Chemical Physics Letters 387 (2004) 377382 www.elsevier.

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Inhibition eects of ethanol on the kinetics of glucose metabolism by S. cerevisiae: NMR and modelling study
Maso Ricci a, Silvia Martini a, Claudia Bonechi a, Lorenza Trabalzini b, Annalisa Santucci b, Claudio Rossi a,*
a

Department of Chemical and Biosystem Sciences, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy b Department of Molecular Biology, University of Siena, Via Fiorentina, 1-53100 Siena, Italy Received 24 September 2003; in nal form 13 February 2004 Published online: 10 March 2004

Abstract Saccharomyces cerevisiae is an important model to understand the eukaryotic cells control mechanisms. In this Letter, an approach based on in vivo 13 C NMR and mathematical modelling was presented to develop a basis for a deeper understanding of eukaryotic responses to environmental stresses. Despite the few metabolites included, the model describes correctly the dynamical behaviour of glucose degradation and ethanol production, providing a qualitative and quantitative description of the response of S. cerevisiae metabolism to stressful concentrations of ethanol. The data and the model highlight that an exogenous stress is able to inuence the cellular activity and the ethanol production rate constant. 2004 Elsevier B.V. All rights reserved.

1. Introduction All living organisms are constantly subject to a variety of physical, chemical, or biological damages usually referred to as stress [1]. Among environmental stresses that yeast cells may undergo, ethanol constitutes the main stress factor during fermentation processes [2,3]. The toxic eects of ethanol on Saccharomyces cerevisiae cells involve the modication of membrane lipid composition and a reduction of metabolic activity, inhibition of glucose up-take, suppression of growth rate, and product formation [4,5]. Literature provides a great variety of studies focused on S. cerevisiae responses to unusual ethanol concentrations, which are represented mainly by synthesis of stress proteins, increases in levels of cellular threalose and glycerol, modulation of ion exchange processes [2,69]. However, a previous paper [2] on the proteome response to ethanol/fermentation stress in S. cerevisiae strain, indicated that alcohol dehydrogenase (ADH)
*

Corresponding author. Fax: +390577234177. E-mail address: rossi@unisi.it (C. Rossi).

expression was not repressed under the fermentation stress and that the same protein is one of the few enzymes of the carbohydrate metabolism which were not proteolyzed (and thus inactivated) within the stress response. Although the large number of studies at the molecular level provided important and detailed information, methods of studying the chemical equilibria and the kinetic eects need to be developed. The behaviour of complex systems, as eukaryotic cells in response to external stimuli, can only be completely understood by considering the complex network of interactions that take place within the system. In the past, cell reactions have been generally analyzed using MichaelisMenten [10,11] dynamics to describe experimental data. This type of analysis is insucient when we need information about interactions between cells, precursors, and products. More recently, other theoretical approaches have been proposed in order to elucidate the dynamics of metabolic reactions [1219]. Although the literature oers many modelling approaches on metabolic processes, involving a large number of parameters, they show a lack of predictivity,

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exibility, and often the calculated parameters are not related to a specic chemical and biochemical meaning. In this work, the eects of increasing concentrations of ethanol on S. cerevisiae glucose degradation have been investigated with a novel approach based on the combined use of in vivo 13 C NMR spectroscopy and mathematical modelling. In vivo NMR spectroscopy, together with 13 C labelled substrates, have been used to follow both the metabolic reactions and ethanol inhibition eects. Because of its non-invasive nature, NMR spectroscopy can be useful in kinetics studies in which several samplings are required during metabolic activity. In fact, selective enriched substrates allow the mapping of the fate of the metabolite during the fermentation process. The tracing out of the pathways enables the determination of the rate of carbon ux through a distinct metabolic route [20]. NMR data were used to develop a mathematical model that describes energy and matter uxes within the system simulating the metabolism of the yeast by the Odum formalism [2123]. The model considers the carbon source, the main end product, and the yeast cells as distinct compartments which interact describing the chemical phenomena involved in the glucose degradation, ethanol production, and feed-back inhibitions. This approach provides chemical and biological information indicating the strategies used by S. cerevisiae in response to exogenous stress. All the parameters describing the state of the system were treated as kinetic parameters and non-linear equations were developed to describe their evolution in time [1215,24]. The model provides a qualitative and quantitative description of the response of S. cerevisiae metabolism to an exogenous stress. The results highlight that the ethanol stress plays a multifarious inhibition on the fermentative process.

2. Experimental Saccharomyces cerevisiae K310 strain was isolated from spontaneous wine fermentation [25]. K310 was grown in yeast peptone dextrose (YPD) at 30 C with rotary shaking up to saturation (cell concentration 1 104 cells/ml). An aliquot of the saturated culture was inoculated in YPD, adjusted to nal pH 4.5 by adding 0.2 mol/l citratephosphate buer and containing 0.55 mol/l unlabelled glucose. The cell suspension was incubated at 25 C without shaking (semiaerobiosis) for 10 h. Stirring or shaking during cell growth should provoke oxidation due to air oxygen that would introduce an additional undesirable shock. It is well known that one of the main eects of fermentation is a sort of oxidation which is exclusively due to ethanol production, being oxygen-derived oxidation ruled out just thanks to semiaerobiosis (growth

without shaking) [2]. Reproducibility of our experimental system was warranted by the monitoring of many parameters during growth and the model proved to be reproducible since it was adopted in several previous studies [2,3,25]. Moreover, proteome maps produced with protein extracts from the same yeast strain grown within NMR tube and larger batches under the same semiaerobiosis conditions showed no signicative dierence (data available upon request), thus proving that protein expression is unaected. This indicated that at least the majority of the cells are subjected to the same treatment both in narrow and larger containers, where, theoretically, the impact of ethanol could be expected to be dierent. This further supported the reproducibility of our experimental system. Samples were then collected and the growth steps were monitored by measuring the absorbance of the culture at 660 nm. For NMR measurements, K310 cell suspension 1 106 cells/ml, indicating an early log phase of growth was centrifuged 5 min at room temperature and 3000g in a Beckman centrifuge model J221 equipped with a JA10 rotor. The supernatant was discarded and the pellet was resuspended in the same medium containing 0.55 mol/l of 1-13 C glucose. 20% (v/v) of D2 0 was added. Three sets of experiments were conducted. In each one the cells were placed in the same batch conditions except for the exogenous ethanol dilution rate which was 0, 0.43 and 1.08 mol/l, respectively. 13 C spectra were collected by a Bruker Avance DRX 600 spectrometer operating at 600 and 150 MHz for proton and carbon nuclei, respectively. Carbon spectra were recorded under continuous broadband decoupling conditions. During the experiment the NMR probe was thermostated at 30 C. The substrate and the end-product concentrations were calculated from the area of NMR peaks through an appropriate calibration. The total ethanol concentration was estimated by an enzymatic assay by SigmaAldrich (kit cod. 332-C). All high purity reagents were from SigmaAldrich (Milano, Italy), Merck Eurolab (Milano, Italy), Carlo Erba (Rodano, MI, Italy), Serva (Heidelberg, D). All the water used was Milli-Q quality (Millipore, Bedford, USA). 1-13 C glucose was from Cambridge Isotope Laboratories (Andover, MA, USA).

3. Results The NMR experiments were performed on three dierent samples with increasing exogenous ethanol concentrations. A parallel growth was carried out during the yeast growth under the NMR monitoring. Samples were collected at time 0 and after 12, 16, 19, 22, 27, 36, 44, 50 and 62 h. At each sampling the pH of the

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cell suspension was checked and growth was monitored by measuring the absorbance of the culture at 660 nm (results not shown). The pH (4.5) never changed during cell growth, as also already reported [2]. The experimental data showing the dynamics of glucose degradation and ethanol production for the three experiments are reported in Fig. 1. In the absence of exogenous ethanol, the cell culture needed 38 h to completely metabolise 0.55 mol/l of glucose and to produce 0.98 mol/l of ethanol. In the presence of 0.43 mol/l of exogenous ethanol the cells needed 43 h to consume the glucose to yield 0.89 mol/l of ethanol (the total concentration of ethanol in the sample, at the and of the experiment, was 1.32 mol/l). With 1.08 mol/l of exogenous ethanol, the fermentation was completed after 55 h yielding to an ethanol concentration of 0.61 mol/l (with a total nal concentration of ethanol in the sample of 1.69 mol/l). Data highlighted that the stress due to the exogenous ethanol aects the kinetics of the fermentation of glucose to ethanol. Increasing the exogenous ethanol concentration, data reveal two main eects on fermentation: a lower fermentation rate and a reduction of the nal concentration of endogenous ethanol. On the basis of the collected information we propose a novel modelling approach able to give a reliable interpretation of experimental data. Due to the high concentration of glucose, we can assume that the main metabolic pathway for the degradation of the substrate was the fermentation. In fact, when yeast grows in the presence of high glucose concentrations, the negative impact exerted by the sugar on the other metabolic pathways, makes the fermentation

the sole degradation pathway [26,27]. Thus, in order to simplify the system, we considered glucose as the unique carbon source and ethanol as the main end product of the fermentative process. In order to develop the S. cerevisiae metabolic model, we took in account four elements: glucose concentration, exogenous ethanol concentration (which represents the source of exogenous stress), endogenous ethanol concentration (the end product) and an index [C] related to the active cells that we dened cellular activity. Due to exogenous stress and to the negative feedback, we assumed a modulation of the cellular activity [C]; in this way [C] was thought as an index related to the total number of cells in the sample having a predominant fermentative metabolism. As described in the Odum diagram reported in Fig. 2, elements in the model are connected to each others by interactions that represent the biochemical routes of yeast metabolism. The model considers three main interactions: (i) The interaction of glucose with active cells which leads to glucose degradation, ethanol production, and an increasing of cellular activity. (ii) The interaction between cells and exogenous ethanol which rules the amount of eectively active cells at the beginning of the fermentation C0 . (iii) The interaction between the total ethanol (both endogenous and exogenous) and the actual cellular activity ([C]),which induces a negative eect determining the amount of inhibited cells during glucose catabolism. Each metabolic route is described by a dierential equation as follows: dG kD GC; dt 1 2

1.0 0.9

dE kP GC; dt

Concentration (mol/L)

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 5 10 15 20 25 30 35 40 45 50 55 60 65

C0

C
kIGC

f (EEX)

kAGC

E END
kPGC

E EX

G
kDGC

time (h)
Fig. 1. Data comparison of glucose and ethanol concentration collected in the presence of increasing exogenous ethanol concentrations. Experiments were performed with 0 g/l of exogenous ethanol [glucose (j) and ethanol ()], 0.43 mol/l of exogenous ethanol [glucose (d) and ethanol (s)] and, 1.08 mol/l of exogenous ethanol [glucose (N) and ethanol (M)].

C IN
Fig. 2. Energy system diagram of the model of fermentative process.

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dC kA GC k1 fEexo Eendo gC; dt Ct0 C0 1 ; 1 hEexo

3 4

where [G], Eendo and Eexo are the concentrations of glucose, endogenous and exogenous ethanol (expressed in mol/l), respectively. [C] is the cellular activity during fermentation, Ct0 is the initial cellular activity, and C0 is the cellular activity in the absence of exogenous ethanol which we assumed to be equal to 1. Both the rates of glucose degradation and ethanol production are related to the glucose concentration and to cellular activity (Eqs. (1) and (2)). Ethanol acts as an inhibition factor modulating the cellular activity [C] (Eq. (3)). ki (with i D; A; P ; I) represents the kinetic constants associated with the degradation of glucose (kD ), production of ethanol kP , cellular activation (kA ) pro-

moted by glucose and cellular inhibition (kI ) due to the total ethanol in the system. The constant h (Eq. (4)) is referred to a function controlling the initial concentration of active cells (Ct0 ) in the presence of exogenous ethanol which reduces the cellular activity at the beginning of the fermentation. When the exogenous stress is absent, the initial cellular activity is assumed to be 1; increasing the concentration of the stress source, the value of Ct0 decreases, which implies a reduced fermentative rate. Cellular activity [C] includes the inhibitory eects exerted by exogenous ethanol at the beginning of the process and the negative feed-back due to the increasing production of ethanol during the fermentation. We supposed that the exogenous ethanol produces a stress on the cell population decreasing the initial cellular activity. The validity of the model was checked against data obtained from the experiments performed with addiction of 0, 0.43, and 1.08 mol/l of exogenous ethanol. The

(a) 1.1
1.0 0.9 0.8

(b) 1 .1
1.0 0.9 0.8

Concentration (mol/L)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 10 20 30 40 50 60

Concentration (mol/L)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 10 20 30 40 50 60

time (h)

time (h)

(c) 1.1
1.0 0.9 0.8

(d)
Cellular Activity (Arbitrary Units)
0 10 20 30 40 50 60

25

20

Concentration (mol/L)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

15

10

0 0 20 40 60 80 100

time (h)

time (h)

Fig. 3. Results of the simultaneous t of data from experiments performed at increasing ethanol concentrations. (ac) Experimental time course of ethanol (s) and glucose (d) are compared with model simulation (continuous line). In (a) S. cerevisiae was placed in a media in the absence of exogenous ethanol. In (b) and (c) the concentration of exogenous ethanol were 0.43 and 1.08 mol/l, respectively. (d) Curves of cellular activity calculated by the model: in the absence of exogenous ethanol (continuous line), at 0.43 and 1.08 mol/l of exogenous ethanol (dashed and dotted lines, respectively).

M. Ricci et al. / Chemical Physics Letters 387 (2004) 377382 Table 1 Estimated values and coecient of variation (CV) of the kinetics parameters Constant kD kP (exogenous ethanol 0 mol/l) kP (exogenous ethanol 0.43 mol/l) kP (exogenous ethanol 1.08 mol/l) KA kI h Value 6.96 10 (s ) 1.26 102 (s1 ) 1.12 102 (s1 ) 0.8 102 (s1 ) 4.42 101 (s1 ) 4.88 102 (s1 ) 3.3 (l/mol)
3 1

381

CV (%) 1.2 1.1 1.1 1.1 0.4 1.3 1.3

constants in each equation were optimised by a nonlinear least-square estimation technique as implemented in ML A B computer program using the Marquardt Levenberg method aiming the best t between measured and predicted values of glucose and produced ethanol concentrations [28]. Data were used simultaneously to calculate the constants kD , kA , kI , and h, which were assumed to be independent from the inhibition contribution of the exogenous ethanol. Assuming a direct action of the ethanol stress on the ethanol production, kP was estimated for each experiment. Figs. 3ac show the experimental and calculated data of glucose consumption and ethanol production measured at dierent concentrations of exogenous ethanol. The agreement between simulated and experimental data was excellent for both glucose and ethanol R2 0:995. The values of the constants calculated from the model are shown in Table 1. Fig. 3d shows the simulated behaviour of cellular activity calculated for each experiment in function of time. The maximum value reached by each curve decreased with increasing ethanol concentration, in agreement with the lower fermentation rate observed in the experimental data. 4. Discussion The mathematical model proposed in this work was conceived to give an interpretation of chemical processes occurring during glucose degradation by S. cerevisiae in the presence of stress ethanol concentrations. The simplications oered by the modelling approach allows to consider the system as composed by elements that interact with each other exchanging energy and matter. Non-linear equations which describe the dynamics of glucose consumption, ethanol production, and cellular activity behaviour (Eqs. (1)(4)), contain the kinetic parameters which are the basis to interpret the observed eects due to the presence of exogenous ethanol within the system. As observed by experimental data shown in Fig. 1, the increasing concentration of exogenous ethanol has two macroscopic eects: a lowering of the degradation rate of glucose, and a reduced production of endogenous ethanol.

The simulated behaviour of cellular activity contributed to evaluate the inhibition eects due to the presence of ethanol. The cellular activity, behaves as the result of two contributions as described in Eq. (3). At the beginning of fermentation the high glucose concentration induces the increase of cellular activity with a rate equal to the activation parameter kA , according to the rst term of Eq. (3). An opposite eect is induced by the produced ethanol which inhibits the fermentation process causing a decrease in the cellular activity with a rate equal to the inhibition parameter kI , according to the second term of Eq. (3). As expected, kA assumes higher values than kI , indicating that the ability of glucose in promoting the fermentation process is greater than the feed-back inhibition due to the presence of ethanol. This causes the curve to be non-symmetrical in respect to time. As shown in Fig. 3d, the value of cellular activity at time zero was not assumed to be constant for all the experiments. We supposed that the exogenous ethanol produced stress eects on cellular population decreasing the initial cellular activity (Ct0 ) according to Eq. (4). This assumption let the model simulate the initial stress which S. cerevisiae cells undergo at the beginning of the fermentation. Although the rate of glucose degradation was aected by the presence of ethanol, experimental data also showed that the cells were able to completely consume the glucose in the sample. This means that the ethanol decreased the cellular activity leading to a lowering in the total fermentation rate. Nevertheless, the ability of single cell to degrade glucose was the same in the presence and in the absence of ethanol. This can be understood analysing the glucose degradation rate constants kD calculated for the three experiments: the same values assumed by KD suggest that the concentration of active cells represent the sole variable able to aect the degradation rate. The variation of the kinetic constant KP was calculated in relation to the exogenous ethanol concentration. It represents an index of how eciently the ethanol was produced in the presence of ethanol stress. In the case of the three discussed experiments the ethanol yielded at the end of the fermentation was dierent. A reduction was observed at higher stress conditions. This is conrmed by the calculated kP values which showed lower values at higher exogenous ethanol concentrations. In the hypothesis that kP does not assume dierent values for each experiment the model is still able to simulate lower fermentation rates but it loses the ability to predict the nal concentration of ethanol. Thus model behaviour and experimental data suggest that the stress induced by the presence of ethanol subtracts fermentative ability reducing the production rate and nal concentration of ethanol.

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M. Ricci et al. / Chemical Physics Letters 387 (2004) 377382 [3] L. Trabalzini, A. Paetti, E. Ferro, A. Scaloni, F. Talamo, L. Millucci, P. Martelli, A. Santucci, Ital. J. Biochem. 52 (4) (2003) 72. [4] T. DAmore, G.G. Stewart, Microb. Technol. 9 (1987) 322. [5] H. Alexandre, V. Ansanay-Galeote, S. Dequin, B. Blondin, FEBS Lett. 498 (2001) 98. [6] F. Estruch, FEMS Microbiol. Rev. 24 (2000) 469. [7] V. Costa, P. Moradas-Ferreira, Mol. Aspects Med. 22 (45) (2001) 217. [8] A.R. Barber, F. Vriesekoop, N.B. Pamment, Enzyme Microb. Technol. 30 (2002) 240. [9] P.W. Piper, FEMS Microbiol. Lett. 134 (1995) 121. [10] J.A. den Hollander, T.R. Brown, K. Ugurbil, R.G. Shulman, Proc. Natl. Acad. Sci. USA 205 (1979) 6096. [11] R.G. Shulman, T.R. Brown, K. Ugurbil, S. Ogawa, S.M. Cohen, J.A. den Hollander, Science 205 (1979) 160. [12] S. Bastianoni, A. Donati, A. Gastaldelli, N. Marchettini, S. Martini, C. Rossi, Chem. Phys. Lett. 310 (1999) 38. [13] S. Bastianoni, A. Gastaldelli, C. Bonechi, C. Mocenni, C. Rossi, Biochem. Biophys. Res. Commun. 227 (1996) 41. [14] S. Bastianoni, A. Donati, A. Gastaldelli, N. Marchettini, D. Renzoni, C. Rossi, Biochem. Biophys. Res. Commun. 227 (1996) 53. [15] C. Rossi, M. Porcelli, C. Mocenni, N. Marchettini, S. Loiselle, S. Bastianoni, Ecol. Model 113 (1998) 157. [16] K.W. Wirtz, J. Biotechnol. 97 (2002) 147. [17] W. Wiechert, J. Biotechnol. 94 (2002) 37. [18] F. Lei, M. Rotbll, S.B. Jrgensen, J. Biotechnol. 88 (2001) 205. [19] B. Teusink, J. Passarge, C.A. Reijenga, E. Esgalhado, C.C. van der Weijden, M. Schepper, M.C. Walsh, B.M. Bakker, K. van Dam, H.V. Westerho, J.L. Snoep, Eur. J. Biochem. 267 (2000) 5313. [20] R.G. Shulman, Sci. Am. 248 (1983) 86. [21] H.T. Odum, in: B.C. Patten (Ed.), System Analysis and Simulation in Ecology, vol. 2, Academic Press, New York, 1972, p. 139. [22] H.T. Odum, in: B.C. Patten (Ed.), Systems Ecology, Wiley, New York, 1983. [23] H.T. Odum, in: C. Rossi, E. Tiezzi (Eds.), Ecological Physical Chemistry, Elsiever, Amsterdam, 1991, p. 25. [24] S. Bastianoni, C. Bonechi, A. Gastaldelli, S. Martini, C. Rossi, in: Chemistry at the beginning of the Third Millennium: Molecular Design, Supramolecules, Nanotechnology and Beyond, Springer, Berlin, 2000, p. 305. [25] A. Santucci, L. Trabalzini, L. Bovalini, E. Ferro, P. Neri, P. Martelli, Electrophoresis 21 (17) (2000) 3717. [26] R.H. De Deken, J. Gen. Microbiol. 44 (1966) 149. [27] A. Fiechter, G.F. Fuhrmann, O. Kappeli, Adv. Microb. Physiol. 22 (1981) 123. [28] B. Bunow, G. Knott, ML A B : A Mathematical Modeling Laboratory, Civilized Software Inc., Bethesda, MD, 1992.

On the other hand the kinetic constant controlling the rate of glucose degradation kD was kept unchanged. Indeed the ethanol stress seems to have no detectable eects on the value of kD .

5. Conclusions The complexity of processes involved in systems as eukaryotic cells are, needs an approach able to describe the network of interactions that take place within the system. In our compartmental model, the degradation dynamics of substrate, the ethanol production, the activation and inhibition processes are strictly coupled. The novel modelling approach proposed in this work is able to clearly describe the various eects induced by the presence of increasing exogenous ethanol concentrations on the glucose metabolism in S. cerevisiae. Despite of the few metabolites included, the model describes correctly the dynamical trends of glucose degradation and ethanol production and allowed to evaluate the cellular activity trend, whose value is not correlated only to the biomass concentration but it depends to the initial exogenous ethanol concentration and to the ethanol stress during the fermentative process. The robustness and exibility of the model enables it to work correctly at dierent initial exogenous ethanol concentrations. Acknowledgements This work was supported by MIUR Fondo per gli Investimenti della Ricerca di Base (FIRB), n. RBAU01JE9A and Italian Inter-University Consortium CSGI. References
[1] R.M. Birch, G.M. Walker, Enzyme Microb. Technol. 26 (2000) 678. [2] L. Trabalzini, A. Paetti, A. Scaloni, F. Talamo, E. Ferro, G. Coratza, L. Bovalini, P. Lusini, P. Martelli, A. Santucci, Biochem. J. 370 (2003) 35.