Identification of proteins with 2 dimensional electrophoresis (2DE) and mass spectrometry in native strain of Saccharomyces cerevisiae with high yield bioethanol production after random mutagenesis.
Growth in the presence stress conditions lead to multigenic responses, that is detectable in proteome structure of S. cerevisiae (22). In this study, random mutagenesis was done by radiation for improvement of the yeast strain. Selection of the mutants after treatment by ultra violet light was done in the presence of furfural. Efficient producers of ethanol in the presence of furfural were chosen for comparative proteome analysis. By 2-DE combined with mass spectrometry (MALDI-TOF/ MS), some differently expressed proteins were detected, one of which was of the antioxidative Stress protein, while the others were associated with the ethanol production and glycolysis/ gluconeogenesis pathways.
MATERIALS AND METHODS
Saccharomyces cerevisiae T12 (PTCC (1) 5315) (21), a native strain that showed good ethanol production and furfural resistance (up to 2 g/l), was used in this study as parent strain.
Ultra violet treatment and selection of mutants
The yeast cells, grown on Potato Dextrose Agar (PDA) Medium, containing (g/l): glucose, 20; potato, 300, for 24 h, were diluted and transferred to YEPD-agar plates, containing (g/l): yeast extract, 10; peptone, 20; dextrose, 20; agar, 15 and freshly distilled furfural 2 g/l; pH 5.5. The plates were mutagenized from a distance of 20 cm by using a Philips[TM] 30 W germicidal U.V lamp for 275 seconds (1,19). The single colonies which were able to grew on these plates, were transferred to YEPD broth (the same ingredients, as above, without agar) containing 4 g/l of furfural, for 48 h. The survived cells were isolated and kept on YEPD agar medium, containing 1.5 g/l furfural, to prevent the loss of resistance against furfural.
After selecting the most resistant mutant strains, ethanol production experiments were performed. In aerobic phase, the resistant yeast cells were cultured in synthetic medium, containing (g/l): glucose, 190; yeast extract, 10; [(N[H.sub.4]).sub.2]S[O.sub.4], 1.2; [(N[H.sub.4]).sub.2]HP[O.sub.4] 0.6; pH 5.3, and placed on rotaryshaker at 150 rev. [min.sup.-1], 30[degrees]C, for 8 h. After this phase, the cultures were aseptically transferred and filled into 25 ml Bijoux bottles for 40 h to complete the anaerobic/ ethanolic fermentation period (17). After completion of fermentation, the cells were removed by centrifugation and the supernatant was distillated to separate the ethanol.
For measurement of ethanol concentration, samples were injected directly into a gas chromatography system (GC-14A, Shimadzu, Japan) with a UV detector and OV17 column (2m x 3.1mm) that had been packed with methyl silicon (Thermo Scientific Pierce, USA). The chromatographic conditions were set to: initial temperature, 50[degrees]C; final temperature, 90[degrees]C; injector temperature, 230[degrees]C; nitrogen as the carrier gas, with a flow rate of about 30 ml/min (12).
Sugar analysis in medium
The amount of glucose in the fermentation samples were measured by an enzymatic glucose reagent (Parsazmun, Karaj, Iran) based on glucose oxidase/ peroxidase reaction. The intensity of colored product (quinoneimine) was measured specrophotometrically at 500 nm.
For comparing ethanol production and glucose consumption in native and mutant strain ANOVA analysis was performed on the results, using SPSS ver. 16 software (SPSS, Inc. USA), and assuming p-value < 0.05 for significance.
Sample preparation for proteomics analysis
The parent T12 (PTCC 5315) and mutant (Fj) strains of S. cerevisiae were grown in PDB medium and incubated on rotary-shaker at 150 rev. [min.sup.-1], 28[degrees]C, for 20 h, in three replicates. Cells were harvested by centrifugation (2800xg, 4[degrees]C, 10 min) in the late mid-exponential phase. The supernatant was removed and the cell pellet was washed by ice-cold deionized water and spun (2800 x g, 4[degrees]C, and 10 min). Yeast cell pellets was collected and grounded to fine powder in liquid [N.sub.2]. The frozen powder was suspended in a lysis buffer (7 M urea, 2 M thiourea, 4% 3-[(cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 40 mM Tris-base). Subsequently, a DNase and RNase solution (1% DNase I, 0.25% RNase A, 50 mM Mg[Cl.sub.2], 0.5 M Tris-HCl, pH 7.0) was added and incubated on ice. Then, 1 mM phenylmethylsulfonyl fluoride was added and the sample was sonicated briefly (6). Cell debris was removed by centrifugation at 10000 x g for 30 min in 15[degrees]C.
For increasing protein concentration and desalting, TCA-Acetone precipitation was done (13). The dried proteins were dissolved in determined rehydration buffer (8 M urea, 2% CHAPS, 20 mM dithiothreitol (DTT), 0.5% immobilized pH gradient (IPG) buffer pH 4-7. Protein content was determined according to the Bradford method using BSA as standard (4). The lysate was either used immediately or aliquoted and kept in -70[degrees]C for further use (6).
Isoelectric focusing was performed on 17-cm IPG strips (BioRad, U SA) with linear pH range of 4-7, which were passively rehydrated overnight by loading the samples, diluted with rehydration buffer containing: 8 M urea, 2% CHAPS, 20 mM DTT, 0.5% immobilized pH gradient (IPG, USA) buffer pH 4-7 and small amounts of bromophenol blue. Isoelectric focusing was carried out using the PROTEAN IEF cell (BioRad, USA) began with linear increase from 0-250 V for 20 min, followed by linear increase to 10000 V 2.5 h, and remained on 10000 V to achieve total 50,000 Vh.
Then, the focused IPG strips were reduced for 20 min at room temperature in equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 1% DTT) and subsequently alkylated for 20 min in equilibration buffer containing 5% iodoacetamide instead of DTT at room temperature. The equilibrated strips were placed on top of 12% SDS-PAGE handmade gels and sealed with 1% agarose. The second dimension electrophoresis was performed using a standard Laemmli buffer system (14) at 16 mA/gel for 30 min and 24 mA/gel for the next 5 h at 20[degrees]C. After 2-DE, for MS identification, proteins in gel were stained by a modified colloidal Coomassie blue method (5).
The stained gels were scanned at a resolution of 300 dpi using the densitometer GS-800 (BioRad, USA). The quantification of spots intensity and statistical evaluation were done on Coomassie blue stained analytical gels using Image Master 2D Platinum Ver. 6.0 (GE healthcare, USA). Statistical analysis of protein variations was carried out in 2-D gels prepared from three replicates (CV<40%) in each group using the student T-test on vol% of matched spots. The statistical significance was assumed less than 0.05 for p-values.
MS analysis, database searching
The protein spots were manually cut from colloidal Coomassie blue stained 2-D gels. Gel pieces were washed two times with 50% aqueous acetonitrile containing 25 mM ammonium bicarbonate, then once with acetonitrile and dried in a vacuum concentrator for 20 min. Sequencing-grade, modified porcine trypsin (Promega, UK) was dissolved in the 50 mM acetic acid supplied by the manufacturer, then diluted 5-fold by adding 25 mM ammonium bicarbonate to give a final trypsin concentration of 0.02 mg/ml gel pieces were rehydrated by adding of trypsin solution, and after 30 min enough 25 mM ammonium bicarbonate solution was added to cover the gel pieces. Digests were incubated overnight at 37[degrees]C.
A 1 mL aliquot of each peptide mixture was applied directly to the ground steel MALDI target plate, followed immediately by adding an equal volume of a freshly-prepared solution of 5 mg/mL of 4-hydroxy-[alpha]-cyano-cinnamic acid (Sigma, UK) in 50% aqueous acetonitrile, containing 0.1%, trifluoroacetic acid.
Bruker flex Analysis software (version 3.3) was used to perform the spectral processing and peak list generation for both the MS and MS/ MS spectra. Tandem mass spectral data were submitted to database searching using a locally-running copy of the Mascot program (Matrix Science Ltd., version 2.1), through the Bruker ProteinScape interface (version 2.1). Search criteria included: Enzyme, Trypsin; Fixed modifications, Carbamido methyl (C); Variable modifications, Oxidation (M); Peptide tolerance, 250 ppm; MS/ MS tolerance, 0.5 Da; Instrument, MALDI-TOF/ TOF Statistical confidence limits of 95% was applied for protein identification.
According to survival curve (not shown) after 275 sec of UV treatment, 99.99% of the yeast cells were killed and this time was selected for next step of mutagenesis. After 275 sec of UV treatment on native strain, the mutant cells that grew were exposed to 4 g/l furfural for isolation of resistant mutants. The obtained mutants were compared regarding production of ethanol in the presence of furfural, where the mutant Fj was isolated among many others.
Comparison of ethanol production and sugar utilization in parent and mutant strains
The mutant strain (Fj) was compared to the parent strain (T12) regarding sugar consumption and ethanol production. As shown in Fig. 1 and 2, differences in residual glucose concentration and ethanol production in mutant and parent strains, until 24 h are negligible, while after this time, utilization of glucose and production of ethanol in mutant strain was increased in relation to the parent. Comparing the production yield (g ethanol/ g initial glucose) showed that this figure was 39% higher in the mutant than the parent.
Proteome differences examination in native and mutant strain
Native and mutant Saccharomyces cerevisiae cells were cultured in the same conditions in three replicates.
Comparative proteomic profiles can help us to understand the reasons for changes in ethanol efficiency after mutation in native strain. Analytical and preparative 2-D gels of proteins extracted from native and mutant strain were carried out on 17cm IPG strips with linear pH range of 4-7 (Fig. 3) (6).
Four protein spots with predefined scale for significant expressional changes were determined (Fig. 4).
After statistical analysis using t-test, 4 proteins were shown to have been over expressed in mutant strain. Differentially expressed proteins were excised from 2-D gels and identified by MALDI-TOF/TOF mass spectrometry (Table 1). The identified proteins were categorized by their known and/or putative functions into three groups. The cellular proteins were involved in Glycolysis pathway, ethanol production pathway and antioxidative stress.
After UV treatment on native strain of saccharomyces cerevisea was caused mutant strain was resistant against 4 g/l of furfural and its ethanol production yield was 3 9% more than parent one For Proteins recognition and identification after mutation and variations consideration in protein pattern in native and mutant strain, a proteomics profiling strategy was utilized (Fig. 3). For extensive variations in protein pattern between native and mutant strain, cell lysis and 2-DE analysis was done repeatedly that final results of protein pattern completely was similar to prior results.
Our findings show that the expression level of ethanol producing enzyme, alcohol dehydrogenase I (ADH1) was increased upon treatment of cells with UV radiation and screening in the presence of furfural (Table 1 and Fig. 4). The result of increased ADH I expression could be the higher yield of ethanol in mutant Fj, compared to its parent strain (Fig. 2). In Saccharomyces cerevisiae, two genes, adhl and adh2, code for two cytoplasmically expressed alcohol dehydrogenases: ADH I and ADH II. ADH I enzyme is involved primarily in ethanol production during fermentation. It is largely responsible for regeneration of [NAD.sup.+] in glycolysis (7). Studies on yeast cells showed that furfural in the range of 1 to 2 g/l, inhibit function of alcohol dehydrogenase 1 enzyme and strongly affect the specific growth rate of the cells (17). Enhancement of the expression of this enzyme in the mutant strain is in good correlation with its behavior, regarding production of more ethanol in the presence of furfural.
Another enzyme, expression of which has shown to be increased, was fructose-1, 6-bisphosphate aldolase (Table 1 and Fig. 4). Fructose-1, 6-bisphosphate aldolase is an essential glycolytic enzyme found in Saccharomyces cerevisiae, which catalyzes the cleavage of fructose 1, 6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (3). Furan derivatives like furfural in the range of 1 to 2 g/l, have reported to inhibit the aldolase enzymes in glycolytic pathway and affect the rate of growth of yeast cells (15).
After protein identification by mass spectrometry, we found an unnamed protein product (homologous to glyceraldehyde-3-phosphate dehydrogenase) with similar ranges of Mr and pI (Table 1). The expression level of this protein was also increased after mutation by UV radiation and screening in the presence of furfural. The glyceraldehydes-3-phosphate dehydrogenase is sensitive to furfural (20), thus its over expression in mutant strain may be consider as a defense against environmental stress. This enzyme still is unnamed in Saccharomyces cerevisiea and is homologous to glyceraldehyde-3-phosphate dehydrogenase.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been considered a classical cytosolic glycolytic protein and in some microorganisms plays some role in cell flocculation (8). This phenomenon has been considered very interesting in ethanol production, by facilitating the separation of the yeast cells from the fermenting medium (18).
Superoxide Mn dismutase (Mn-SOD), another protein which was found in higher level in the mutant, than the parent strain, catalyzes the conversion of superoxide anions to hydrogen peroxide and oxygen, thus provides the protection against toxic intracellular radicals that are produced under oxidative stresses (10). The manganese-superoxide dismutase (Mn-SOD) is encoded by the gene sod 2 and is located in the mitochondrial matrix (11). The presence of furfural causes known metabolic stresses, against which the over expression of Mn-SOD, may be of great significance. In previous reports it has been shown that furfural and phenolic compounds, such as vanillin, reduce and even inhibit the Mn-SOD activity and consequently cause yeast cells death (9).
Our results showed that after random mutation by UV, pattern of expression of proteins was altered meaningfully since identified proteins that had over expressed in mutant strain, played an important role in resistance against metabolic stress and metabolism of energy. Designing recombinant strains after exploration of related genes could be an interesting subject for further investigation.
This work was financed by Iranian Research Organization for Science and Technology by grant No. 10121857. Authors gratefully acknowledge Dr. Behrouz Vaziri (Department of Biotechnology, Pasteur Institute of Iran, Tehran, Iran) for his effective scientific assistance and technical advising on 2-DE experiments, Dr. Adam Dowle (Proteomics Technology Facility, Department of Biology, University of York, United Kingdom), for his critical review on mass spectrometry analysis, and SinaClon Bioscience (Tehran, Iran) for facilitating the exchange of data. We also thank Farnaz Zandi and Fatemeh Torkashvand for their valuable technical and supportive assistance.
(1.) Baltz HR, Mutagenesis in Streptomyces spp. In: Manual of Industrial Microbiology and Biotechnology, eds. Demain A L & Solomon N A, Washington D. C, 1986; pp. 185-190.
(2.) Banerjee N, Bhatnagar P, Viswanathan L, Inhibition of glycolysis by Furfural in Saccharomyces cerevisiae. Eur J Appl Microbiol Biotechnol, 1981; 3: 24-28.
(3.) Berry A and Marshall K E, Identification of zinc-binding ligands in the Class II fructose-l, 6-bisphosphate aldolase of Escherichia coli. FEBS Lett, 1993; 318: 11-16.
(4.) Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976; 72: 248-254.
(5.) Candiano G, Bruschi M, Musante L, Santucci L and et al, Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis, 2004; 25: 1327-1333.
(6.) Cheng J, Qiao B, Yuan Y, Comparative proteome analysis of robust Saccharomyces cerevisiae insights into industrial continuous and batch fermentation. Appl Microbiol Biotechnol, 2008; 81: 327-338.
(7.) Ciriacy M, Genetics of alcohol dehydrogenase in Saccharomyces cermisiae. I. Isolation and analysis of adh mutants. Mutat Res, 1975; 29: 315-325.
(8.) Delgado ML, O'Connor JE, Azorin I, Renau-Piqueras J, Gil ML and Gozalbo D, The glyceraldehyde-3-phosphate dehydrogenase polypeptides encoded by the Saccharomyces cerevisiae TDH1, TDH2 and TDH3 genes are also cell wall proteins. Microbiology, 2001; 147: 411-417.
(9.) Endo A, Nakamura T, Ando A, Tokuyasu K, Shima J, Genome-wide screening of the genes required for tolerance to vanillin, which is a potential inhibitor of bioethanol fermentation, in Saccharomyces cerevisiae, Biotechnology for Biofuels, 2008;1:3.
(10.) Farr SB, D'Ari R, Touati D, Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase. Proc Natl Acad Sci USA, 1986; 83: 8268-8272.
(11.) Galiazzo F and Labbe-Bois R, Regulation of Cu, Zn- and Mn-superoxide dismutase transcription in Saccharomyces cerevisiae. FEBS Lett, 1993; 315: 197-200.
(12.) Grob RL, Barry EF (2004) Modern practice of gas chromatography, Fourth Edition. John Wiley & Sons, Inc Hoboken, New Jersey. Published simultaneously in Canada.
(13.) Jiang L, Lin He Land Fountoulakis M, Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. Journal of Chromatography A, 2004; 1023: 317-320.
(14.) Laemmli UK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970; 227: 680-685.
(15.) Modig T, Liden G, Taherzadeh MJ, Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. Biochem J, 2002; 363: 769-776.
(16.) Olsson L and Hahen-Hagerdal B (1996) Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microb Technol 18: 312-331.
(17.) Palmqvist E, Almeida JS, Hahn-Hagerdal B, Influence of furfural on anaerobic glycolitic kinetics of Saccharomyces cerevisiae in batch culture. Biotechnol. Bioeng, 1999; 62: 447-454.
(18.) Sieiro C, Reboredo NM, Villa TG, Flocculation of industrial and laboratory strains of Saccharomyces cerevisiae. J Ind Microbiol, 1995; 14: 461-466.
(19.) Sridhar M, Sree NK, Rao LV, Effect of UV radiation on thermotolerance, ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains. Bioresour Technol, 2002; 83: 199-202.
(20.) Taherzadeh MJ, Gustafsson L, Niklasson C and Liden G, Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae. Appl Microbiol Biotechnol, 2000; 53: 701-708.
(21.) Tofighi A1, Azin M, Mazaheri Assadi M, Assadi-rad MHA, Nejadsattari T and Fallahian MR, Inhibitory Effect of High Concentrations of Furfural on Industrial Strain of Saccharomyces cerevisiae. Int J Environ Res, 2010; 4: 137-142.
(22.) Zuzuarregui A, Monteoliva L, Gil C, del Olmo M, Transcriptomic and proteomic approach for understanding the molecular basis of adaptation of Saccharomyces cerevisiae to wine fermentation. Appl Environ Microbiol, 2006; 72:836-847.
Bahareh Rahimian Zarif
Department of Biology, Sanandaj branch, Islamic Azad University, Sanandaj, Iran.
(Received: 18 March 2015; accepted: 21 June 2015)
* To whom all correspondence should be addressed. E-mail: Bahareh_r_z@yahoo.com
Table 1. Summary of protein identification, using MALDI-TOF mass spectrometric analysis Spot Protein name MOWSE Accession Theoretical no. score no. * Mr/pI 1 Alcohol 577 gi|1168350 37.2/6.26 dehydrogenase I 2 Fructose 1, 755 gi|6322790 39.8/5.51 6-bisphosphate aldolase 3 unnamed protein product 239 gi|50288681 35.95/6.46 homologous to glyceraldehyde-3- phosphate dehydrogenase) 4 dismutase, Mn superoxide 260 gi|223570 22.7/5.8 Spot Observed Sequence Student Expressional no. Mr/pI coverage (%) -T fold change (mutant/parent) 1 46/6.4 22% 6.63 4.6 2 41/5.75 29% 8.62 2.0 3 28/6.1 8% 11.25 3.7 4 28/6.3 13% 39.4 9.6
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|Author:||Zarif, Bahareh Rahimian|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Sep 1, 2015|
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