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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2009, p. 3765–3776 Vol. 75, No. 110099-2240/09/$08.00�0 doi:10.1128/AEM.02594-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Comparative Proteomic Analysis of Tolerance and Adaptation ofEthanologenic Saccharomyces cerevisiae to Furfural, a

Lignocellulosic Inhibitory Compound�†Feng-Ming Lin, Bin Qiao, and Ying-Jin Yuan*

Key Laboratory of Systems Bioengineering, Ministry of Education and Department of Pharmaceutical Engineering, School ofChemical Engineering & Technology, Tianjin University, P.O. Box 6888, Tianjin 300072, People’s Republic of China

Received 13 November 2008/Accepted 1 April 2009

The molecular mechanism involved in tolerance and adaptation of ethanologenic Saccharomyces cerevisiae toinhibitors (such as furfural, acetic acid, and phenol) represented in lignocellulosic hydrolysate is still unclear.Here, 18O-labeling-aided shotgun comparative proteome analysis was applied to study the global proteinexpression profiles of S. cerevisiae under conditions of treatment of furfural compared with furfural-freefermentation profiles. Proteins involved in glucose fermentation and/or the tricarboxylic acid cycle wereupregulated in cells treated with furfural compared with the control cells, while proteins involved in glycerolbiosynthesis were downregulated. Differential levels of expression of alcohol dehydrogenases were observed. Onthe other hand, the levels of NADH, NAD�, and NADH/NAD� were reduced whereas the levels of ATP and ADPwere increased. These observations indicate that central carbon metabolism, levels of alcohol dehydrogenases,and the redox balance may be related to tolerance of ethanologenic yeast for and adaptation to furfural.Furthermore, proteins involved in stress response, including the unfolded protein response, oxidative stress,osmotic and salt stress, DNA damage and nutrient starvation, were differentially expressed, a finding that wasvalidated by quantitative real-time reverse transcription-PCR to further confirm that the general stressresponses are essential for cellular defense against furfural. These insights into the response of yeast to thepresence of furfural will benefit the design and development of inhibitor-tolerant ethanologenic yeast bymetabolic engineering or synthetic biology.

Bioethanol produced from renewable resources such aslignocelluloses is considered to be an attractive alternative tofossil fuels, for it is renewable, can make use of fast-rotationplants, produces fewer emissions, and generates no net carbondioxide. Nevertheless, there are some barriers in the lignocel-lulosic-to-ethanol conversion process, including inhibitor tol-erance, ethanol tolerance, and utilization of xylose (62). Inhib-itors formed by acid-catalyzed hydrolysis of lignocelluloses,which include furan derivatives, weak acids, and phenolic com-pounds, reduce both the growth rate and fermentation of eth-anologenic Saccharomyces cerevisiae (2). The mechanisms ofinhibition acting upon yeast during fermentation of lignocel-lulosic hydrolysate have been studied intensively, but mainlywith traditional methods such as metabolite analysis, enzymeactivity analysis, metabolic flux analysis, and kinetic analysis(50). Furfural is one of the major inhibitors for lignocellulosichydrolysates. Previous studies have shown that in most cases,furfural can be converted by yeast to furfural alcohol (12, 30).Sometimes furoic acid (64), furoin and furil (47), and acyloinproducts (28, 61) can also be detected in the medium underdifferent sets of cultivation conditions. The genetic mecha-

nisms involved in furfural tolerance have been investigated byscreening an S. cerevisiae disruption library to find potentialrelative genes (18). Through gene cloning and enzyme activitystudy, Liu et al. found that the conversion of furfural is cata-lyzed by multiple aldehyde reductases (40).

The traditional methods described above can analyze onlyone or a few metabolites, proteins, or genes and are unable toglobally assess the inhibition issue, which is complex and sys-tematic. Moreover, previous work mainly focused on extracel-lular metabolites and the activity of some key enzymes,whereas what happens inside yeast cells in response to inhib-itors remains a “black box” to us. Integration of different “om-ics” tools, including those of transcriptomics, proteomics, andmetabolomics, into the study of systems biology is a potentiallypowerful approach to address these challenges (61, 68). Manyproteomic, transcriptomic, and/or metabolomic studies of S.cerevisiae have provided us with an increasingly rich under-standing of the response of this organism to various environ-mental perturbations. Investigation of genomic expressionprofiles of the ethanologenic yeast S. cerevisiae to HMF (5-hydroxymethylfurfural) stress conditions showed that up toseveral hundred genes were differentially expressed signifi-cantly in response to HMF treatment (41, 42, 58). Comparativelipidomics analysis has been applied to study the ethanologenicyeast response to different inhibitors, such as furfural, aceticacid, and phenol (69). The results of comparative proteomeanalysis (8, 23) and small-molecule metabolite profiling of eth-anologenic yeast during industrial fermentation (13) have beenpreviously reported, enhancing the molecular understanding ofphysiological adaptation of industrial strains for optimizing the

* Corresponding author. Mailing address: Key Laboratory of Sys-tems Bioengineering, Ministry of Education and Department of Phar-maceutical Engineering, School of Chemical Engineering & Technol-ogy, Tianjin University, P.O. Box 6888, Tianjin 300072, People’sRepublic of China. Phone: 86-22-87401546. Fax: 86-22-27403888.E-mail: [email protected].

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 10 April 2009.

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performance of industrial bioethanol fermentation. However,the specifics of global protein expression in response to thepresence of biomass conversion inhibitors have not yet beenquantitatively measured for ethanologenic yeast.

Quantitative proteomics, i.e., quantifying protein expressionlevels in different sets of complex biological samples on a largescale, is critical for our understanding of biological systems andpathways as a whole. It is considered likely to be a potentialcornerstone of systems biology in the near future (56). Currentquantitative proteomic methods fall into three categories: thetraditional two-dimensional (2D) electrophoresis, stable iso-tope labeling, and nonlabeling methods (45). Each of the dif-ferent labeling methods undergoing development has its ad-vantages and disadvantages (see reference 45 for reviews).Among quantitative proteomic methods, 18O stable isotopelabeling is convenient to use, low in cost, highly specific interms of specific 18O C-terminal modifications, and capable intheory of labeling proteins globally. The 18O-labeling methodhas demonstrated its applicability in differential comparativeproteomics with biological applications performed using Por-phyromonas gingivalis strain W50 (4), the human plasma pro-teome (55), breast cancer cells (5), and the low-molecular-weight serum proteome (26). 18O labeling is becoming apowerful labeling strategy for quantitative proteomics ap-plication.

To give insights into the tolerance and adaptation of eth-anologenic yeast to biomass conversion inhibitors at the pro-tein level, comparative shotgun proteomic investigations com-bining 18O labeling with 2D liquid chromatography-tandemmass spectrometry (2D-LC-MS/MS) were performed here tosystematically identify proteins by the use of an industrialstrain of S. cerevisiae and to quantify cells treated with furfuralcompared with control cells under aerobic batch culture con-ditions. Quantitative real-time reverse transcription-PCR (RT-PCR) and metabolite analysis were utilized to provide orthog-onal evidence for the comparative proteome results.

MATERIALS AND METHODS

Yeast strain. An industrial strain of S. cerevisiae, purchased from Angel YeastCo., Ltd. (Hubei, People’s Republic of China), in the form of alcohol instantactive dry yeast, was utilized in this study. This industrial strain has the advan-tages of thermal resistance (38 to 42°C), low-acid tolerance (pH 2.5), and highglucose tolerance (60%) and can tolerate 13% (vol/vol) ethanol.

Cultivation conditions. After recovery from a lyophilized form, S. cerevisiaewas maintained on agar slants containing YEPD medium (2% glucose, 2% yeastextract, 1% peptone, and 2% agar). S. cerevisiae was initially grown in 250-mlconical flasks containing 50 ml of YEPD medium (2% glucose, 2% yeast extract,and 1% peptone) on a rotary shaker at 30°C and 160 rpm for 12 h. Subsequently,the 50-ml seed cultures were transferred into 2-liter conical flasks containing 450ml of YEPD medium on a rotary shaker at 30°C and 90 rpm for approximately12 h and grown to an optical density (OD) of about 3. Cells for the controlexperiment and the furfural treatment experiment were harvested from the sameinoculation culture. An initial OD of 0.35 was used for aerobic bath culturesperformed at 30°C in 2-liter conical flasks containing 450 ml of medium, with astirrer speed of 90 rpm. The aerobic bath culture medium was composed of 10%glucose, 2% yeast extract, and 1% peptone. During exponential growth in therespiratory-fermentative phase, when the OD was approximately 3 to 4, 50-mlvolumes of aerobic bath culture media containing 0 and 7.33 ml of furfural wereintroduced into the medium for the control experiment and the furfural treat-ment experiment, respectively. Samples for subsequent protein extraction werecollected by centrifugation at 5,000 � g for 10 min at 4°C from furfural-treatedand control cultures at 20 min and 2 h after the addition of furfural, respectively.

The concentration of furfural in lignocellulosic hydrolysates can range from0.5 to 11 g/liter, and there are a broad range of other compounds that have

inhibitory effects on microbial fermentation (2). Usually, the inhibitor concen-trations used to test their effects on fermentation were 10 to 100 times larger thanthe concentration found in the hydrolysates (31). Therefore, based on our pri-mary fermentation experiments and the pertinent literature, the final furfuralconcentration used was set at 17 g/liter for the study of the response of yeast tothe presence of furfural under an extreme set of conditions.

Analysis of fermentation parameters. Cell growth was determined by measur-ing the absorbance of the culture at 600 nm with a spectrometer (model 722grating spectrometer; Shanghai No. 3 Analysis Equipment Factory, Shanghai,China). The concentrations of glucose, ethanol, glycerol, and furfural were mea-sured by high-performance LC. Samples from an aerobic bath culture were firstfiltered through 0.22-�m-pore-size sterile filters and loaded onto an AminexHPX-87H ion-exchange column (Bio-Rad, Hercules, CA) operated at 65°C andwere then eluted with 5 mM H2SO4 at 0.6 ml/min. A refractive index detectorwas used. Cell viability was assessed by methylene-blue staining and a fluores-cence microscope (Eclipse E800; Nikon, Japan).

Protein extraction, 16O/18O labeling, and 2D-LC-MS/MS analysis. The extrac-tion of whole-yeast-cell proteins was conducted as described by Wang and Yuan(66), with minor modifications. After reduction and alkylation, the proteins wereprecipitated again by using cold organic solvent (ethanol/acetone/acetic acid,50:50:0.1) overnight at �25°C, followed by centrifugation and lyophilization. Thepellets were stored at �25°C until use.

Protein samples from the control experiment and the furfural treatment ex-periment were dissolved in digestion buffer (1 M urea, 100 mM NH4HCO3)made using 16O and 18O water (Isotec, Miamisburg, OH) (95%), with eachsolution maintained at a concentration of 1 �g/�l, and were digested with trypsin(Promega, Madison, WI) at a ratio of 50:1 at 37°C for 24 h. Then, additionaltrypsin was added to achieve a final ratio of 20:1, and the incubation wasmaintained at 37°C for another 18 h. Digestions were terminated by addingformic acid to the final volume concentration of 5%. The corresponding 16O- and18O-labeled samples from the same time point were combined immediatelybefore 2D-LC-MS/MS analysis.

Nanoflow LC-MS/MS analysis was performed by the use of a LCQ DecaXPMaxa mass spectrometer (Thermo Finnigan, Palo Alto, CA) under the control ofthe Xcalibur data system (Thermo Finnigan, Palo Alto, CA) as described byWang and Yuan (66). There were some modifications in terms of salt steps andthe elution gradient. A 14-step separation from the strong cation exchangerfollowed by a gradient elution from the reverse-phase chromatography resultswas utilized to separate the peptides. The 14 salt steps used 0, 25, 40, 50, 75, 100,150, 200, 250, 300, 400, 500, 700, and 1,000 mM ammonium chloride, respec-tively. The elution gradient for the reverse-phase chromatography consisted of 1min of 100% buffer A (5% [vol/vol] acetonitrile–0.1% [vol/vol] formic acid inwater); a 70-min gradient to 30% buffer B (0.1% [vol/vol] formic acid in aceto-nitrile); a 20-min gradient to 50% buffer B; a 10-min gradient to 95% buffer B;5 min of 95% buffer B; an 8-min gradient to 100% buffer A; and 7 min ofre-equilibration at 100% buffer A.

Identification and relative quantification of proteins. MS/MS data weresearched using the SEQUEST algorithm, and a database of S. cerevisiae openreading frames was downloaded from the Saccharomyces Genome Database on9 March 2007. The parameters used for the analysis of MS/MS spectra weredetailed in a previous study (66). In this study, carboxyl-terminal double 18Oswere designated for use in variable modification. Then, SEQUEST output fileswere submitted to the PeptideProphet and ProteinProphet websites of the Se-attle Proteomics Center (http://tools.proteomecenter.org) for statistical assess-ment of peptide and protein sequence matches, respectively (65). INTERACTsoftware was utilized to organize and display the results. The accepted error ratefor peptide and protein identification was controlled to remain below 10%.

The abundance ratios (18O/16O) for labeled peptide pairs were calculatedusing reconstructed ion chromatograms and the following equation, which issimilar to one previously reported (70) but with slight modifications:

Ratio18O16O �

I0 �M4

M0� I2�1 �

M2

M0� � �1 �

M2

M0�M2

M0I0

I0(1)

where I0, I2, and I4 represent the measured relative intensities for the first twoisotopic variants of unlabeled peptides, monolabeled peptides, and dilabeledpeptides, respectively, and M0, M2, and M4 represent the sums of the theoreticalrelative intensities for the first two isotopic variants of unlabeled peptides, mono-labeled peptides, and dilabeled peptides, respectively. The natural isotopic dis-tribution was calculated using the peptide sequence and the MS-isotope program(http://prospector.ucsf.edu/). The isotope distribution pattern for the 18O-la-beled peptide was assumed to be the same as for the unlabeled peptide. The

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labeled and unlabeled peptide pair ratios were first sorted by protein locus andfiltered using Dixon’s test to omit the outliers. The remaining peptide ratios wereused to calculate the protein means and standard deviations.

RT-PCR. Total RNAs from three biological replicates were isolated usingTrizol reagent (Invitrogen) for RT-PCR. Each biological replicate was analyzedthrice. Quantitative real-time PCR assays were performed using SYBR greenPCR master mix (ABI) and an ABI 7300 real-time PCR system (ABI). Thesequences of primers used for quantitative PCR are described in Table S1 in thesupplemental material. The cycling program was as follows: an initial cycle of 2min at 50°C and 10 min at 95°C, followed by 40 cycles of 10 s at 95°C and 30 sat 60°C. The disassociation analysis was carried out in a routine fashion byacquiring fluorescent readings for 1°C increases from 55 to 95°C. Data wereanalyzed using system 7300 SDS software to calculate threshold cycle (CT)values. Subsequently, the relative expression ratios for the genes were deter-mined according to the following equations:

Sample �CT � CTSample � CTTDH2 (2)

��CT � sample �CT � control �CT (3)

Severalfold increase (sample versus control) � 2��CT (4)

NAD�/NADH and ATP/ADP assays. Rapid sampling, quenching, and metab-olite extraction of biomass (in approximately 10 ml of culture broth) wereperformed according to the method of Luo et al. (44). All LC-MS experimentswere carried out using an LCQ DecaXP Max mass spectrometer (Thermo Finni-gan, Palo Alto, CA) under the control of the Xcalibur data system (ThermoFinnigan, Palo Alto, CA). A sample of 20 �l was loaded onto an Atlantis T3 4.6-by 250-mm column (Waters, Ireland) (5 �m pore size), which was equilibratedfor 30 min before loading with 98% solution A (5 mM NH4HCO3 in water) and2% solution B (80% methanol–5 mM NH4HCO3 in water). A 20-min gradient to60% solution A and a 5-min gradient to 60% solution A were used. The columneluent was electrosprayed directly into a mass spectrometer. The mass spectrom-eter was operated in the negative-ion and selected-reaction-monitoring mode.The optimized parameters were as follows: ion-spray voltage, �4.5 kV; sheathgas and auxiliary gas, 33 and 5 (arbitrary units), respectively. The capillarytemperature was 300°C, and the scan range was 140 to 850 m/z. The standardcurve was obtained by analyzing standard solutions (NADH and NAD�; Sigma,St. Louis, MO) at five concentrations (50, 10, 1, 0.5, and 0.1 �g) selected forNADH and NAD� concentration calculations. The levels of NADH/NAD� andATP/ADP were calculated according to the ratios of the integrated ion currents.Three biological replicate experiments were performed, with two injections foreach sample.

Data analysis. The functional category and subcellular localization determi-nations for proteins were carried out with FunCatDB software (http://mips.gsf.de/projects/funcat), and biochemical pathways were classified and reconstructedby reference to the KEGG database (http://www.genome.jp/kegg/) and the Sac-charomyces Genome Database (http://www.yeastgenome.org/). The Saccharo-myces Genome Database was also utilized to obtain information on proteins.Proteins quantified with respect to two or more peptides were considered to besignificantly changed when one of the following three criteria was satisfied: (i)expression changed by no less than �1.5-fold and with relative standard devia-tions (RSDs) below 40%; (ii) determination of an 18O/16O ratio higher than 3 orlower than 0.33 and an RSD below 50%; or (3) all corresponding peptideabundances changed by no less than �1.5-fold without regard to RSD values.

RESULTS AND DISCUSSION

Physiological profile of S. cerevisiae treated with furfural orleft untreated. The physiological effect of furfural on the in-dustrial S. cerevisiae strain was studied by comparison of cul-tivation with the addition of furfural (17 g/liter) into aerobicbatch cultivations during exponential growth in the respiratory-fermentative phase to the control cultivations grown under thesame conditions without the introduction of furfural. The re-sults are presented in Fig. 1. The addition of furfural almostcompletely suppressed cell growth, leading to very slow cellgrowth in the first 2 h after addition of furfural and a halt inbiomass formation after that. Upon extended incubation for9 h, no increase in growth was observed. Both ethanol forma-tion and glucose consumption were inhibited, showing slow

rates in the first 4 h and then stopping at 4 h after the additionof furfural. Glycerol formation was halted immediately, andthere was no change in the glycerol concentration after theaddition of furfural. In contrast, the furfural concentration inthe medium decreased sharply during the first 4 h and slowly inthe next 4 h and then showed no change with a residual con-centration of about 6 g/liter, indicating that furfural had beenconverted by yeast cells to compounds of lesser toxicity. Usingthe methylene blue technique for cell viability determinationsduring the experiment, we observed that yeast cells were notable to recover from the inhibition caused by furfural and thatall the cells died at 8 h after the addition of furfural (see Fig.S1 in the supplemental material).

It is obvious that the introduction of furfural into aerobicbatch yeast cultivations had an immediate and drastic effect onthe physiological behavior of yeast and that the response ofyeast to the presence of furfural was a continued dynamicprocess. Confronted with furfural, the industrial yeast cellsshowed a notable decrease in cell growth, glucose consump-tion, ethanol production, and glycerol production but still triedto detoxify furfural to alleviate its inhibitory effects. Theseresults may reflect a metabolic rearrangement in yeast to copewith furfural detoxification and with diverse effects caused bythe presence of furfural, such as the arrest of cell growthand/or the accumulation of acetaldehyde, underscoring theneed for utilization of systems biology approaches for further

FIG. 1. Comparison of cell growth rates and metabolite concentra-tions during aerobic batch cultivation of S. cerevisiae in the presence(filled symbols) or absence (empty symbols) of furfural. (A) Concen-tration of furfural (diamond) and glucose (circle); (B) biomass (trian-gle), ethanol (square), glycerol (circle). All experiments were per-formed in duplicate. Furfural (17 g/liter) was added when the cultureshad reached an OD of 3 to 4 (denoted by time 0).

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research into the effect of furfural on the industrial S. cerevisiaestrain.

Proteins differentially expressed in response to furfural. Tostudy the proteomic response of the industrial S. cerevisiaestrain under conditions of treatment with furfural, a shotgunyeast comparative proteome investigation has been carriedout. Two equivalent whole-yeast extracts acquired from thecontrol experiment and the furfural treatment experimentwere trypsinized into peptides in H2

16O and H218O, respec-

tively. Samples from the same time point (the 20-min or 2-htime point) were combined in a 1:1 ratio and subjected to threerounds of analysis using 2D-LC-MS/MS. Proteins were identi-fied through database searching, and relative protein expres-sion levels were determined by calculating corrected 18O/16Opeptide ratios and using peak areas. A total of 2,037 and 3,655peptides (corresponding to 205 and 309 proteins, respectively)derived from three replicate experiments run for both 20 minand 2 h were quantified manually. Of those proteins, 175 werequantified for both of the datasets. According to the data-mining criteria described above (see Materials and Methods)as used for analysis of proteins that are differentially expressed,70 proteins were upregulated and 6 proteins were downregu-lated at 20 min, whereas 31 proteins were upregulated and 35proteins were downregulated at 2 h in response to the presenceof furfural.

To get an overview of the differentially expressed proteinsand guide subsequent data analysis, determinations of the sub-cellular localizations and functional categories of the differen-tially expressed proteins were carried out using FunCatDBsoftware. The numbers of differentially expressed proteins for

each cellular compartment or functional category for timepoints 20 min and 2 h are shown in the form of 100% stackedcolumns, as displayed in Fig. 2.

Subcellular localization distributions of differentially ex-pressed proteins at 20 min were notably different from thoseseen at 2 h. Proteins with an abundance change at 20 min werenot localized to the bud, golgi, or peroxisome compartments,whereas proteins with an abundance change at 2 h did notoriginate from the cell wall. The other subcellular compart-ments were well represented and had different proportions ofdifferentially expressed proteins in each data set. The diversesubcellular localization distribution profiling results for 20 minof cultivation versus 2 h demonstrate that furfural has differenteffects on cellular compartments over time. When introducedinto the aerobic batch culture, furfural first entered into theyeast cell through the cell wall and plasma membrane andaffected yeast gene expression in as little as 10 min (39). As aresult, the numbers of differentially expressed proteins local-ized to the cell wall, plasma membrane, and nucleus weremuch greater at 20 min than at 2 h. On the other hand, morecompartments, including the bud, golgi, and peroxisome com-partments, were affected by furfural at 2 h than at 20 min.

The function distribution of proteins with altered expressionin response to the presence of furfural at 20 min and 2 h ispresented in Fig. 2B. As a whole, differentially expressed pro-teins at 20 min were overrepresented compared to those seenat 2 h for most function groups, with the exception of theregulation of metabolism and protein function, unclassifiedprotein, and development function groups. At 20 min of cul-tivation, furfural may affect yeast more severely and result in

FIG. 2. Subcellular localization distribution (A) and functional categories (B) of 76 and 65 proteins differentially expressed in response to thepresence of furfural at 20 min and 2 h, respectively. The 100% stacked column charts are used to compare the numbers of differentially expressedproteins in each cellular compartment or function category at 20 min and 2 h. The total number of differentially expressed proteins from the twotime points represents 100%, while the individual numbers of differentially expressed proteins at 20 min and 2 h compose the two stacked elementsthat make up one column. (B) PF, protein fate; CRDV, cell rescue, defense, and virulence; CCDP, cell cycle and DNA processing; IE, interactionwith the environment; BCC, biogenesis of cellular components; CTD, cell type differentiation; CTTFTR, cellular transport, transport facilities, andtransport routes; PS, protein synthesis; PBFCR, protein with binding function or cofactor requirement; CF, cell fate; CC/STM, cellular commu-nication/signal transduction mechanism; UP, unclassified proteins; RMPF, regulation of metabolism and protein function.



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more proteins with changes in abundance than would be seenat 2 h, due to the higher concentration of furfural at the earliertime point (15.9 g/liter and 10.5 g/liter at 20 min and 2 h,respectively; see Fig. 1). Yeast cells can convert furfural toless-toxic compounds, resulting in a decreased concentrationof furfural over time. A dose-dependent response of ethanolo-genic yeast to the presence of furfural and HMF at concentra-tions from 10 to 120 mM has been characterized by Liu et al.(43). Proteins with abundance changes caused by the presenceof furfural were localized to most compartments and werefound to be involved in almost all the functions and pathwaysin yeast cells, revealing that the response of yeast to furfural isglobal and systematic. Furthermore, our observations also sug-gest that the response of yeast to furfural is a continued dy-namic and complex process.

Glycolysis pathway. As implied by the KEGG pathway anal-ysis of the differentially expressed proteins, furfural may havea great impact on the glycolysis pathway. After the addition offurfural into the aerobic batch cultures of the industrial S.cerevisiae strain, 12 out of 16 proteins quantified in the glyco-lysis pathway had been dramatically upregulated at 20 min,while only 1 of 17 proteins had been upregulated and 3 pro-teins downregulated at 2 h (Fig. 3). Evidently, the glycolysis ofS. cerevisiae had first been rapidly induced by the addition offurfural and had then reached control levels at 2 h compared tothe control experiment results (verified by NADH/NAD� andATP/ADP assays). This gives further evidence that the re-sponse of yeast to the presence of furfural is a continuingdynamic process, as described above. It is reasonable to believethat the expression levels of proteins catalyzing the reactions of

FIG. 3. Relative expression levels of proteins involved in central carbon metabolism, including glycolysis, the TCA cycle, glycerol biosynthesis,and the PPP. The 18O/16O ratios of proteins are presented for the 20-min time point (the first value in parentheses) and the 2-h time point (thesecond value in parentheses). The underlined 18O/16O ratios were obtained on the basis of analysis of one peptide. The 20-min time point dataare missing because proteins were not detected at 20 min by 2D-LC-MS/MS.



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glycolysis decrease over time, for glucose consumption hadalmost halted and wash out of the cell cultures had occurred at8 h under the conditions that included treatment with furfural.The idea of the activation of glycolysis in the presence offurfural is supported by other studies. Taherzadeh et al. (61)determined that glycolysis inducing changes in abundance wasimmediately affected by furfural and that the reduction offurfural relied on active glycolysis by investigating conversionof furfural in aerobic and anaerobic bath cultures of S. cerevi-siae CBS 8066 growing on glucose. Analysis of the metabolicflux distributions for aerobic steady-state cultures with andwithout furfural in the medium showed that the presentationof furfural in the medium resulted in an increase of 30% in thespecific rate of glycolysis compared to the rate seen withfurfural-free medium (27). Through metabolite analysis,Palmqvist et al. (48) found that furfural decreased cell repli-cation without inhibiting cell activity and had a twofold effecton the kinetics of glucose metabolism in S. cerevisiae (i.e., theglucose metabolism rate was inhibited but the final ethanolyield was slightly increased at a nonlethal concentration offurfural) (48).

In contrast to the results seen with activated glycolysis, thegrowth and fermentation performance of the industrial S. cer-evisiae strain were significantly retarded. However, notable re-duction of furfural was observed in the first 4 h, demonstratingthat activated glycolysis may correlate with the conversion offurfural. Presumably, the reduction of furfural to furfural al-cohol may be catalyzed by alcohol dehydrogenases (ADHs),with NADH as a cofactor (12, 46, 67), which was evidencedhere by the upregulation of Adh5p and Adh1p as describedbelow. It is possible that glycolysis is activated by furfural toprovide as much NADH as is required for the reduction offurfural, whereas ethanol production is reduced, because thereduction of furfural competes with the reaction of acetalde-hyde to ethanol for both NADH and ADHs.

Glycerol biosynthesis. Downregulated levels of Gpd1p (at20 min, not quantified; at 2 h, 0.46), Rhr2p (0.58; 0), andHor2p (not quantified; 0.43) catalyzing glycerol biosynthesiswere observed in furfural-treated cells compared to controlcells (Fig. 3). Furthermore, as shown in Fig. 1, the glycerolconcentration stayed constant after the introduction of furfuralto the aerobic bath cultures of yeast, indicating that furfuralseverely inhibits glycerol formation. It has been demonstratedthat the reduction of furfural to furfural alcohol is preferred toglycerol production as a redox sink, subsequently resulting inthe replacement of glycerol formation by furfural alcohol pro-duction (48, 61). Palmqvist et al. (49) and Taherzadeh et al.(61) observed that glycerol production was decreased in thepresence of furfural under anaerobic conditions. Glycerol bio-synthesis acts as a redox sink, providing additional reoxidationof cytosolic NADH. At the same time, NADH is the majorcofactor required for reduction of furfural to furfural alcohol.As a result, glycerol production and furfural reduction com-pete for a shared pool of NADH, as is also observed with thereaction of acetaldehyde to ethanol and furfural reduction. Inthe presence of a high concentration of furfural, yeast reducesthe production of glycerol to meet the urgent requirement ofNADH for the conversion of furfural to furfural alcohol (seeFig. 8).

TCA cycle. As a central metabolic pathway, the tricarboxylicacid (TCA) cycle provides precursors for many compounds,including some amino acids, and generates useful amounts ofATP and NADH under aerobic conditions. None of the en-zymes in the TCA cycle was identified at 20 min, whereasCit1p, Aco1p, Aco2p, Idh1p, and Mah1p were identified andquantified at 2 h and all displayed a trend toward increasedproduction, with Aco2p and Mdh1p noticeably upregulated inthe presence of furfural. Analysis of the metabolic flux distri-butions for the aerobic steady-state cultures with and withoutfurfural in the medium showed that the presence of furfural inthe medium resulted in a 50% increase in the specific rate ofthe TCA cycle compared to the rate seen with furfural-freemedium (27). The upregulation of enzymes at 2 h reveals thatthe TCA cycle can be activated in the presence of furfural toproduce more NADH for the reduction of furfural (see Fig. 8).

PPP. The pentose phosphate pathway (PPP) is an importantcarbohydrate metabolism pathway, oxidizing glucose to gener-ate NADPH for reductive biosynthesis reactions within cellsand ribose-5-phosphate for the synthesis of the nucleotides andnucleic acid. However, the expression levels of Gnd1p (at 20min, 1.46; at 2 h, 1.08), Tkl1p (0.91; 0.98), and Tal1p (1.23;1.30) involved in the PPP were not affected by the presence offurfural at either time point (Fig. 3). This observation is con-sistent with the results of a previous study showing that al-though selective-deletion mutants coded by genes in the PPPshowed growth deficiencies in the presence of furfural, thesemutants were inefficient in reducing furfural to furfural alcohol(18). Therefore, it is suggested that the PPP may have no directcorrelation with furfural conversion under the conditions stud-ied here.

Since it has been reported that the central carbon metabo-lism of S. cerevisiae is controlled to a large extent via posttran-scriptional mechanisms in chemostat studies (11, 32, 33), quan-titative RT-PCR was not carried out to substantiate ourfindings concerning the involvement of the central carbon me-tabolism in cellular response to the presence of furfural. In-stead, the levels of intracellular NADH, NAD�, NADH/NAD�, and ATP/ADP were measured by LC/MS (Fig. 4). Theintracellular concentrations of NAD� and NADH were de-creased at least twofold and fourfold, respectively, leading to alower NADH/NAD� ratio in furfural-treated cells comparedwith control cells at both time points. In contrast, the ATP/ADP ratio was increased significantly at 20 min but not at 2 h.The urgent requirement of furfural reduction caused a severeshortage of NADH, leading to the upregulation of some en-zymes in the central carbon metabolism to provide as muchNADH and ATP as possible in cells treated with furfuralcompared with control cells at 20 min and 2 h (although theeffect was less significant at 2 h). The need for ATP may besmaller than that for NADH in furfural-treated cells. Thus, thelevels of intracellular NAD�, NADH, and NADH/NAD�

were decreased whereas the ATP/ADP ratio was increasedafter the addition of furfural. These results corroborate thecomparative proteome results described above. NAD� andNADH are involved in various biological processes, includingaging, apoptosis, cell death, energy metabolism, mitochondrialfunctions, calcium homeostasis, antioxidation-generation ofoxidative stress, gene expression, and so on (71). It has beensuggested that NAD� depletion mediates poly(ADP-ribose)



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polymerase-1-induced cell death (1). Previous work has sug-gested that NAD� and NADH may be involved in apoptosis: itwas reported that selective inhibitors of NAD� synthesis caninduce apoptosis (24) and that NADH/NADPH depletion is anearly event in apoptosis (52). Thus, the decrease in bothNAD� and NADH levels in yeast cells upon treatment withfurfural, resulting from an inhibition of synthesis of these com-pounds or from accelerated degradation, may be ultimatelyresponsible for the cell death observed. Furthermore, our datasuggest that acetaldehyde likely accumulates in the cultureduring furfural reduction due to a decreased NADH concen-tration in the cell. In a previous study, the accumulation ofacetaldehyde after the addition of furfural was observed andthe effect of furfural on cell replication was shown to be relatedto acetaldehyde formation (48). Acetaldehyde has been foundto exert inhibition effects on yeast growth (59), which is con-sistent with the arrest of growth observed in this study. Obvi-ously, the presence of furfural caused several secondary effects,including the drop in both NAD� and NADH levels, the ac-cumulation of acetaldehyde, and a contribution to the general

stress environment, which in turn may affect the cellular me-tabolism in yeast cells treated with furfural.

The altered expression levels of most proteins catalyzing thereactions of central carbon metabolism (with the exception ofthose involved in the PPP) revealed that the addition of fur-fural led to a central carbon metabolism rearrangement inyeast cells in order to induce toleration of furfural. Glycolysisand/or the TCA cycle were stimulated to provide sufficientNADH for efficient conversion of furfural. Also, NADH wasdiverted from glycerol synthesis and ethanol production intofurfural alcohol formation. Thus, the formation of ethanol andglycerol was inhibited during the adaptation of yeast to furfural(see Fig. 8).

ADHs. Among the seven ADHs in yeast cells, three werequantified at 20 min and six at 2 h (Fig. 5). When 17 g offurfural/liter of medium was used, the expression of Adh5p wasmarkedly upregulated (i.e., it was more than 4 times higherthan that seen in furfural-free medium at both time points)whereas translational levels showed no significant changes.This discrepancy for Adh5p at the protein level and the tran-script level has been reported by other groups (6, 7, 9). Adh5pcan catalyze the conversion of acetaldehyde to ethanol, withactivity apparent only in an adh1 and adh3 double-deletionstrain (57), and this conversion can be induced by the presenceof dimethyl sulfoxide (72). Adh6p was significantly upregulatedat 20 min (with one peptide quantified) and showed no changeat 2 h. Larroy et al. reported that Adh6p is an NADPH-dependent ADH of broad substrate specificity that is able toreduce aldehydes, including cinnamaldehyde, veratraldehyde,and furfural (34). In addition, the reduction of 5-hydroxy-methyl furfural and furfural with NADPH as a cofactor wasincreased in cell-free crude extracts from Adh6p-overexpress-ing strains (51). Adh1p, the major enzyme catalyzing the reac-tion of acetaldehyde to ethanol, displayed mediate increases atboth time points, which were verified by quantitative RT-PCR(see Fig. 7). Adh1p has been suggested in previous studies tobe an enzyme possibly catalyzing the reduction of furfural tofurfural alcohol (21). The overexpression of Adh1p can in-crease the formaldehyde resistance of S. cerevisiae (20). Sinceethanol formation was severely inhibited at both time points,the enhancement of ADHs was not related to the reaction of

FIG. 4. Intracellular NADH and NAD� levels (A), ATP/ADP ra-tios (B), and NAD�/NADH ratios (C) in furfural (fur)-treated cells(gray bars) and control (con) cells (black bars) at 20 min and 2 h. Threebiological replicate experiments were performed along with two injec-tions for each sample. The P values indicate the statistical significanceof the observed differences, with P � 0.05 considered to be statisticallysignificant and P � 0.05 to be not statistically significant, as determinedby a two-tailed Student t test. CDW, cell dry weight.

FIG. 5. Histogram showing six members of the ADH gene familyand their respective change ratios at 20 min and 2 h. The filled circlesindicate that proteins were not detected at that time point.



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acetaldehyde to ethanol. Furthermore, it has been reportedthat the reduction of furfural to furfural alcohol is likely cat-alyzed by ADHs (12, 46, 67). Thus, in the context of theliterature and our experiments, it is possible that Adh5p,Adh1p, and Adh6p may catalyze the reduction of furfural tofurfural alcohol. However, further validation studies are nec-essary to confirm this hypothesis, since the induction of Adh5p,Adh1p, and Adh6p by furfural may be associated with otherbiological processes.

Adh2p, Adh4p, and Sfa1p were detected and quantified onlyat 2 h. Adh2p and Sfa1p had decreased expression levels,whereas the expression abundance of Adh4p was not affectedby the presence of furfural. Adh2p, unlike other ADHs, cata-lyzes the reaction of ethanol to acetaldehyde and is repressedin the presence of glucose (9). Addition of furfural inhibitedthe glucose consumption and led to higher glucose concentra-tions in the furfural treatment experiment (Fig. 1), and this inturn repressed the expression of Adh2p. The downregulationof Sfa1p is consistent with the lower ethanol concentration inthe medium with furfural compared to the furfural-free me-dium at 2 h, as it had been reported that Sfa1p is induced bythe presence of ethanol (17). The decreased levels of expres-sion of Adh2p and Sfa1p suggest that the ripple effect imposedby the presence of furfural exists and becomes more significant

with the passage of time after the addition of furfural. Thus,ADHs may play a role in the tolerance and adaptation ofethanologenic yeast to furfural.

Proteins related to stress response. Although it has beenmentioned before that furfural may result in the accumulationof reactive oxygen species, vacuole and mitochondrial mem-brane damage, and chromatin and actin damage in S. cerevisiae(39), there has been no previous study focusing on the stresseffects caused in S. cerevisiae by the presence of furfural. Thelevels of abundance of 23 proteins related to stress responsedisplayed significant changes either at one time point or atboth time points (Fig. 6A). Analysis with respect to functionalcategory showed that these proteins are involved in the re-sponse of yeast to unfolded proteins, oxidative stress, osmoticand salt stress, DNA damage, and nutrient starvation.

There are eight proteins related to unfolded protein re-sponse (UPR), including five HSP70 proteins: Ssb1p, Ssb2p,Ssc1p, Ssz1p, and Kar2p. At 20 min after the addition offurfural, proteins related to the folding, sorting, and translo-cation of newly synthesized polypeptide chains such as Egd2p(16), Hsp10p (25), and Ssb1p were upregulated. At 2 h, nochange in the expression of Egd2p and Ssb1p was seen,whereas Hsp10p was slightly downregulated, as shown by theresults of quantification of one peptide. Instead, another group

FIG. 6. Proteins related to stress response that exhibited significant changes in abundance in this study (A) and ribosomal proteins quantifiedin this study (B). The filled circles indicate that proteins were not detected at that time point.



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of proteins (Ssb2p, Ssc1p, and Ssz1p) related to the folding,sorting, and translocation of newly synthesized polypeptidechains displayed increased expression levels at 2 h after theaddition of furfural. It is worth noting that Kar2p was upregu-lated at 2 h, since Kar2p not only is induced by UPR but is alsoinvolved in the regulation of UPR through interaction withIre1p. The induction of these proteins related to the UPRimplies that the addition of furfural may lead to the accumu-lation of unfolded proteins, subsequently resulting in triggeringof the UPR. Further evidence was observed when the results ofstimulation of ribosome proteins were recorded (Fig. 6B). At20 min, all of the 32 ribosomal proteins quantified in this studyexhibited a trend to increased regulation changes as a group;17 of those proteins were significantly upregulated in responseto furfural. At 2 h, 16 of 34 ribosomal proteins quantifiedshowed distinctly increased expression levels, while 1 proteinwas slightly downregulated. At both time points, 27 proteinswere quantified, with 11 proteins upregulated. The main func-tion of ribosome is to organize protein synthesis. The upregu-lation of ribosomal proteins suggests that protein synthesis inthe aerobic batch culture containing furfural may have beenaccelerated compared to that seen with furfural-free culturesunder the same conditions. The acceleration of protein syn-thesis may lead to the accumulation of unfolded proteins in theendoplasmic reticulum (ER) lumen, in turn activating theUPR to restore protein-folding capacity and adapt to newconditions caused by the presence of furfural. The UPR canprotect cells against ER stress, but when this objective cannotbe achieved within a certain time period or when ER stress isprolonged, the UPR can initiate cell death or apoptosis. Theconcentration of furfural applied in this study was so high thathigh-intensity and long-term ER stress existed, and washout ofcultures occurred at 8 h.

Proteins that respond to oxidative stress represent the sec-ond-largest group among the stress-response-related proteinsthat showed abundance changes in the presence of furfural.The thioredoxins Trx1p and Trx2p, heat shock protein Hsp12p,and superoxide dismutase Sod1p were significantly upregu-lated at 20 min but showed no abundance change at 2 h inresponse to the presence of furfural. Expression of Tsa1p, ahousekeeping thioredoxin peroxidase, was first notably up-regulated at 20 min and then slightly downregulated at 2 h. Theexpression level of Ahp1 was downregulated at both timepoints after the addition of furfural into aerobic batch culturesof S. cerevisiae. Ahp1p is a thiol-specific peroxiredoxin protect-ing cells from oxidative damage by reducing hydroperoxides(35). Yhb1p, a nitric oxide oxidoreductase detoxifying nitricoxide (38), was quantified only at 2 h and showed an increasedexpression level. The oxidative stress may be related to thedecrease of the NAD�, NADH, and NADH/NAD� levels,since it has been suggested that NAD� and NADH influenceantioxidation and the generation of oxidative stress (71).

At both time points, furfural induced the expression levels ofRps3p (at 20 min, 1.70; at 2 h, 1.42) and Stm1p (3.25; 1.87),which respond to DNA damage. Genetic analyses have sug-gested that Stm1p, a G4 quadruplex and purine motif triplexnucleic acid-binding protein, participates in several biologicalprocesses, including interaction with ribosome and subtelo-meric Y DNA (63), telomere maintenance by interaction withCdc13p, and apoptosis (25, 36). Furthermore, its accumulation

induces cell death (36). Like the UPR, the upregulation ofStm1p at both time points may be involved in the washout ofcultures at 8 h. In contrast, Rps3p is essential for viability (15)and is involved in DNA damage processing and with apurinic-apyrimidinic endonuclease activity (29). Thus, the upregula-tion of expression of these two proteins reveals that furfuralcauses DNA damage in S. cerevisiae.

The Pkc1p–mitogen-activated protein kinase (Pkc1p-MAPkinase) pathway regulates cell wall maintenance and integrity,which are essential for the growth and the integrity of prolif-erating cells (60). The Pkc1p-MAP kinase pathway is reportedto be negatively regulated by Zeo1p (at 20 min, 2.3; at 2 h, 1.3)(19) and Lsp1p (at 20 min, not quantified; at 2 h, 2.83) (73), sothe upregulation of expression of Zeo1p at 20 min and ofLsp1p at 2 h revealed that this pathway is inactivated in thepresence of furfural, leading to lack of cell wall integrity anddefective cell growth. This hypothesis is further supported bythe downregulation of Rho1p (at 20 min, not quantified; at 2 h,0.31), which is required for the Pkc1p localization to sites ofpolarized growth throughout the cell cycle and also to regionsof cell wall damage (3, 54). Rho1p also regulates cell wallsynthesizing enzyme 1, also called 3-beta-glucan synthase(Fks1p and Gsc2p) (14, 54). Expression of Act1p, a structuralconstituent of the cytoskeleton that is involved in cell polar-ization, endocytosis, and many other cytoskeletal functions(53), was significantly upregulated at 20 min and unchanged at2 h. The altered expression of proteins that are involved in cellintegrity, growth, and survival strongly implies that the pres-ence of furfural had caused a rearrangement of cell structureand damage to yeast cell integrity, growth, and survival. More-over, Pho2p, which participates in nutrient starvation, was sig-nificantly upregulated at 20 min. PHO2 is known as a tran-scriptional activator of PHO5 and PHO81 (phosphateutilization), HIS4 (histidine biosynthesis), CYC 1, TRP4, andHO; it also activates expression of the ADE1, ADE2, ADE5,ADE7, and ADE8 genes, which are involved in the metabolicpathway of purine nucleotide biosynthesis (10, 37).

To confirm the protein expression data obtained by 18Olabeling, the transcript levels of the 10 selected stress-relatedproteins described above were measured by quantitative RT-PCR at 20 min and 2 h (Fig. 7). The quantitative RT-PCRresults for Act1p, Hsp10p, Hsp12p, Pho2p, Tsa1p, and Zeo1pat 20 min and for Lsp1p and Stm1p at 2 h are consistent withthe relative quantitative protein expression results obtainedusing 18O labeling. Furthermore, the levels of expression ofZeo1p, Hsp10p, and Hsp12p changed in the same direction atboth the protein level and the transcription level, although thechanges at the transcript level were statistically significantwhereas those at the protein level were not. Generally, therewere discrepancies between quantitative RT-PCR results andthe relative quantitative protein expression results such asthose seen with Ahp1p at both time points. These discrepan-cies may have been due to the fact that, apart from the effectdetermined by the amount of mRNA present, the proteinexpression level is influenced by protein turnover and post-translational modifications. What is more, the mRNA mole-cules may be relatively unstable compared to proteins in gen-eral, contributing to the difference in turnover rates betweenmRNA and protein, as reported in a previous study using S.cerevisiae (22). The quantitative RT-PCR results provide or-



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thogonal evidence of the reliability of the relative quantitativeprotein expression results determined using 18O labeling.

Clearly, furfural not only influences yeast with respect toprimary carbonate metabolic pathways and protein synthesisbut also causes the formation of a complex stress environmentin yeast cells. The expression levels of proteins involved incommon stress responses, including the UPR, oxidative stress,osmotic and salt stress, DNA damage, and nutrient starvation,were altered due to the complex stress environment formed bythe addition of furfural to the aerobic batch culture. The re-direction of resources toward stress defense may lead to di-minishing amounts of free available energy supplied by catab-olism for cell growth and insufficient ATP for phosphorylationof glucose to form glucose-6-phosphate, which is critical for theutilization of glucose. Thus, cell growth and glucose consump-tion are inhibited by furfural. With the passage of time, yeast

cells first display a lag phase and eventually adapt to furfuralstress, but when the concentration of furfural is too high andrequires too much NADH, the yeast cell is severely damagedand washout occurs (Fig. 8).

Conclusion. The addition of high concentrations of furfuralto the aerobic batch cultures of an industrial strain severelyinhibited biomass growth, glucose consumption, ethanol pro-duction, and glycerol production. Relative quantitative pro-teomic data here provide a deeper understanding of the mo-lecular mechanism involved in the response of S. cerevisiae tofurfural under aerobic batch conditions. Together with upregu-lation of Adh5p and Adh1p, activation of glycolysis and/or theTCA cycle and repression of glycerol biosynthesis were ob-served, suggesting that the reduction of furfural to furfuralalcohol catalyzed by Adh5p and Adh1p with NADH as a co-factor may be a potential pathway for the conversion of fur-

FIG. 7. A set of 13 transcript levels was measured by quantitative RT-PCR. Ratios between furfural-treated and control cell levels at 20 minand 2 h are shown. Three biological replicate experiments were performed along with three technical analyses for each biological replicate. Astatistical analysis of independent culture replicates was performed using a two-tailed Student t test. The proteins coded by genes that weresignificantly transcriptionally regulated are depicted (*, P � 0.05).

FIG. 8. Model depicting the effects of the presence of furfural on ethanologenic yeast under aerobic batch conditions. Under conditions thatincluded treatment with furfural, glycolysis and/or the TCA cycle was activated (green) whereas glycerol and ethanol production (red) wererepressed to provide as much NADH as required for furfural reduction to furfural alcohol. On the other hand, a general stress response (purple)was caused by furfural. As a result, yeast cells displayed a lag phase to tolerate and adapt to furfural, but when the concentration of furfural wastoo high, leading to a requirement for too much NADH and resulting in severe damage to yeast cells, washout occurred.



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fural to compounds of lesser toxicity. What is more, proteinsinvolved in stress response were also differentially expresseddue to the complex stress environment whose formation wascaused by the presence of furfural. The redirection of re-sources toward furfural conversion and stress defense may leadto the inhibition of yeast cell growth and fermentation. Quan-titative RT-PCR and metabolite analysis (of the levels ofNADH, NAD�, NADH/NAD�, and ATP/ADP) were utilizedto provide orthogonal evidence supporting the comparativeproteomics results. Secondary effects due to the presence offurfural were observed and may have been related to the in-hibitory effects of furfural. These insights into the response ofyeast to furfural will benefit the design and development ofinhibitor-tolerant ethanologenic yeast strains for lignocellu-lose-bioethanol fermentation, which is one of the significantchallenges for cost-competitive bioethanol production.

ACKNOWLEDGMENTS

We are very grateful for the financial support from the NationalScience Fund of China for Distinguished Young Scholars (project20425620), the Key Program (project 20736006), National Basic Re-search Program 973 of China (grant 2007CB714301), the InternationalCollaboration Project of MOST (grant 2006DFA62400), and KeyProjects in the National Science & Technology Pillar Program (grant2007BAD42B02).

REFERENCES

1. Alano, C. C., W. H. Ying, and R. A. Swanson. 2004. Poly(ADP-ribose)polymerase-1-mediated cell death in astrocytes requires NAD� depletionand mitochondrial permeability transition. J. Biol. Chem. 279:18895–18902.

2. Almeida, J. R. M., T. Modig, A. Petersson, B. Hahn-Hagerdal, G. Liden, andM. F. Gorwa-Grauslund. 2007. Increased tolerance and conversion of inhib-itors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J. Chem.Technol. Biotechnol. 82:340–349.

3. Andrews, P. D., and M. J. R. Stark. 2000. Dynamic, Rho1p-dependentlocalization of Pkc1p to sites of polarized growth. J. Cell Sci. 113:2685–2693.

4. Ang, C. S., P. D. Veith, S. G. Dashper, and E. C. Reynolds. 2008. Applicationof O-16/O-18 reverse proteolytic labeling to determine the effect of biofilmculture on the cell envelope proteome of Porphyromonas gingivalis W50.Proteomics 8:1645–1660.

5. Brown, K. J., and C. Fenselau. 2004. Investigation of doxorubicin resistancein MCF-7 breast cancer cells using shot-gun comparative proteomics withproteolytic O-18 labeling. J. Proteome Res. 3:455–462.

6. Chong, P. K., A. M. Burja, H. Radianingtyas, A. Fazeli, and P. C. Wright.2007. Proteome analysis of Sulfolobus solfataricus p2 propanol metabolism. J.Proteome Res. 6:1430–1439.

7. Chong, P. K., A. M. Burja, H. Radianingtyas, A. Fazeli, and P. C. Wright.2007. Proteome and transcriptional analysis of ethanol-grown Sulfolobussolfataricus P2 reveals ADH2, a potential alcohol dehydrogenase. J. Pro-teome Res. 6:3985–3994.

8. Chong, P. K., A. M. Burja, H. Radianingtyas, A. Fazeli, and P. C. Wright.2007. Translational and transcriptional analysis of Sulfolobus solfataricus P2to provide insights into alcohol and ketone utilisation. Proteomics 7:424–435.

9. Ciriacy, M. 1975. Genetics of alcohol dehydrogenase in Saccharomyces cer-evisiae. II. Two loci controlling synthesis of the glucose-repressible ADH II.Mol. Gen. Genet. 138:157–164.

10. Daignan-Fornier, B., and G. R. Fink. 1992. Coregulation of purine andhistidine biosynthesis by the transcriptional activators BAS1 and BAS2. Proc.Natl. Acad. Sci. USA 89:6746–6750.

11. DeLuna, A., A. Avendano, L. Riego, and A. Gonzalez. 2001. NADP-gluta-mate dehydrogenase isoenzymes of Saccharomyces cerevisiae—purification,kinetic properties, and physiological roles. J. Biol. Chem. 276:43775–43783.

12. Diaz de Villegas, M. E., P. Villa, M. Guerra, E. Rodriguez, D. Redondo, andA. Martinez. 1992. Conversion of furfural into furfuryl alcohol by Saccharo-myces cerevisiae. Acta Biotechnol. 12:351–354.

13. Ding, M.-Z., J.-S. Cheng, W.-H. Xiao, B. Qiao, and Y.-J. Yuan. 2009. Com-parative metabolomic analysis on industrial continuous and batch ethanolfermentation processes by GC-TOF-MS. Metabolomics 5:229–238.

14. Drgonova, J., T. Drgon, K. Tanaka, R. Kollar, G. C. Chen, R. A. Ford,C. S. M. Chan, Y. Takai, and E. Cabib. 1996. Rho1p, a yeast protein at theinterface between cell polarization and morphogenesis. Science 272:277–279.

15. Fingen-Eigen, M., H. Domdey, and K. Kohrer. 1996. The ribosomal proteingene RPS3 is an essential single copy gene of the yeast Saccharomycescerevisiae. Biochem. Biophys. Res. Commun. 223:397–403.

16. George, R., T. Beddoe, K. Landl, and T. Lithgow. 1998. The yeast nascentpolypeptide-associated complex initiates protein targeting to mitochondriain vivo. Proc. Natl. Acad. Sci. USA 95:2296–2301.

17. Gompel-Klein, P., M. Mack, and M. Brendel. 1989. Molecular characteriza-tion of the two genes SNQ and SFA that confer hyperresistance to 4-nitro-quinoline-N-oxide and formaldehyde in Saccharomyces cerevisiae. Curr.Genet. 16:65–74.

18. Gorsich, S. W., B. S. Dien, N. N. Nichols, P. J. Slininger, Z. L. Liu, and C. D.Skory. 2006. Tolerance to furfural-induced stress is associated with pentosephosphate pathway genes ZWF1, GND1, RPE1, and TKL1 in Saccharomy-ces cerevisiae. Appl. Microbiol. Biotechnol. 71:339–349.

19. Green, R., G. Lesage, A. M. Sdicu, P. Menard, and H. Bussey. 2003. Asynthetic analysis of the Saccharomyces cerevisiae stress sensor Mid2p, andidentification of a Mid2p-interacting protein, Zeo1p, that modulates thePKC1-MPK1 cell integrity pathway. Microbiology 149:2487–2499.

20. Grey, M., M. Schmidt, and M. Brendel. 1996. Overexpression of ADH1confers hyper-resistance to formaldehyde in Saccharomyces cerevisiae. Curr.Genet. 29:437–440.

21. Gutierrez, T., M. L. Buszko, L. O. Ingram, and J. F. Preston. 2002. Reduc-tion of furfural to furfuryl alcohol by ethanologenic strains of bacteria and itseffect on ethanol production from xylose. Appl. Biochem. Biotechnol. 98:327–340.

22. Gygi, S. P., Y. Rochon, B. R. Franza, and R. Aebersold. 1999. Correlationbetween protein and mRNA abundance in yeast. Mol. Cell. Biol. 19:1720–1730.

23. Hansen, R., S. Y. Pearson, J. M. Brosnan, P. G. Meaden, and D. J. Jamieson.2006. Proteomic analysis of a distilling strain of Saccharomyces cerevisiaeduring industrial grain fermentation. Appl. Microbiol. Biotechnol. 72:116–125.

24. Hasmann, M., and I. Schemainda. 2003. FK866, a highly specific noncom-petitive inhibitor of nicotinamide phosphoribosyltransferase, represents anovel mechanism for induction of tumor cell apoptosis. Cancer Res. 63:7436–7442.

25. Hayashi, N., and S. Murakami. 2002. STM1, a gene which encodes a guaninequadruplex binding protein, interacts with CDC13 in Saccharomyces cerevi-siae. Mol. Genet. Genomics 267:806–813.

26. Hood, B. L., D. A. Lucas, G. Kim, K. C. Chan, J. Blonder, H. J. Issaq, T. D.Veenstra, T. P. Conrads, I. Pollet, and A. Karsan. 2005. Quantitative analysisof the low molecular weight serum proteome using O-18 stable isotopelabeling in a lung tumor xenograft mouse model. J. Am. Soc. Mass Spectrom.16:1221–1230.

27. Horvath, I. S., C. J. Franzen, M. J. Taherzadeh, C. Niklasson, and G. Liden.2003. Effects of furfural on the respiratory metabolism of Saccharomycescerevisiae in glucose-limited chemostats. Appl. Environ. Microbiol. 69:4076–4086.

28. Horvath, I. S., M. J. Taherzadeh, C. Niklasson, and G. Liden. 2001. Effectsof furfural on anaerobic continuous cultivation of Saccharomyces cerevisiae.Biotechnol. Bioeng. 75:540–549.

29. Jung, S. O., J. Y. Lee, and J. Kim. 2001. Yeast ribosomal protein S3 has anendonuclease activity on AP DNA. Mol. Cells 12:84–90.

30. Klinke, H. B., A. B. Thomsen, and B. K. Ahring. 2004. Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl. Microbiol. Biotechnol. 66:10–26.

31. Klinke, H. B., A. B. Thomsen, and B. K. Ahring. 2001. Potential inhibitorsfrom wet oxidation of wheat straw and their effect on growth and ethanolproduction by Thermoanaerobacter mathranii. Appl. Microbiol. Biotechnol.57:631–638.

32. Kolkman, A., P. Daran-Lapujade, A. Fullaondo, M. M. A. Olsthoorn, J. T.Pronk, M. Slijper, and A. J. R. Heck. 2006. Proteome analysis of yeastresponse to various nutrient limitations. Mol. Syst. Biol. 2:2006.0026.

33. Kolkman, A., M. M. A. Olsthoorn, C. E. M. Heeremans, A. J. R. Heck, andM. Slijper. 2005. Comparative proteome analysis of Saccharomyces cerevisiaegrown in chemostat cultures limited for glucose or ethanol. Mol. Cell. Pro-teomics 4:1–11.

34. Larroy, C., M. R. Fernandez, E. Gonzalez, X. Pares, and J. A. Biosca. 2002.Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) geneproduct as a broad specificity NADPH-dependent alcohol dehydrogenase:relevance in aldehyde reduction. Biochem. J. 361:163–172.

35. Lee, J., D. Spector, C. Godon, J. Labarre, and M. B. Toledano. 1999. A newantioxidant with alkyl hydroperoxide defense properties in yeast. J. Biol.Chem. 274:4537–4544.

36. Ligr, M., I. Velten, E. Frohlich, F. Madeo, M. Ledig, K. U. Frohlich, D. H.Wolf, and W. Hilt. 2001. The proteasomal substrate Stm1 participates inapoptosis-like cell death in yeast. Mol. Biol. Cell 12:2422–2432.

37. Liu, C., Z. Y. Yang, J. Yang, Z. X. Xia, and S. H. Ao. 2000. Regulation of theyeast transcriptional factor PHO2 activity by phosphorylation. J. Biol. Chem.275:31972–31978.

38. Liu, L. M., M. Zeng, A. Hausladen, J. Heitman, and J. S. Stamler. 2000.Protection from nitrosative stress by yeast flavohemoglobin. Proc. Natl.Acad. Sci. USA 97:4672–4676.

39. Liu, Z. L. 2006. Genomic adaptation of ethanologenic yeast to biomassconversion inhibitors. Appl. Microbiol. Biotechnol. 73:27–36.



http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

40. Liu, Z. L., J. Moon, B. J. Andersh, P. J. Slininger, and S. Weber. 2008.Multiple gene-mediated NADPH-dependent aldehyde reduction is a mech-anism of in situ detoxification of furfural and 5-hydroxymethylfurfural bySaccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 81:743–753.

41. Liu, Z. L., and P. J. Slininger. 2005. Development of genetically engineeredstress tolerant ethanologenic yeasts using integrated functional genomics foreffective biomass conversion to ethanol, p. 283–294. In J. Outlaw, K. Collins,and J. Duffield (ed.), Agriculture as a producer and consumer of energy.CAB International, Wallingford, United Kingdom.

42. Liu, Z. L., and P. J. Slininger. 2006. Transcriptome dynamics of ethanolo-genic yeast in response to 5-hydroxymethylfurfural stress related to biomassconversion to ethanol, p. 679–684. In A. Marzal, M. I. Reviriego, C. Navarro,F. de Lope, and A. P. Møller (ed.), Recent research developments in mul-tidisciplinary applied microbiology, understanding and exploiting microbesand their interactions—biological, physical, chemical and engineering as-pects. Wiley, New York, NY.

43. Liu, Z. L., P. J. Slininger, B. S. Dien, M. A. Berhow, C. P. Kurtzman, andS. W. Gorsich. 2004. Adaptive response of yeasts to furfural and 5-hydroxy-methylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethlfuran. J. Ind. Microbiol. Biotechnol. 31:345–352.

44. Luo, B., K. Groenke, R. Takors, C. Wandrey, and M. Oldiges. 2007. Simul-taneous determination of multiple intracellular metabolites in glycolysis,pentose phosphate pathway and tricarboxylic acid cycle by liquid chroma-tography-mass spectrometry. J. Chromatogr. A 1147:153–164.

45. Miyagi, M., and K. C. S. Rao. 2007. Proteolytic O-18-labeling strategies forquantitative proteomics. Mass Spectrom. Rev. 26:121–136.

46. Modig, T., G. Liden, and M. J. Taherzadeh. 2002. Inhibition effects offurfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvatedehydrogenase. Biochem. J. 363:769–776.

47. Morimoto, T. H. S., and N. Matutani. 1969. Studies on fermentation productfrom furfural by yeast. III. Identification of furoin and furil. J. Ferment.Technol. 47:486–490.

48. Palmqvist, E., J. S. Almeida, and B. Hahn-Hagerdal. 1999. Influence offurfural on anaerobic glycolytic kinetics of Saccharomyces cerevisiae in batchculture. Biotechnol. Bioeng. 62:447–454.

49. Palmqvist, E., H. Grage, N. Q. Meinander, and B. Hahn-Hagerdal. 1999.Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoicacid on growth and ethanol productivity of yeasts. Biotechnol. Bioeng. 63:46–55.

50. Palmqvist, E., and B. Hahn-Hagerdal. 2000. Fermentation of lignocellulosichydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Tech-nol. 74:25–33.

51. Petersson, A., J. R. M. Almeida, T. Modig, K. Karhumaa, B. Hahn-Hagerdal,M. F. Gorwa-Grauslund, and G. Liden. 2006. A 5-hydroxymethyl furfuralreducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene con-veys HMF tolerance. Yeast 23:455–464.

52. Petit, P. X., M. C. Gendron, N. Schrantz, D. Metivier, G. Kroemer, Z.Maciorowska, F. Sureau, and S. Koester. 2001. Oxidation of pyridine nucle-otides during Fas- and ceramide-induced apoptosis in Jurkat cells: correla-tion with changes in mitochondria, glutathione depletion, intracellular acid-ification and caspase 3 activation. Biochem. J. 353:357–367.

53. Pruyne, D., and A. Bretscher. 2000. Polarization of cell growth in yeast. II.The role of the cortical actin cytoskeleton. J. Cell Sci. 113:571–585.

54. Qadota, H., C. P. Python, S. B. Inoue, M. Arisawa, Y. Anraku, Y. Zheng, T.Watanabe, D. E. Levin, and Y. Ohya. 1996. Identification of yeast Rho1pGTPase as a regulatory subunit of 1,3-beta-glucan synthase. Science 272:279–281.

55. Qian, W. J., M. E. Monroe, T. Liu, J. M. Jacobs, G. A. Anderson, Y. F. Shen,R. J. Moore, D. J. Anderson, R. Zhang, S. E. Calvano, S. F. Lowry, W. Z.Xiao, L. L. Moldawer, R. W. Davis, R. G. Tompkins, D. G. Camp, R. D.Smith, and the Inflammation and the Host Response to Injury Large ScaleCollaborative Research Program. 2005. Quantitative proteome analysis ofhuman plasma following in vivo lipopolysaccharide administration usingO-16/O-18 labeling and the accurate mass and time tag approach. Mol. Cell.Proteomics 4:700–709.

56. Ramos-Fernandez, A., D. Lopez-Ferrer, and J. Vazquez. 2007. Improved

method for differential expression proteomics using trypsin-catalyzed O-18labeling with a correction for labeling efficiency. Mol. Cell. Proteomics6:1274–1286.

57. Smith, M. G., S. G. Des Etages, and M. Snyder. 2004. Microbial synergy viaan ethanol-triggered pathway. Mol. Cell. Biol. 24:3874–3884.

58. Songi, M. Z., and Z. L. Liu. 2007. A linear discrete dynamic system model fortemporal gene interaction and regulatory network influence in response tobioethanol conversion inhibitor HMF for ethanologenic yeast. Syst. Biol.Comput. Proteomics 4532:77–95.

59. Stanley, G. A., T. J. Hobley, and N. B. Pamment. 1997. Effect of acetaldehydeon Saccharomyces cerevisiae and Zymomonas mobilis subjected to environ-mental shocks. Biotechnol. Bioeng. 53:71–78.

60. Sussman, A., K. Huss, L. C. Chio, S. Heidler, M. Shaw, D. Ma, G. X. Zhu,R. M. Campbell, T. S. Park, P. Kulanthaivel, J. E. Scott, J. W. Carpenter,M. A. Strege, M. D. Belvo, J. R. Swartling, A. Fischl, W. K. Yeh, C. Shih, andX. S. Ye. 2004. Discovery of cercosporamide, a known antifungal naturalproduct, as a selective Pkc1 kinase inhibitor through high-throughput screen-ing. Eukaryot. Cell 3:932–943.

61. Taherzadeh, M. J., L. Gustafsson, C. Niklasson, and G. Liden. 1999. Con-version of furfural in aerobic and anaerobic batch fermentation of glucose bySaccharomyces cerevisiae. J. Biosci. Bioeng. 87:169–174.

62. U.S. Department of Energy. 2006. Breaking the biological barriers to cellu-losic ethanol: a joint research agenda. DOE/SC-0095. U.S. Department ofEnergy Office of Science and Office of Energy Efficiency and RenewableEnergy Efficiency and Renewable Energy, Washington, DC.

63. Van Dyke, M. W., L. D. Nelson, R. G. Weilbaecher, and D. V. Mehta. 2004.Stm1p, a G(4) quadruplex and purine motif triplex nucleic acid-bindingprotein, interacts with ribosomes and subtelomeric Y DNA in Saccharomy-ces cerevisiae. J. Biol. Chem. 279:24323–24333.

64. Villa, G. P., R. Bartroli, R. Lopez, M. Guerra, M. Enrique, M. Penas, E.Rodríquez, D. Redondo, I. Iglesias, and M. Díaz. 1992. Microbial transfor-mation of furfural to furfuryl alcohol by Saccharomyces cerevisiae. ActaBiotechnol. 12:509–512.

65. von Haller, P. D., E. Yi, S. Donohoe, K. Vaughn, A. Keller, A. I. Nesvizhskii,J. Eng, X. J. Li, D. R. Goodlett, R. Aebersold, and J. D. Watts. 2003. Theapplication of new software tools to quantitative protein profiling via iso-tope-coded affinity tag (ICAT) and tandem mass spectrometry—II. Evalua-tion of tandem mass spectrometry methodologies for large-scale proteinanalysis, and the application of statistical tools for data analysis and inter-pretation. Mol. Cell. Proteomics 2:428–442.

66. Wang, Y. X., and Y. J. Yuan. 2005. Direct proteomic mapping of Streptomycesluteogriseus strain 103 and cnn1 and insights into antibiotic biosynthesis. J.Proteome Res. 4:1999–2006.

67. Weigert, B., K. Klein, M. Rizzi, C. Lauterbach, and H. Dellweg. 1988.Influence of furfural on the aerobic growth of the yeast Pichia stipitis. Bio-technol. Lett. 10:895–900.

68. Xia, J. M., F. M. Lin, and Y. J. Yuan. 2007. Systematic analysis of yeastresponse to inhibitors. Prog. Chem. 19:1159–1163.

69. Xia, J. M., and Y. J. Yuan. 2009. Comparative lipidomics of four strains ofSaccharomyces cerevisiae reveals different responses to furfural, phenol, andacetic acids. J. Agric. Food Chem. 57:99–108.

70. Yao, X. D., A. Freas, J. Ramirez, P. A. Demirev, and C. Fenselau. 2001.Proteolytic O-18 labeling for comparative proteomics: model studies withtwo serotypes of adenovirus. Anal. Chem. 73:2836–2842. (Erratum, 73:2675.)

71. Ying, W. H. 2008. NAD�/ NADH and NADP�/NADPH in cellular functionsand cell death: regulation and biological consequences. Antioxid. RedoxSignal. 10:179–206.

72. Zhang, W., D. L. Needham, M. Coffin, A. Rooker, P. Hurban, M. M. Tanzer,and J. R. Shuster. 2003. Microarray analyses of the metabolic responses ofSaccharomyces cerevisiae to organic solvent dimethyl sulfoxide. J. Ind. Mi-crobiol. Biotechnol. 30:57–69.

73. Zhang, X. P., R. L. Lester, and R. C. Dickson. 2004. Pil1p and Lsp1pnegatively regulate the 3-phosphoinositide-dependent protein kinase-likekinase Pkh1p and downstream signaling pathways Pkc1p and Ypk1p. J. Biol.Chem. 279:22030–22038.



http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/