dimension was performed by SDS polyacrylamide gel electrophoresis on 12.5% polyacrylamide gels using an Ettan Daltsix vertical electrophoresis system (GE Healthcare Bioscience). Electrophoresis was conducted at 12℃ using 2 W/gel for 1 h and followed by 15 W/gel until the dye front reached the bottom of the gel. After electrophoresis, the analytical gels were fixed in 40% (v/v) methanol and 10% (v/v) acetic acid for 1 h and stained with colloidal Coomassie Blue G250 [6] .
- Image and Data Analyses
The stained gels were digitalized by scanning (300 dpi, reflection mode, monochrome) using Image Master LabScan (GE Healthcare Biosciences). The images were analyzed with PDQuest 8.0.1 software (Bio-Rad). The landmarks for the group of gels were assigned manually, and the spots in different gels were detected and matched by the PDQuest software. The intensity of the spots was normalized according to the “total quantity of valid spots” method. The over- and under-expression of the potential protein spots was defined as these spot intensities that were 1.5-fold below or above comparing with the matched spots in different 2-DE gels. The quantifications of spots were calculated as the mean ± standard deviation (SD). A master gel was created by the combination of every shared spot on the 2-DE gel images of each sample under low and high osmotic pressure conditions to a virtual gel with the PDQuest software [25] .
- Mass Spectrometry (MALDI-TOF/TOF)
The interesting spots in the 2-DE gels were manually excised and decolorized in a destainer (100 mmol/l NH 4 HCO 3 , 30% (v/v) acetonitrile, and double distilled H 2 O) for 0.5 h. Subsequently, the distained gel particles were dehydrated using 100% (v/v) acetonitrile twice, and then the dried gel particles were soaked with 10 ng/µl trypsin at 4℃ for 60 min. Thereafter, the samples were digested in 25 mmol/l NH 4 HCO 3 (pH 8.0) for at least 20 h at 37℃. The collected supernatant peptide solution (1 µl) was spotted onto an Anchorchip target (Bruker-Daltonics, Bremen, Germany) and air-dried at room temperature for 15 min. After that, the same volume of a matrix (10 mg/ml α-cyano-4-hydroxy- trans -cinnamic acid, 50% (v/v) acetonitrile, and 0.1% (v/v) trifluoroacetic acid) was used to cover the dried peptide spots and air-dried at room temperature for another 15 min. Finally, MS analyses of the peptide spots were performed on an Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker-Daltonics). All mass spectra were calibrated using the Bruker peptide calibration standard II (Bruker-Daltonics).
- Database Searches
In accordance with the method for database searches of the protein spots [25] , the fungal taxonomy of NCBInr at the National Center for Biotechnology Information was used in the Mascot server ( http://www.matrixscience.com ). The parameters used for the search were as follows: Database, NCBInr; enzymatic digestion, trypsin; allow up to one missed cleavage; Taxonomy, Fungi; fixed modifications, carbamidomethyl (C) (cysteine carbamidomethylation); variable modifications, oxidation (M) (methionine oxidation); peptide tolerance, 100 ppm; MS/MS tolerance, 0.5 Da; peptide charge, +1; monoisotopic peptide values; and instrument, MALDI-TOF-TOF. For the search results, a score calculated by the Mowse scoring algorithm in MASCOT was considered as significant ( p < 0.05), which indicated identity or extensive homology.
Results and Discussion
- Osmotic Pressure Tolerance and Erythritol Production byY. lipolytica
Hitherto, there is no prior published data on the osmotic pressure tolerance of Y. lipolytica under hyperosmotic conditions. Growth test on the hyperosmotic agar plates containing various concentrations of NaCl (0-70 g/l) was performed. As shown in Fig. 1 , there was no apparent inhibition on the growth of Y. lipolytica below 3.85 osmol/kg. When the osmotic pressure was higher than 4.15 osmol/kg, the inhibitory effect on cell growth made the yeast lawns increasingly smaller and thinner. The growth of Y. lipolytica was almost ceased when 70 g/l NaCl was used at 5.59 osmol/kg of osmotic pressure. Fig. 2 shows the erythritol production under various osmotic pressure values in shake-flask experiments. With the osmotic pressure increased from 3.17 to 4.15 osmol/kg, significant increase in erythritol production was achieved. The maximum concentration of 98.9 g/l of erythritol was obtained. However, the erythritol concentration decreased when the initial osmotic pressure was higher than 4.15 osmol/kg, indicating that an optimal osmotic pressure was required for erythritol biosynthesis by osmophilic yeast. Accordingly, the osmotic pressure values of 3.17 and 4.15 osmol/kg were chosen as the low and high initial osmotic pressure for further testing in the batch fermenter.
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Growth test of Y. lipolytica on hyperosmotic agar plates with 200 g/l glycerol and NaCl (0-70 g/l) at various levels of osmotic pressure.
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Final erythritol concentrations under various levels of osmotic pressures from the 250 ml shake-flask experiments.
Erythritol production was determined under low and high osmotic pressure by comparing the fermentation kinetics with and without the addition of 30 g/l NaCl. Time profiles are shown in Fig. 3 . The cell growth rate and maximum biomass decreased as the initial osmotic pressure increased. Under high osmotic pressure of 4.21 osmol/kg, 98.5 g/l of erythritol was produced, which was 44.9 g/l higher than that at low osmotic pressure of 3.17 osmol/kg (53.6 g/l). The reason might be that the high osmotic pressure increased the carbon flux directed towards erythritol biosynthesis, serving as a compatible solute.
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Fermentation profiles of Y. lipolytica under low ( ▲ ) and high ( ■ ) osmotic pressure. (A) Biomass, (B) Glycerol, and (C) Erythritol. Each point represents the mean (n = 3) ± standard deviation.
- 2-DE Map ofY. lipolyticaCellular Proteins Under Osmotic Stress
To investigate the effect of osmotic pressure on Y. lipolytica during the production of erythritol, a comparative proteomic analysis of the intracellular proteins expression level of Y. lipolytica under low and high osmotic pressure was carried out. The 4.21 and 3.17 osmol/kg levels of osmotic pressure were selected as the high and low osmotic pressure in the proteomics experiment in accordance with our previous research [60] . Under various osmotic pressure conditions, about 1,100 Coomassie blue-stained protein spots were detected using the pH range of 3-10 NL IPG strips. The molecular mass of the corresponding proteins ranged from about 10 to 97.4 kDa, and the pI was in the range of 3-10 ( Fig. 4 ). In addition, the experimental results of the 2-DE were highly reproducible.
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The 2-dimensional electrophoresis map of Y. lipolytica extracted total protein. All of the identified protein spots are numbered according to Table 1.
Comparing the abundance of protein expression of the detected spots under different osmotic pressure levels, approximately 325 proteins passed the 1.5-fold expression criterion, whereas most of the protein spots were low-abundant proteins that were faint on the 2-DE gels to be excised and identified. Finally, 54 protein spots corresponding to 44 different proteins were successfully identified, and are summarized in Table 1 . The identified protein spots are numbered on the 2-DE map ( Fig. 4 ). In accordance with protein functions cataloged by the MIPS Comprehensive Yeast Genome Database (CYGD), all the identified proteins were classified into four categories (energy, metabolism, cell rescue and stress response, and miscellaneous) ( Table 1 ). It is interesting that several different protein spots located on the 2-DE gel indicated one identical enzyme. For example, gi|50555229 and gi|50550873 representing triosephosphate isomerase and malate dehydrogenase were identified in two spots. In addition, gi|50556354 and gi|50553214, representing aldo-keto reductase and serine hydroxymethyltransferase, were found in three spots, respectively. A similar condition also appeared in other research on Y. lipolytica [39] , P. pastoris [16] , and Candida albicans [20] . Conceivably, the reasons might be that these proteins are regulated by post-translational glycosylation and phosphorylation modifications [2 , 33 , 41] or possessing highly similar isoforms.
Expressed proteins identified in the 2-DE map ofY. lipolyticaat different osmotic pressure levels.
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aScores >60 indicate identity or extensive homology (p < 0.05), and the matched peptides are summarized in the supplementary data. bAv. ratio represents the average fold change of the expressed protein abundance for comparison under high and low osmotic pressure set points.
- Effect of Osmotic Pressure on theY. lipolyticaIntracellular Proteome
Comparing the expression levels of the identified protein spots in 2-DE gels between low and high osmotic pressure conditions, 20 spots representing 18 types of proteins were up-regulated in low osmotic pressure, and 34 spots representing 26 types of proteins were up-regulated in high osmotic pressure ( Fig. 5 ). Most of these proteins are related to carbohydrate metabolism, protein biosynthesis, cell rescue, and osmotic pressure response.
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Summary of various expression levels of Y. lipolytica total protein extracted under low (A) and high (B) osmotic pressures levels. Symbols: overexpressed proteins under low (circle) and high (square) levels of osmotic pressure.
- Impact of Osmotic Stress on Energy Metabolism-Related Proteins
Two proteins had up-regulated expression under high osmotic pressure, identified as triosephosphate isomerase, which catalyzed dihydroxyacetone phosphate to glyceraldehyde-3-phosphate (spot 1 in Table 1 and Fig. 6 ). In yeast, glycerol is usually synthesized as a compatible solute under high osmotic pressure. Inactivation of triosephosphate isomerase activity leads to accumulation of dihydroxyacetone phosphate, the first metabolite in glycerol synthesis, which affects the metabolic flux in the direction of glycerol accumulation [10] . However, during erythritol production by Y. lipolytica , glycerol was consumed as the sole carbon source. High osmotic pressure induces Y. lipolytica to produce more erythritol to counteract the damages due to osmotic stress. More glucose-6-phosphate generated through glycerol dissimilation and gluconeogenesis was needed to synthesize erythritol using the pentose phosphate pathway (PPP). In this case, triosephosphate isomerase was up-regulated in high osmotic pressure ( Fig. 6 ). Moreover, triosephosphate isomerase was also induced by drought and salt stress in rice [58] .
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Enlarged sectors of the 2-DE gels including the identified proteins related to erythritol biosynthesis under low (left) and high (right) osmotic pressure levels. The graphs show the normalized amount of each protein spot. Each value is the mean ± SD in triplicate assays.
Enolase is the key enzyme in glycolysis responsible for converting 2-phosphoglycerate to phosphoenolpyruvate. There are two isoenzymes, Eno1p and Eno2p, which are the products of genes ENO1 and ENO2 . It was proven that Eno1p increased in abundance in S. cerevisiae [43] , C. magnolia [26] , and mammalian TALH cells [15] in high osmotic stress environment. However, the opposite regulation of Eno2p was observed in this study (spot 2 in Table 1 ). Eno2p was down-regulated approximately 1.6-fold under high osmotic pressure, which was similar to the phenomenon of Eno2p being repressed about 2-fold under 1.5 M NaCl in S. cerevisiae [43] and markedly down-regulated in 150 mM NaCl in rice root cells [58] . The reason might be (i) enolase is regulated at the transcriptional, post-transcriptional, translational, and post-translational levels [58] ; and (ii) ENO2 contains a cis-acting regulatory region mediating glucose-dependent induction of gene expression, and Eno2p is the main form of enolase expressed during non-stressed growth in glucose-containing medium [9] . When yeast cells are grown on gluconeogenic carbon sources, the expression of Eno2p is more sensitive to high osmotic stress. In addition, the contrasting regulatory response of ENO1 (up) and ENO2 (down) genes under salt stress was also found in S. cerevisiae [43] .
Among the up-regulated proteins, mitochondrial cis -aconitate hydratase and malate dehydrogenase are responsible for maintaining the necessary high energy and energy balance under high osmotic pressure condition in the tricarboxylic acid pathway [27] . Additionally, cis -aconitate hydratase is highly inducible in the absence of glutamate, and the induction is further increased when both glutamate and lysine are absent [18] , which was in agreement with the decrease of glutamate dehydrogenase expression in our study. Moreover, overexpressed lactate dehydrogenase and malate dehydrogenase under high osmotic pressure have an activation effect on the gluconeogenesis pathway in which erythritol is the major metabolite produced [15] .
The significant overexpression of transketolase protein (2.60-fold) was observed under high osmotic pressure condition (spot 8 in Table 1 and Fig. 6 ). Transketolase is an important enzyme in the PPP, which is responsible for producing high levels of intermediates, such as sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate, and for erythrose-4-phosphate biosynthesis [49] . Consequently, transketolase was induced to generate more erythritol as the compatible solute to resist the high osmotic stress in Y. lipolytica . Similar results were reported in other microorganisms such as P. pastoris [16] and Escherichia coli [54] .
For proteins involved in the respiration and fermentation categories, gi|50551783 representing potassium-activated aldehyde dehydrogenase (Ald4p) was up-regulated by 1.8-fold under high osmotic pressure condition. Two types of characterized aldehyde dehydrogenase, Mg 2+ -activated, NADP + -linked and k + -activated, NAD + -linked, locate in the cytosol and mitochondrial matrix of the yeast cell, respectively [4] . At high osmotic pressure, Y. lipolytica overexpressed Ald4p conceivably to contribute to the regeneration of NADPH and maintain the reduced state in the mitochondria to protect the cells against reactive oxygen species (ROS). The mitochondrial NAD + kinase reaction followed by the NADP + -dependent acetaldehyde dehydrogenase reaction catalyzed by Ald4p served as the source of mitochondrial NADPH, and the ALD4 gene was up-regulated under H 2 O 2 stress in S. cerevisiae [36] . Cytochrome c oxidase subunit 4 that belongs to the respiratory chain was overexpressed about 1.6-fold (spot 11 in Table 1 ). There is no report about cytochrome c oxidase subunit 4 expression affected by osmotic stress, but the COX4 gene was induced 1.4-fold higher when S. cerevisiae was exposed to sorbic acid stress [13] . Additionally, mitochondrial cytochrome- b5 reductase was also overexpressed by 1.83-fold, which is similar to the report that the expression profile of the MCR1 gene was induced more than double during response to osmotic shock under 0.5 mol/l NaCl [28] . The possible reason is that additional energy (for heat production and the ATP pool) was needed for yeast cells to maintain the metabolic activity to adapt to the high osmotic condition [30] . Interestingly, the mitochondrial ATP synthase gamma subunit responsible for generating ATP was down-regulated in Y. lipolytica under high osmotic stress. The reasons might be that yeast cells adapted their metabolism to regulate cell growth and essential biosynthetic pathways, and fortification of the metabolic pathway to produce compatible solutes was needed. Consequently, the ATP pool was maintained at a relatively low level.
- Impact of Osmotic Stress on Cell Component-Related Proteins
Further changes appeared to occur in the metabolism of amino acids, proteins, and lipids. MET6 encodes 5-methyltetrahydropteroyltriglutamate-homocysteine methyl-transferase that catalyzes the transfer of the methyl group from 5-methyltetrahydrofolate to homocysteine, resulting in methionine formation [35] . We found that Met6p was induced by 2.13-fold under high osmotic pressure. Conceivably, the rationale for the overexpression of Met6p under high osmotic pressure is that methionine is synthesized as the intermediate of the osmoprotection to counteract the damage of hyperosmotic pressure. However, in S. cerevisiae , the formation of sulfhydryl groups is necessary for the production of glutaredoxin and thioredoxin, and the expression of the MET6 gene was severely repressed in 0.7 mol/l NaCl or 0.95 mol/l sorbitol medium [45] . Another important osmotic response of amino acid synthetase, glutamate dehydrogenase encoded by the GDH1 gene, was repressed more than 1.8-fold, similar to the identical protein in S. cerevisiae [43] .
Another enzyme, serine hydroxymethyltransferase (SHMT), plays an important role in single-carbon metabolism involving the biosynthesis and degradation of some amino acids (Ser, Gly, and Met), purines, sugars, and organic acids [11] . We found that SHMT expression was drastically inhibited or terminated under hyperosmotic condition. The down-regulated expression of SHMT under high osmotic pressure was probably due to metabolic changes rather than direct induction or repression as a result of environmental stress. In addition, microorganisms need to accumulate some typical amino acids (Gly, Glu, or Pro) for osmotic protection, to maintain the membrane integrity and protein stability. Consequently, the expression of the enzymes related to amino acid catabolism was reduced. Similar results on the repression of SHMT under various stresses such as formate, acetate, pyruvate, or methanol, were also reported in higher plants [24] .
From the experimental results of our previous report, yeast cell growth was arrested when exposed to high osmotic pressure [60] . The reasons might be that the enzymes related to cellular biosynthesis, such as the expression of the 60S ribosomal protein, translation elongation factor 1-alpha, and seryl-tRNA synthetase, were down-regulated at various levels under high osmotic stress condition. Diminished expression of such proteins upon hyperosmotic stress reflects the growth arrest of Y. lipolytica , leading to the decrease of protein and nucleotide synthesis [45] .
- Impact of Osmotic Stress on Cell Rescue and Stress Response-Related Proteins
The proteins related to cell rescue and stress response were the most significant response factors in the proteomics. There were 21 spots representing 16 types of proteins meeting the criterion in the 2-DE map ( Fig. 4 ). Among these enzymes, the aldo-keto reductases (AKRs) family, heat shock proteins (HSPs) family, and peroxiredoxins (PRXs) family were more inducible to osmotic pressure when the yeast was exposed to hyperosmotic stress ( Table 1 ). The AKRs are the superfamily of the enzymes catalyzing the NADPH-dependent conversion of various carbonyl (aldehydes and ketones) compounds into corresponding alcohols. The established microbial AKRs superfamily contains many enzymes such as erythrose reductase, arabinose dehydrogenase, xylose reductases, glycerol dehydrogenase, 2,5-diketo-D-gluconic acid reductases, and beta-keto ester reductases [17] . AKRs played some crucial roles in stress responses, such as to osmotic stress [19] , heat shock [7] , and oxidative stress [8] , as well as rescued cell growth under stressful conditions.
In this proteomic analysis, the expression of AKRs was up-regulated significantly under high osmotic pressure, especially in the gi|50546136 (5.12-fold) protein. Aldose reductase synthesis rate increased 15-fold when mammalian renal medullary cells switch to a hypertonic environment and the degradation of aldose reductase protein is slow [40] . When Y. lipolytica is exposed to a high osmotic environment, the yeast cell will accumulate a large number of polyols to balance the aw between the intracellular and extracellular environments, to prevent dehydration of the cell. The protection roles of polyols produced by yeast under high osmotic pressure has been proven [16] . In addition, our previous report demonstrated the hyperosmotic stress response of Y. lipolytica CICC 1675, which accumulates a large amount of erythritol as the main polyol to counteract hyperosmotic damage. High osmotic pressure could induce erythrose reductase activity significantly, which catalyzes erythrose reduction to erythritol [60] .
Another category of stress response proteins are the heat shock proteins (HSPs), which play crucial roles in protein folding and prevent protein aggregation. Several stresses, especially heat stress, induce overexpression of HSPs [5] . These proteins are highly conserved during evolution, and their expression is modulated when exposed to diverse temperature [12] . The up-regulated proteins Hsp12 and Hsp20 belong to the small heat shock proteins family, which is characterized by a low molecular mass ranging from 12 to 30 kDa. A significant level of Hsp12 is induced in yeast cells when exposed to heat shock, osmotic stress, oxidative stress, and high concentration of alcohol as well as in early-stationary-phase cells [53] . It was reported that Hsp12 gene expression was induced by 114-fold when exposed to 0.7 mol/l NaCl in S. cerevisiae [45] . In this proteomic analysis, the expression of Hsp12 and Hsp20 were up-regulated by 1.69- and 2.04-fold, respectively. Another heat shock protein, STI1p, is a 66 kDa protein encoded by the STI1 gene, which is not homologous to the other conserved HSP70 family members in yeast, despite similarities in size and regulation. When S. cerevisiae was exposed to heat stress (from 23℃ to 39℃), STI1 was increased approximately 10-fold in RNA level [42] . The transcription of STI1 was enhanced only under heat shock stress, but not in response to oxidative, osmotic, or starvation stresses [44] . STI1p was overexpressed by 1.57-fold in Y. lipolytica under hyperosmotic pressure in our study. That could be because the overexpression of HSPs enhanced the survival of stressed cells by acting as molecular chaperones to prevent irreversible protein denaturation [15] .
The 78 kDa glucose-regulated protein (GRP78), belonging to the HSP70 family, is an endoplasmic reticulum (ER) molecular chaperone involving in ER Ca 2+ binding, misfolded protein degradation, and controlling the activation of transmembrane ER stress sensors [31] . GRPs were induced by ER stress to prevent cells from damage under oxidative stress, Ca 2+ ionophores, heat stress, and drug treatments [15 , 46] . However, the interesting finding was that GRP78 involved in osmotic stress resistance was down-regulated by 3.22-fold. Conceivably, the decreasing expression of GRP78 under hyperosmolarity is because the down-regulation of these Ca 2+ binding proteins causes an increase of free Ca 2+ , which upsets the calcium homeostasis between the inside and outside of the cytoplasm membrane [15] . In addition, the unfolded protein response (UPR), including HSPs and chaperones, plays an essential role in response to osmotic stress [23] . UPR induction was demonstrated to be important under salt stress in Rhodotorula mucilaginosa [29] . The co-chaperone mitochondrial DnaJ homolog Mdj1p was up-regulated (1.54-fold) under high osmotic pressure, suggesting that Y. lipolytica required increased chaperone capacity to deal with the increased amount of unfolded proteins.
Oxidation stress is also a general stress accompanying osmotic stress. Microorganisms are also prone to oxidative stresses such as an increased level of hydrogen peroxide under osmotic stress [32] . The up-regulated spots representing the proteins peroxiredoxin, superoxide dismutase, and catalase T, which are known to be involved in protecting cells from oxidative damage induced by hydrogen peroxide, were induced at various levels in Y. lipolytica under high osmotic pressure ( Fig. 6 and Table 1 ). The relationship between osmotic stress and oxidation stress is currently not well defined. The hypothetical reasons may be explained as follows: (i) There are various stress conditions existing simultaneously in nature, and induced protein responses are stimulated by other stress conditions [1] . (ii) The operation of the electron transport chain may be discrupted by osmotic stress, leading to the generation of ROS. In eukaryotic cells, respiration is thought to be the main source of ROS generation in the mitochondria through oxidative phosphorylation. Under the stress of xenobiotics, carcinogens, and UV and ionizing radiation, more ATP is needed to counteract the stress damage to cellular organelles [38] . Under this possible scenario, peroxiredoxin and catalase T are induced significantly to carry out the detoxification of ROS to maintain the redox balance and resist the oxidative damage on the cells [45 , 55] .
In addition, the spots of gi|50547013 that represented the two proteins of cerevisin (proteinase from yeast) were up-regulated by 1.70- and 1.41-fold in our proteomic analysis. Cerevisin is the product of the prb1 gene, which is repressed by glucose at the level of transcription. The capabilities of protein degradation triggered by the vacuolar proteinase yscB increased considerably under the condition of specific carbon complement (acetate) and nitrogen deprivation [51] . The false proteins may be synthesized under extreme environmental stress. The degradation of the false proteins is necessary for the cell to prevent the accumulation of wasted protein. The proteins-induced protein degradation is elevated considerably [21 , 51] . Conceivably, it may be the cause of the up-regulated expression of cerevisin under high osmotic pressure.
Sphingolipid long-chain bases responsive protein (Lsp1p) is the signaling molecule regulating cell growth, heat stress responses, endocytosis, cell wall synthesis and repair, as well as repolarization of the actin cytoskeleton in response to stresses [14] . Lsp1p negatively influences heat stress resistance of the logarithmic and stationary phase cells. Deletion of the LSP1 gene leads to nearly 4-fold higher resistance as compared with the wild type of S. cerevisiae under heat stress [61] . In our proteomic research, the gi|50548497 spots representing the two proteins of Lsp1p were down-regulated by 2.20- and 1.29-fold under hyperosmolarity. The reason might be that osmotic stress exhibits a similar effect on sphingolipid metabolism as heat stress.
Y. lipolytica has been recognized as an excellent model organism to study high osmotic stress response. To the best of our knowledge, this is the first report on the 2-DE proteomic analysis of the expression of soluble intracellular proteins under osmotic stress in Y. lipolytica . The expression of some novel proteins involved in osmoregulation and osmotic stress response has been identified. Moreover, we have compared soluble cell extracts from Y. lipolytica yeast exposed to different osmotic pressure levels and detected that 54 spots representing 44 proteins exhibited statistically significant changes in expression level as compared under high and low osmotic pressure. Most of the expressed proteins have been identified and found to be involved in the energy, metabolism, cell rescue, and stress response pathways. Among these expressed proteins, the AKRs family and catalase T were the most overexpressed enzymes related to regulation of response to higher osmolarity. It is conceivable that the introduction of these crucial proteins into other non-osmophilic microorganisms would significantly improve their osmotic tolerance, leading to substantial improvement of fermentation processes with these improved microorganisms under high substrate concentration.
Acknowledgements
This study was supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), and the Jiangsu provincial “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program, National Natural Science Foundation of China (31171640, 31301408), National High-tech R&D Program of China (2012AA021505, 2012AA021201), and National Key Technologies R&D Program of China (2011BAD23B04).
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