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Energetic and metabolic transient response ofSaccharomyces cerevisiae to benzoic acidM. T. A. P. Kresnowati*, W. A. van Winden, W. M. van Gulik and J. J. HeijnenDepartment of Biotechnology, Delft University of Technology, The NetherlandsBenzoic acid is important for the food industries.Along with other weak acids such as sulfite and sulfurdioxide, sorbic acid, acetic acid, propionic acid andlactic acid, benzoic acid is used on a large scale as afood preservative, preventing microbial spoilage infoods and beverages.The optimum condition for this type of preservativesis a low pH. In acidic media, particularly at pH valueslower than the pKa(the dissociation constant) of theweak acid, the acid is present mostly in its non-dissoci-ated form, which is able to permeate cell membranes.Because of the high intracellular pH (6.4–7.5) [1–5],the intruding non-dissociated acid will dissociate intoits anion with the release of a proton. This results inintracellular acidification [6] which affects the homeo-stasis of metabolism such that substantial energy isrequired to overcome acidification by actively pumpingout protons. This energy-consuming process leads to adecrease in biomass yield, as observed previously [7].At sufficiently high concentrations, benzoate has beenreported to inhibit glycolysis [6,8,9] leading to a cessa-tion of growth. Furthermore, it is also reported tocause oxidative stress in aerobically cultivated yeast[10].However, some yeasts such as Saccharomyces cerevi-siae and Zygosaccharomyces bailii, both of which areknown to be important food spoilage yeasts, are ableto adapt to the presence of these weak acids with alarge energy expenditure and hence are able to increasetheir tolerance to these weak acids up to a certainKeywordsadaptation; benzoic acid; chemostat;transient; yeastCorrespondenceJ. J. Heijnen, Department of Biotechnology,Delft University of Technology, Julianalaan67, 2628 BC Delft, The NetherlandsFax: +31 15 278 2355Tel: +31 15 278 2342E-mail: j.j.heijnen@tudelft.nl*Present addressMicrobiology and Bioprocess TechnologyLaboratory, Department of Chemical Engi-neering, Bandung Institute of Technology,Indonesia(Received 2 January 2008, revised 29August 2008, accepted 4 September 2008)doi:10.1111/j.1742-4658.2008.06667.xSaccharomyces cerevisiae is known to be able to adapt to the presence ofthe commonly used food preservative benzoic acid with a large energyexpenditure. Some mechanisms for the adaptation process have been sug-gested, but its quantitative energetic and metabolic aspects have rarely beendiscussed. This study discusses use of the stimulus response approach toquantitatively study the energetic and metabolic aspects of the transientadaptation of S. cerevisiae to a shift in benzoic acid concentration, from 0to 0.8 mm. The information obtained also serves as the basis for furtherutilization of benzoic acid as a tool for targeted perturbation of the energysystem, which is important in studying the kinetics and regulation of cen-tral carbon metabolism in S. cerevisiae. Using this experimental set-up, wefound significant fast-transient (< 3000 s) increases in O2consumptionand CO2production rates, of $ 50%, which reflect a high energy require-ment for the adaptation process. We also found that with a longer expo-sure time to benzoic acid, S. cerevisiae decreases the cell membranepermeability for this weak acid by a factor of 10 and decreases the cell sizeto $ 80% of the initial value. The intracellular metabolite profile in thenew steady-state indicates increases in the glycolytic and tricarboxylic acidcycle fluxes, which are in agreement with the observed increases in specificglucose and O2uptake rates.AbbreviationsCER, CO2production rate; OUR, O2uptake rate.FEBS Journal 275 (2008) 5527–5541 ª 2008 The Authors Journal compilation ª 2008 FEBS 5527concentration. This implies that, in order to signifi-cantly inhibit the growth of these yeasts, a high doseof weak acids would be required for food preservation,whereas a low maximum concentration is permitted.It has been reported that these yeasts adapt to thepresence of weak acids by inducing an ATP-bindingcassette transporter, Pdr12, to actively expel the accu-mulated ‘dissociated’ weak acids [11,12], and by adapt-ing membrane permeability to these acids [13]. Thisreduces the passive diffusion of non-dissociated acid,limits the influx of these weak acids and reduces theireffects on cell metabolism. An overview of these adap-tation mechanisms is shown in Fig. 1.The fact that the presence of benzoic acid introducesan independent ATP drain in cell metabolism may alsobe of interest to those studying the regulation of cellenergetics and metabolism. It offers the possibility toperturb, in a targeted way, the ATP pool, which isimportant in the in vivo kinetic evaluation of centralcarbon metabolism. However, to be able to performthis kind of experiment, quantitative information onthe effect of benzoic acid on cell energetics and metab-olism is required.Although some mechanisms for the adaptation tobenzoic acid have been suggested, little quantitativedata on this mechanism have been presented. More-over, studies have mostly been performed in shakeflask cultures [13,14], where the environment cannot betightly controlled or monitored. Thus, changesobserved in the metabolism may be caused by changesin multiple experimental parameters which complicateinterpretation of the results. Also steady-state chemo-stat studies have been performed to examine the ener-getic aspects of growth in the presence of benzoic acid[7,15]. However, adaptation is best revealed by a tran-sient study.This study presents the combined use of a well-defined, tightly controlled aerobic, glucose-limitedchemostat system and the application of a stimulus–response approach to quantitatively study the tran-sient adaptation of S. cerevisiae to benzoic acid. Anaerobic glucose-limited steady-state chemostat cultureof S. cerevisiae was suddenly exposed to a certainextracellular benzoic acid concentration (a stepchange perturbation from 0 to 0.8 mm benzoic acid,at pH 4.5) after which the transient response of theculture was monitored. The analysis focuses on thequantitative energetic aspects of the transient adapta-tion, to reveal metabolic regulation and the perturba-tion of the central carbon metabolism. To completethe analysis, fermentation characteristics and intra-cellular metabolite distributions in the two steady-state conditions, with and without benzoic acid, werealso compared.TheoryBenzoic acid transport modelIn solution, benzoic acid attains a pH-dependent equi-librium between the non-dissociated and dissociatedforms,AB CFig. 1. The general response of S. cerevisiae to benzoic acid. (A) Benzoic acid enters cell via passive diffusion, the released proton isexpulsed by an energy-consuming H+-ATPase (Pma1), whereas the dissociated benzoic acid may still introduce some toxicity; (B) inductionof ATP-binding cassette transporter Pdr12 to actively expel benzoate, the expulsion of benzoate causes a futile cycle of benzoic acid diffu-sion and subsequent active export; (C) changes in membrane characteristics to limit the influx of benzoic acid into the cell.Transient response to benzoic acid M. T. A. P. Kresnowati et al.5528 FEBS Journal 275 (2008) 5527–5541 ª 2008 The Authors Journal compilation ª 2008 FEBSHB Ð Hþþ BÀK ¼CHþCBÀCHBð1Þin which K is the benzoic acid dissociation constant,HB is the non-dissociated form of the acid and B)isthe dissociated form (benzoate). Thus, for a certain mea-surable total benzoate concentration (CB¼ CBÀþ CHB),the fraction of the non-dissociated (protonated) statecan be calculated asfHB¼11 þðK=CHþÞð2ÞCell membranes are normally permeable to the non-dissociated form of relatively apolar weak acids, there-fore such molecules can passively diffuse through cellmembranes. By assuming that benzoic acid is trans-ported by passive diffusion only, which holds when thebenzoate exporter is not induced [1], the uptake rate ofbenzoic acid (qHB; molÆkgDW)1Æs)1) can be modeledas:qHB¼ k6VxdxðCHBexÀ CHBinÞð3Þin which k (mÆs)1) is the membrane permeability coeffi-cient for benzoic acid, CHBexand CHBin(molÆm)3) arethe extracellular and intracellular non-dissociated ben-zoic acid concentration, Vx(m3ÆkgDW)1) is the cellvolume per gram dry weight of biomass and dx(m) isthe cell diameter. The term (6 · Vx⁄ dx) actually consti-tutes the specific surface area of the cell (AX;m2ÆkgDW)1). The values used in the calculation aredx=5· 10)6m [16], Vx=2· 10)3m3ÆkgDW)1andk = 0.92 · 10)5mÆs)1[1].At a steady-state and in the absence of an activeexporter, the intracellular non-dissociated benzoicacid is in equilibrium with the extracellular non-dissociated benzoic acid and thus their concentra-tions are equal. Hence, following the dissociationequation (Eqn 1) the ratio of total intracellular tototal extracellular benzoate concentration reflectsthe difference in the intracellular and extracellularpH asCBinCBex¼10pHinÀpKþ 110pHexÀpKðÞþ 1ð4ÞIt is known that benzoic acid is not metabolized byyeast cells [1,17]. Under this condition, the accumu-lation of total benzoate inside the cells (CBin) can becalculated from the total benzoate mass balance. Con-sidering that the fraction of the total cell volume isnegligible compared with the total broth volume,CxÆVx> V, the total concentration of intracellularbenzoate can be calculated asCBin¼CB0À CBexCxVxð5Þin which CB0is the initial total benzoate concentrationin the medium (CB0). By combining Eqns (4,5) we cancalculate the intracellular pH (pHin) from theadded ⁄ initial total benzoate in the medium, the mea-sured extracellular total benzoate concentration, thebiomass concentration and the extracellular pH (pHex)CB0À CBexCxVxCBex¼10ðpHinÀpKaÞþ 110ðpHexÀpKaÞþ 1ð6ÞIn the presence of a benzoate exporter, such asPdr12, intracellular benzoate is actively exported, andthis process consumes energy. This leads to an increasein the extracellular total benzoate concentration, adecrease in the intracellular total benzoate concentra-tion and additional O2consumption. To maintain theintracellular charge balance, a proton is activelyco-transported. Assuming that 1 ATP is consumed forthe export of each of these species, the defined P ⁄ Oratio = 1.46 [17], and all benzoic acid which entersthe cell via passive diffusion is exported, the influx ofbenzoic acid via passive diffusion can be related to theadditional O2consumption (OUR – OUR0) as:OUR À OUR0ÀÁÂ 2P=O ¼ 2qHBCxVLð7ÞHere OUR0is the O2consumption rate (molÆs)1)inthe absence of benzoate. Equation 7 shows that theexport of 1 mol of benzoate leads to an extra O2con-sumption of 1 ⁄ (P ⁄ O) = 0.68 mol. If the exporter wereto export benzoic acid instead of the benzoate anion,which does not lead to an intracellular charge imbal-ance, the export would lead to 0.34 mol of additionalO2consumption per mol of benzoate.ResultsThe benzoic acid shift experiment was performed usingan abrupt change in the total benzoate concentrationin the fermentor from 0 to 0.8 mm at a constant pH of4.5. The steady-state characteristics of the fermentationprior to the shift experiment are shown in Table 1. Itwas calculated that the carbon and degree of reductionbalances agree closely, with 97.6% carbon recoveryand 96.1% degree of reduction recovery.Transient responses in the culture to the shift in ben-zoic acid concentration were followed in terms ofextracellular metabolite concentrations, dissolved O2and CO2concentrations, off-gas O2and CO2concen-trations, biomass concentration (CX) and cell morphol-ogy. Thereafter, a new steady-state was reached,characterized by a significantly lower biomass concen-M. T. A. P. Kresnowati et al. Transient response to benzoic acidFEBS Journal 275 (2008) 5527–5541 ª 2008 The Authors Journal compilation ª 2008 FEBS 5529tration and significantly higher specific rates of glucoseand O2consumption (Table 1).Transient benzoic acid profileWithin 20 s of the shift in the benzoic acid concentra-tion, the total extracellular benzoate concentrationdecreased to 250 lm, which is 30% of the added con-centration in the medium (Fig. 2A). After $ 1 h, theextracellular total benzoate concentration slowly startsto increase and reaches a stable-steady concentrationof $ 650 lm, which is 80% of the added total benzo-ate in the feed medium. This steady-state is reached24–30 h after the start of the transient response.Transient O2and CO2profilesO2and CO2concentrations (Fig. 2B,C) responddynamically to the shift in benzoic acid concentration.Shortly after the shift, the O2concentrations in boththe liquid and gas phases decrease rapidly. After aminimum value is reached, within 1000 s of the benzo-ate shift, the O2concentrations in both phases arerestored, overshoot and then slowly stabilize. The newsteady-state condition, however, is only achieved$ 30 h after the shift. Opposing transient profiles areobserved for the CO2concentrations (Fig. 2C).Transient extracellular metabolite profilesConsistent with the carbon-limited condition for thechemostat culture, the residual glucose concentrationremains low after the shift in the benzoic acid concen-tration. Within 1000 s of the shift, the ethanol concen-tration increases from a very low residualconcentration of < 5–15 mgÆL)1(Fig. 2D) and isshortly followed by an increase in the acetic acid con-centration to 10 mgÆL)1(Fig. 2E). After 1000 s theseconcentrations return to the steady-state valuesmeasured before the shift and remain low.Transient cell morphologyWe also observed changes in cell morphology followingthe shift in benzoic acid concentration in the medium(Table 2). Cell-image analysis of broth samples takenduring the transient condition at 18.7, 48.4 and 72.1 hfollowing the shift in benzoic acid concentration indi-cates a negative trend in the cell-equivalent diameter,albeit not statistically significant due to the large stan-dard deviation. Moreover, the cells elongate. The lattercan be inferred from the increase in the cell roundnessindex (the roundness is defined as the perimeter2⁄[4 · p · area], and the roundness of a circle = 1) andthe increase in the cell aspect ratio index (the cellaspect ratio is defined as the ratio between the twoaxial diameters of the object, the aspect ratio of acircle = 1) (Fig. 3).Transient O2uptake, CO2production andbiomass production ratesThe observed rapid decrease in O2concentration inboth the gas and liquid phases and the rapid increasein CO2concentration in both phases following theshift in benzoic acid concentration reflect a rapidincrease in both the O2uptake rate (OUR) and theCO2production rate (CER) (Fig. 4A,B).The maximum increase in the OUR calculated fromthe liquid-phase mass balance is 1.5-fold (from 80 to120 mmolÆh)1), whereas a 1.8-fold increase (from 80 to146 mmolÆh)1) is calculated from the combined liquid-and gas-phase balances. As discussed in Experimentalprocedures, the OUR calculated from the liquid-phasemass balance is a better description of the fastdynamic condition. Virtually the same dynamic patternis obtained for CER, which also increases 1.8-foldcompared with the steady-state value, within 600 s ofthe shift. Thereafter both OUR and CER slowlydecrease to close to their previous steady-state values.However, from $ 3000 s after the shift, the OUR andCER are observed to slowly increase again. At the endof the observation window, $ 72 h after the start ofthe transient, new steady values of 117 mmolÆh)1forboth OUR and CER (i.e. a 1.5-fold increase comparedwith the initial steady-state values) are calculated.During the observation the respiration quotient (RQ)is always close to 1.Long-term OUR and CER profiles indicate a signifi-cant decrease in the biomass production rate(rX) (Fig. 4C), such that at the new steady-state theTable 1. Characterization of steady-state fermentation prior to andafter the shift in benzoic acid concentration. CX, biomass concentra-tion; l, specific growth rate; qO2, specific O2consumption rate;qCO2, specific CO2production rate, qS, specific glucose consump-tion rate.Benzoic acid concentrationin the medium (mM) 0 0.8Fermentation characteristicsBiomass concentration (kgDWÆm)3) 14.09 ± 0.17 7.81l (h)1) 0.05 0.05qO2(mmolÆgDW)1Æh)1) 1.46 ± 0.06 3.76qCO2(mmolÆkgDW)1Æh)1) 1.45 ± 0.04 3.72qSglucose (mmolÆgDW)1Æh)1) 0.53 ± 0.01 0.96Transient response to benzoic acid M. T. A. P. Kresnowati et al.5530 FEBS Journal 275 (2008) 5527–5541 ª 2008 The Authors Journal compilation ª 2008 FEBSbiomass production rate is calculated to be $ 65% ofthe initial steady-state value (110–170 mmolÆXÆh)1).Accordingly, the calculated biomass concentration hasdecreased from 14.9 to 9.6 kgDWÆ m)3(Fig. 4D). Thisis confirmed by the measured biomass concentrations(Fig. 4D) which decrease by 10% from 14.1 to12.7 kgDWÆm)3within 5.3 h of the shift and by 55%,i.e. to 7.8 kgDWÆm)3, at 72 h after the shift, when theexperiment was finished. The calculated biomassconcentrations are 4–25% higher than the measuredvalues. However, it should be realized that the calcu-lated recoveries of carbon and the degree of reductionduring the transient, using Eqns (14,15) and the experi-mental data of the biomass concentrations, OUR andCER are found to deviate respectively by 5–11 andABCDEFig. 2. Transient responses to the shift in benzoic acid concentration (the timing of the shift is marked by a dashed vertical line). (A) Benzoicacid profile, (B) O2profiles in the liquid (gray solid line) and gas phase (black dashed line), (C) CO2profiles in the liquid (gray solid line) andgas phase (black dashed line), (D) ethanol concentration profile, (E) acetic acid concentration profile.Table 2. Response in cell morphology following the shift in benzoicacid concentration in the medium.Age (h)Equivalentdiametera(lm) RoundnessbAspectratiocSamplenumber0 4.94 ± 1.30 1.12 ± 0.11 1.25 ± 0.18 61518.7 4.31 ± 1.24 1.14 ± 0.11 1.32 ± 0.21 47448.4 4.38 ± 0.96 1.15 ± 0.12 1.36 ± 0.24 118072.1 4.06 ± 0.91 1.15 ± 0.11 1.47 ± 0.29 911aEquivalent diameter is the diameter of the cell if the cell isassumed to be spherical.bRoundness measures the shape of theobject, it is defined as (perimeter2· 1000) ⁄ (4 · p · area). Theroundness of a circle = 1.cAspect ratio gives the ratio betweenthe two axes of the object. The aspect ratio of a circle is similar tothe aspect ratio of a square = 1.M. T. A. P. Kresnowati et al. Transient response to benzoic acidFEBS Journal 275 (2008) 5527–5541 ª 2008 The Authors Journal compilation ª 2008 FEBS 55315–28%. Furthermore, the observed changes in cellmorphology and adaptation to benzoic acid may alsochange cell structure and composition. Hence theassumption of constant biomass molecular mass maynot have been valid and may have introduced errors inthe calculated biomass concentration. If this is thecase, the discrepancy in the total carbon balance indi-cates up to 25% deviation in the cell molecular mass,which is highly unlikely. Another possible source ofthe discrepancy in the total carbon and degree ofreduction balance is byproduct formation. However,the biomass production rates (rX) calculated from bothcarbon and degree of reduction balances agree whichdoes not point to significant byproduct formation. Thisleaves us the possibility of systematic measurementerrors, particularly during the transient.During the entire observation period of 72 h afterthe shift in benzoic acid concentration, the increase inOUR and the decrease in biomass concentration in thechemostat result in a strong and steady increase in thebiomass specific O2consumption rate (qO2) (seeFig. 4E), reaching a final value which is 2.2-fold higherthan the initial steady-state value. During the first hourafter the shift, the biomass concentration does notchange significantly and the therefore the qO2profile issimilar to the OUR profile.The final steady-state increase in the specific O2andglucose consumption rates found in this experimentare comparable with the increase in specific O2andglucose consumption rates between the chemostatABFig. 3. Microscopic cell image of S. cerevisiae (A) before and (B)after (72.1 h) the addition of benzoic acid to the culture.ABCDEFig. 4. Transient responses to the shift in benzoic acid concentra-tion (the timing of the shift is marked by a dashed vertical line). (A)O2consumption rate (OUR; the gray curve represents the short-term transient response OUR calculated from the liquid-phase bal-ance only), (B) CO2production rate (CER), (C) calculated biomassproduction rate (rX), (D) calculated and measured biomass concen-trations (CX), black circles represent the measured values; for (C)and (D) both the calculated values from the total carbon balance(black lines) and from the degree of reduction balance (gray lines)are shown. (E) Specific O2consumption (qO2, steady-state value isindicated by a gray dashed line).Transient response to benzoic acid M. T. A. P. Kresnowati et al.5532 FEBS Journal 275 (2008) 5527–5541 ª 2008 The Authors Journal compilation ª 2008 FEBSculture without benzoic acid and the chemostat culturewith a residual total benzoate concentration of 2 mm[7]. Although the total benzoate concentration in thelatter experiment is higher than in this study, thoseexperiments were performed at an extracellular ofpH 5.0, at which the non-dissociated benzoic acid frac-tion is lower than at pH 4.5 as in the present study.The non-dissociated benzoic acid concentration whichcorresponds to this condition is 0.27 mm, only 25%higher than the non-dissociated benzoic acid concen-tration of 0.21 mm in the present study with a totalbenzoate concentration of 0.64 mm at an extracellularpH of 4.5. In our experiments (l = 0.05 h)1) themeasured specific O2consumption increases from 1.46to 3.76 mmolÆh)1(2.6-fold), whereas in Verduyn’sexperiments (at l = 0.1 h)1) it increases from 2.5 to6 mmolÆh)1(2.4-fold).Steady-state intracellular metabolite profilesIntracellular concentrations of the intermediates of theglycolytic, tricarboxylic acid cycle, and pentose phos-phate pathway, as well as storage carbohydrate andadenine nucleotides, were measured during the twosteady-state conditions, with and without benzoic acidin the feed medium (Table 3). The values presented areaverages of six independent samples, each of whichwas measured in duplicate. The calculated standarddeviations of $ 5% indicate the quality of the sample-processing method and the analysis.In the presence of benzoic acid, we observed signifi-cantly lower amounts of ATP, ADP and AMP whichlead to a slightly higher energy charge level, respec-tively 0.87 ± 0.004 and 0.85 ± 0.005 with and with-out benzoic acid (Table 3). This is remarkableconsidering the much higher ATP fluxes due to thehigher specific O2consumption in the presence ofbenzoate.For the glycolytic intermediates, we observed thatthe presence of benzoic acid leads to increased levelsof fructose 1,6 bisphosphate (twofold) and glyc-erol 3-phosphate (fivefold), as well as decreased levelsof the phospho-enol-pyruvate and 2-phosphoglycer-ate + 3-phosphoglycerate pools, respectively, to 65and 75% of their concentration in the absence ofbenzoic acid (Table 3).One striking difference between the two steady-statesis that the concentrations of the weak acids in thetricarboxylic acid cycle (pyruvate, citrate, a-ketogluta-rate, succinate, fumarate and malate) in the presenceof benzoic acid are all significantly higher (1.4–9.9-fold) than those concentrations without the presenceof benzoic acid (Table 3).DiscussionTo study the transient behavior following the shift inbenzoic acid concentration further, the analysisfocused on two different time windows: short-termresponses (0–3000 s) and long-term responses(> 3000 s). To complete the overview, comparisonbetween the two steady-state conditions, with andwithout benzoic acid is presented first.Steady-state comparison with and withoutbenzoic acid – increase in catabolismComparison between the steady-state fermentationcharacteristics in the presence or the absence ofbenzoic acid shows that in general the presence ofbenzoic acid results in higher specific O2consumptionand glucose uptake rates, as well as a decrease inthe biomass concentration. These observations areTable 3. Intracellular metabolite concentrations measured duringthe steady-state without and with 0.8 mM benzoic acid in the medi-um, values are presented in lmolÆgDW)1, except for the energycharge and adenylate kinase mass action ratio which are dimen-sionless. The values are an average of six independent samples.Benzoic acid concentrationin the medium (mM) 0 0.8ATP 7.94 ± 0.30 6.61 ± 0.23ADP 1.74 ± 0.03 1.35 ± 0.03AMP 0.64 ± 0.02 0.37 ± 0.02SAXP 10.32 ± 0.31 8.33 ± 0.24Adenylate kinasemass action ratioa0.59 ± 0.03 0.74 ± 0.06Energy chargeb0.85 ± 0.00 0.87 ± 0.00Glucose 6-phosphate 1.74 ± 0.08 1.59 ± 0.08Fructose 6-phosphate 0.27 ± 0.01 0.25 ± 0.026-Phosphogluconate 0.20 ± 0.01 0.29 ± 0.02Glucose 1-phosphate 0.29 ± 0.01 0.34 ± 0.02Mannose 6-phosphate 0.70 ± 0.02 0.71 ± 0.05Trehalose 6-phosphate 0.22 ± 0.01 0.19 ± 0.00Fructose 1,6-bisphosphate 0.16 ± 0.00 0.31 ± 0.01Phosphoenolpyruvate 0.66 ± 0.02 0.43 ± 0.032-Phosphoglycerate ⁄3-phosphoglycerate0.81 ± 0.03 0.61 ± 0.03Glucose 3-phosphate 0.01 ± 0.00 0.05 ± 0.00Glyoxylate 0.01 ± 0.00 0.04 ± 0.00Pyruvate 0.09 ± 0.01 0.24 ± 0.01Citrate 5.26 ± 0.16 7.26 ± 0.36a-ketoglutarate 0.06 ± 0.00 0.25 ± 0.01Succinate 0.04 ± 0.01 0.34 ± 0.02Fumarate 0.04 ± 0.00 0.39 ± 0.02Malate 0.21 ± 0.01 2.02 ± 0.10aAdenylate kinase mass action ratio = (ADP)2⁄ (ATPÆAMP).bEnergycharge = (ATP + 0.5 ADP) ⁄ (ATP + ADP + AMP).M. T. A. P. Kresnowati et al. Transient response to benzoic acidFEBS Journal 275 (2008) 5527–5541 ª 2008 The Authors Journal compilation ª 2008 FEBS 5533supported by intracellular metabolite measurements.The observed patterns of the glycolytic intermediates,i.e. a higher level of fructose 1,6-biphosphate andlower levels of phospho-enol-pyruvate and the 2-phos-phoglycerate + 3-phosphoglycerate pool in the pres-ence of benzoic acid compared with in the absence ofbenzoic acid (Table 3), are also commonly observedas a response to a glucose pulse [18–20] and indicatean increase in glycolytic flux in the presence ofbenzoic acid. The increase in glycolytic flux is consis-tent with the calculated increase in the specificglucose uptake rate (Table 1). Interestingly, the pres-ence of benzoic acid also leads to a higher level ofglycerol 3-phosphate (fivefold), which may indicate ahigher cytosolic NADH ⁄ NAD ratio. The higherNADH ⁄ NAD ratio is verified by calculation of thisratio from the lumped reactions of aldolase, triosephosphate isomerase, glyceraldehydes-3-phosphatedehydrogenase, phosphoglycerate kinase and phospho-glycerate mutase, which gives a 1.7-fold increase inthe NADH ⁄ NAD ratio in the presence of benzoicacid. The higher NADH ⁄ NAD ratio is consistentwith higher glycolytic flux and also the higher specificO2consumption rate, which is probably stimulated bythe higher NADH ⁄ NAD ratio. By contrast, theobserved higher concentrations of the weak acids inthe tricarboxylic acid cycle in the presence of benzoicacid reflect the much higher tricarboxylic acid cycleflux.Overall, intracellular metabolite profiles show that inthe presence of benzoic acid cells accelerate theircatabolism to generate more energy to overcome theATP drain for exporting benzoate and protons. It con-firms the black box energetic observations of theincreased specific O2consumption and glucose uptakerates.Transient benzoic acid profile indicates thetiming of benzoic acid transporter inductionFermentation was started without benzoic acid in themedium. In this condition, we expect that the benzoatetransporter, such as Pdr12p, is absent and benzoic acidwill be in equilibrium inside and outside the cellfollowing the intracellular and extracellular pHdifference, as described in Eqn (4). Accordingly, intra-cellular pH can be calculated from the transient totalbenzoate profile. Within the first 3000 s following theshift in the benzoic acid concentration intracellular pHis calculated to be 6.44–6.65. This is in agreement withthe reference value of steady-state intracellular pH forthis yeast species [1], which shows that, within thistime window, the benzoate transporter is not presentand only equilibration by passive diffusion occurs.In the longer term, > 1 h following the shift, weobserve that the extracellular total benzoate concentra-tion increases (Fig. 5A). Accordingly, the intracellulartotal benzoate concentration, which is calculated fromthe measured extracellular total benzoate concentra-tion, decreases (Fig. 5B). This may be explained by adecrease in intracellular pH, which shifts the distribu-tion of benzoic acid towards the extracellular compart-ment. However, considering the tightly controlled pHhomeostasis, it is not likely that cells permanentlylower their intracellular pH. Because the decrease inintracellular total benzoate concentration coincideswith an increase in the O2uptake rate (Fig. 4A), it ismore likely that this is caused by induction of the ben-zoate exporter. If this is the case, the time required toinduce the benzoate exporter observed in this studywould be $ 3000 s, which is much faster than the pre-viously reported value of 28 h [5] at which the extru-sion of benzoic acid became apparent. The observedABFig. 5. Benzoic acid concentration profile inresponse to the shift in the benzoic acidconcentration (the timing of the shift ismarked by a dashed vertical line). (A) Mea-sured extracellular total benzoate concentra-tion, (B) calculated intracellular totalbenzoate concentration.Transient response to benzoic acid M. T. A. P. Kresnowati et al.5534 FEBS Journal 275 (2008) 5527–5541 ª 2008 The Authors Journal compilation ª 2008 FEBScontinuous increase in extracellular total benzoate con-centration, from 3000 s to $ 24–30 h after the mediumshift, may indicate the slow completion of the induc-tion of this transporter.Long-term transient response following theshift in benzoic acid concentration – adaptationsin membrane properties and cell size to thepresence of benzoic acidIn order to study the adaptation of cells to benzoicacid, we use the transient O2consumption profile toreconstitute the dynamics in benzoic acid transport, assummarized in Fig. 6. By assuming that the increase inO2consumption is the result of additional ATP pro-duction needed to export protons and benzoate fromthe cells, and that all the benzoic acid entering the cellvia passive diffusion is exported back into the medium,the net influx of benzoic acid is reconstructed follow-ing Eqn (7). As a comparison, the total, fermentorscale, benzoic acid influx profile via passive diffusion(=qHBÆCXÆVL) is also calculated from the availableextracellular and intracellular benzoic acid concentra-tion profiles following Eqn (3), using the previouslydetermined membrane permeability value of benzoicacid for S. cerevisiae unadapted to benzoic acid,0.92 · 10)5mÆs)1[1] and by assuming that intracellularpH is constant at 6.5, which is the averaged intracellu-lar pH calculated during the short-term dynamics, asdiscussed previously.In Fig. 7 we show the calculation step by step.Figure 7A shows the driving force for the benzoic acidpassive diffusion (CHBexÀ CHBin). Figure 7B shows thetotal membrane surface area (=AXÆCXÆVL) availablefor the benzoic acid transport during the transientobservation based on the measured changes in the cellconcentration (Fig. 4D) and cell diameter (Table 2),and by assuming a constant biomass dry weight spe-cific volume (Vx=m3ÆkgDW)1) and that cells arespherical. Figure 7C shows the expected total benzoicacid influx via passive diffusion. Figure 7D shows theadditional O2consumption due to the addition ofbenzoic acid.Fig. 6. Benzoic acid and benzoate transport model, at pseudosteady-state condition the uptake rate of benzoic acid (qHB) and theexpulsion rate of benzoate (qB) are equal. k, membrane permeabil-ity coefficient for benzoic acid; CHBrmex, extracellular non-dissociatedbenzoic acid concentration; CHBin, intracellular non-dissociatedbenzoic acid concentration; Vx, cell volume per g dry weight ofbiomass; dx, cell diameter; OUR, O2consumption rate; OUR0,O2consumption rate in the absence of benzoate; CX, biomass con-centration; VL, liquid volume in the fermentor.Fig. 7. Modeling the long-term cellular response to benzoic acid.(A) Undissociated extracellular (solid line) and intracellular (dashedline) benzoic acid concentrations profile, (B) changes in total cellsurface area in the fermentor, (C) benzoic acid influx rate profile cal-culated via passive diffusion, (D) additional OUR profile, (E) appar-ent membrane permeability for benzoic acid.M. T. A. P. Kresnowati et al. Transient response to benzoic acidFEBS Journal 275 (2008) 5527–5541 ª 2008 The Authors Journal compilation ª 2008 FEBS 5535Figure 7C,D shows that the total benzoate influxescalculated using the two methods do not agree. Theratio between the calculated benzoate influx via passivediffusion and the additional O2consumption rate is$ 11 mol O2per mol benzoate exported, which ismuch higher than the expected value of 1.46 (seeEqn 7). This discrepancy is very likely caused bychanges in cell membrane properties, which arereflected by the change in membrane permeability forbenzoic acid. The apparent membrane permeabilityconstant of benzoic acid (Fig. 7E), which was calcu-lated from the measured additional O2consumption(Fig. 7D), transient total membrane surface area(Fig. 7B) and the driving force for the passive diffusionof benzoic acid (Fig. 7A), is much lower than the pre-viously reported value estimated from unadapted cells,and shows interesting dynamics, particularly within thefirst 20 h ($ 1 generation time) of the transientresponse. It is remarkable that such a decrease inmembrane permeability is achieved only within 1 gen-eration time and points to the associated genetic regu-lation of the synthesis of membrane molecules, suchthat the membrane composition of the adapted cell isless permeable for benzoic acid. This calls for an anal-ysis of the transcript distribution and the analysisof membrane composition during the adaptation tobenzoic acid.It is important to notice that the above calculationwas performed based on the assumption of a constantbiomass dry weight specific volume (Vx). As the cellsize decreases the cell reduces its organic mass (cellularmachinery) proportional to the cube of its diameter,and reduces its surface area, which is proportional tothe square of the diameter. This may indicate that,along with the decrease in benzoic acid influx, which isproportional to the cell surface area, the cell alsodecreases its cellular machinery which may implydecreases in metabolic flux. This would make thedecrease in cell diameter a counterintuitive response.To verify what actually happens in the transition,accurate measurement of cell volume distribution andcell mass distribution are required.Overall, these long-term responses show that cells areable to adapt to benzoic acid by decreasing their specificsurface area and their membrane permeability, in agree-ment with previous observations by Warth [13].Short-term transient response following the shiftin benzoic acid concentration–boost in energygenerationThe observed rapid increase in OUR and CER shortlyafter the shift in benzoic acid concentration(Fig. 4A,B) indicates a fast flux rearrangement insidethe cell. It implies that more glucose is used for energygeneration and that the glycolytic flux increases tempo-rarily. This is supported by the observed transientincrease in extracellular ethanol, which was followedby a transient increase in extracellular acetic acid(Fig. 2D,E). It is reported that ethanol production inS. cerevisiae is a direct consequence of the accumula-tion of pyruvate, which is the end product glycolysis[3]. It should be noted that the increase in extracellularethanol and acetate concentration is transient and thelevel is relatively small. Thus, it may be safely assumedthat the energy is generated from respiration.The timing of the previously discussed observationsalso provides other information about cell regulation.The fact that the increase in ethanol concentration isobserved before the increase in acetic acid concentra-tion and OUR, suggests that cells can rapidly increasethe glycolytic flux, whereas the adjustment of respira-tion is slower. As a consequence of the rapid increasein glycolytic flux, the NADH concentration is rapidlybuilt up, which triggers an increase in the rate of reac-tions consuming NADH, e.g. alcohol dehydrogenasethat synthesizes ethanol and oxidative phosphoryla-tion. The increase in ethanol concentration shows therequirement of the cell to balance the fast NADHaccumulation, which could not be directly accommo-dated by the oxidative phosphorylation. The capacityof the latter process increases later, and is observed asan increase in OUR and CER as well as an increasein acetate (ethanol is converted back to acetate andproduces $ 2 NADH per mol ethanol).It is interesting to note that under carbon-limitedconditions S. cerevisiae is rapidly able to increase therate of O2consumption by 1.5-fold. It shows that,despite the constant feed rate of glucose in the glucose-limited chemostat, the cell can rapidly increase glucosecatabolism. Quantitative explanation of this phenom-enom is summarized in Fig. 8. There are two possibleexplanations for the origin of the transient increase inglucose catabolism: a decrease in the biomass produc-tion rate allowing an increased channeling of glucosetowards catabolism, or a temporary mobilization ofstorage carbohydrates.It is even more interesting to see that after the initialincrease, O2consumption is seen to rapidly decreaseagain, at $ 500 s after the shift experiment, almostreaching its initial steady-state value (Fig. 4A). Theobserved dynamic pattern of the O2consumptionprofile during this short transient of 0–3000 s, i.e. atemporary increase followed by a decrease in the O2consumption profile, is therefore most likely related tothe mobilization of storage carbohydrate compoundsTransient response to benzoic acid M. T. A. P. Kresnowati et al.5536 FEBS Journal 275 (2008) 5527–5541 ª 2008 The Authors Journal compilation ª 2008 FEBS[...]... et al Transient response to benzoic acid Observed total short tr ransient increase in ol OUR of about 45 mmo O2 (equivalent to additional 0.037 C-mol of sugar metabolism) o Possible source: Fig 8 Quantification of short-term transient response following the shift in benzoic acid concentration Possible target: Utilization of storage carbohydrate (total availability 0.25 C-mol) such as trehalose and glycogen,... of H+ATPase Assuming that the level of Pdr12 that needs to be synthesized is 35% of the total amount of plasma membrane proteins, in comparison with the level of H+-ATPase which represents 20–50% of the total amount of plasma membrane proteins [23,24], and assuming that the total amount of plasma membrane proteins composes 5% of the total protein; the abundance of Pdr12 was calculated to be 1.75% of. .. intracellular pH homeostasis via activation of a proton exporter, H+-ATPase, during the fast intrusion of benzoic acid by passive diffusion It is calculated that the total influx of benzoic acid within the first 3000 s is $ 2 mmol for the total 4 L fermentor scale However, assuming a P ⁄ O ratio of 1.46, the estimated additional O2 consumption for the active export of 2 mmol of protons would be 1.4 mmol O2, which... during the transient of 45 mmol is equivalent to the catabolic consumption of 0.037 CÆmol of storage carbohydrates which is only 15% of the total storage carbohydrate available Overall, the increase in O2 consumption rate reflects the high energy requirement of the cells upon sudden exposure to benzoic acid The remaining question is why the cells need the energy A possible answer is that cells need to maintain... regulation of respiration and alcoholic fermentation Yeast 8, 501–517 8 Francois JM, van Schaftingen E & Hers HG (1986) Effect of benzoate on the metabolism of fructose-2,6bisphosphate in yeast Eur J Biochem 154, 141–145 9 Pearce AK, Booth IR & Brown AJP (2001) Genetic manipulation of 6-phosphofructo-1-kinase and fructose 2,6-bisphosphate levels affects the extent to which benzoic acid inhibits the growth of. .. the observed extracellular total benzoate concentration profile, which shows that the benzoate transporter is induced within the first 3000 s of the transient response In order to verify this hypothesis further, measurement of transcript and protein levels during this short transient response will be necessary In conclusion, the adaptation of aerobic S cerevisiae by benzoic acid has been investigated... respiratory capacity of the cell The critical respiratory capacity of S cerevisiae is obtained at a residual benzoate concentration of 10 mm, at pH 5.0 [7] Simultaneous with the medium switch, sodium benzoate solution of pH 4.5 was rapidly injected, via a pneumatic system, into the fermentor to give an almost instantaneous final total benzoate concentration of 0.8 mm Sampling methods Samples to determine... CÆmolÆkgDW)1) of trehalose [21] These levels correspond to $ 0.25 CÆmol of carbohydrates in a fermentor scale containing 4 L of broth with the observed biomass concentration level, which is more than enough to explain the total additional consumption of O2 during the first 3000 s of the transition Assuming that 1 CÆmol of glucose can generate 3.5 mol ATP and a P ⁄ O value of 1.46 [17], the additional fermentor... evaluation of sampling techniques for residual glucose determination in carbon-limited chemostat culture of Saccharomyces cerevisiae Biotechnol Bioeng 83, 395–399 28 Lange HC, Eman M, van Zuijlen G, Visser D, van Dam JC, Frank J, de Mattos MJT & Heijnen JJ (2001) Transient response to benzoic acid Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae. .. temperature (K) and universal gas constant [barÆm)3Æmol)1ÆK)1), respectively Combining Eqns (8,9) yields: dCO2 þ /G;in xO2;g;in dt dxO2 À NG dt OUR ¼ À/L CO2 À VL À /G;out xO2;g ð12Þ time of the off-gas measurement, for which the contribution of the dilution in the fermentor headspace, the length of tubing connecting the fermentor with the off-gas analyzer and the response time of the off-gas analyzer . Energetic and metabolic transient response of Saccharomyces cerevisiae to benzoic acid M. T. A. P. Kresnowati*, W. A. van Winden, W. M. van Gulik and. Geneticmanipulation of 6-phosphofructo-1-kinase and fructose2,6-bisphosphate levels affects the extent to which benzoic acid inhibits the growth of Saccharomyces cerevisiae.
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