Hxt-Carrier-Mediated Glucose Efflux upon Exposure of Saccharomyces cerevisiae to Excess Maltose

When wild-type Saccharomyces cerevisiae strains pregrown inmaltose-limited chemostat cultures were exposed to excess maltose,release of glucose into the external medium was observed. Controlexperiments confirmed that glucose release was not caused bycell lysis or extracellular maltose hydrolysis. To test thehypothesis that glucose efflux involved plasma membrane glucosetransporters, experiments were performed with an S. cerevisiaestrain in which all members of the hexose transporter (HXT)gene family had been eliminated and with an isogenic referencestrain. Glucose efflux was virtually eliminated in the hexose-transport-deficientstrain. This constitutes experimental proof that Hxt transportersfacilitate export of glucose from S. cerevisiae cells. Afterexposure of the hexose-transport-deficient strain to excessmaltose, an increase in the intracellular glucose level wasobserved, while the concentrations of glucose 6-phosphate andATP remained relatively low. These results demonstrate thatglucose efflux can occur as a result of uncoordinated expressionof the initial steps of maltose metabolism and the subsequentreactions in glucose dissimilation. This is a relevant phenomenonfor selection of maltose-constitutive strains for baking andbrewing.

INTRODUCTION

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Introduction

The disaccharide maltose is an important carbon source for Saccharomycescerevisiae during beer fermentation and leavening of dough (3,10, 20, 42). In addition to having applied significance, themaltose regulon in S. cerevisiae serves as a paradigm for metabolicregulation in eukaryotes (18, 23, 36, 37).

In S. cerevisiae, an intracellular maltase ( ${alpha}$ -glucosidase; EC3.2.1.20) hydrolyzes maltose to glucose. The transport of maltoseover the cell membrane differs from the transport of glucose(Fig. 1). In S. cerevisiae, glucose uptake occurs exclusivelyvia facilitated diffusion (29, 43). Facilitated diffusion ofglucose involves the 17 members of the HXT gene family (61),which encode hexose transporters that differ with respect tokinetic properties (12), transcriptional regulation, and intracellularlocalization (39). As Hxt-mediated glucose transport does notrequire input of metabolic energy, alcoholic fermentation ofglucose by S. cerevisiae results in a net yield of two ATP moleculesper glucose molecule (30). Conversely, maltose is taken up viaa maltose-one-proton symport mechanism (49). Extrusion of thesymported proton via the plasma membrane ATPase costs one ATPmolecule per proton (53, 59). Consequently, the net ATP yieldfrom alcoholic fermentation of one maltose molecule is onlythree ATP molecules (21).

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FIG. 1. Schematic representation of glucose and maltose transport in S. cerevisiae. (A) Facilitated diffusion of glucose, driven by the concentration gradient of the sugar. (B) Maltose-proton symport driven by the proton motive force and the sugar concentration gradient. ATP hydrolysis by the plasma membrane ATPase is required to expel the protons that enter the cell together with the maltose. For each maltose molecule transported into the cell, one ATP molecule is hydrolyzed (53, 59). 1, Hxt transporter; 2, maltose permease; 3, H⁺-ATPase complex.

The genes encoding the maltose permease are located in fivehighly homologous loci (MAL1, MAL2, MAL3, MAL4, and MAL6) (2,8). The number and identity of MAL loci is strain dependent(35). Each MAL locus consists of three genes. The first gene(MALx1) encodes the maltose-proton symporter (9). Maltase isencoded by the MALx2 gene (13, 22). The third MAL gene (MALx3)encodes a DNA-binding, maltose-dependent transcriptional activatorthat specifically controls expression of the MALx1 and MALx2genes (7, 21).

Maltose metabolism in S. cerevisiae is strongly downregulatedby glucose. At the transcriptional level, glucose repressestranscription of the MALx1 and MALx2 genes via binding of thetranscriptional repressor Mig1p in the MAL intergenic region(26, 27, 62). Moreover, glucose causes rapid catabolite inactivationof maltose permease activity (5). This glucose-induced inactivationcan involve two different signaling pathways (32). The firstpathway uses Rgt2p as a sensor of extracellular glucose andinduces degradation of the maltose permease protein. This degradationrequires ubiquitination and endocytic internalization of themaltose transporter protein to the vacuole, where proteolysistakes place (32-34, 45). The second pathway depends on glucosetransport and causes very rapid inactivation of maltose transportactivity, followed by degradation of the maltose permease (24,25). Which signal triggers this catabolite inactivation is stilla matter of debate. Some authors have proposed that hexose transportvia Hxt transporters is required for this pathway (24, 25),whereas other authors have stated that galactose and even maltosecan also elicit catabolite inactivation (40, 46). In addition,trehalose and/or trehalose 6-phosphate have recently been mentionedas possible signals for catabolite inactivation (4). The glucose-inducedloss of maltose transport activity is generally much fasterthan the loss expected from mere proteolytic degradation ofthe maltose transporter. This observation has been explainedby glucose-induced phosphorylation of the maltose transporterthat precedes proteolytic breakdown and immediately reducestransport activity (5).

Despite this multilayer regulation of maltose metabolism, severalreports have indicated that S. cerevisiae has difficulty copingwith sudden changes in the extracellular maltose concentration.Exposure of aerobic, maltose-limited chemostat cultures to excessmaltose has even been reported to result in maltose-accelerateddeath (41). The loss of viability, accompanied by the releaseof glucose into the medium, was interpreted to be caused bynonrestricted maltose uptake and hydrolysis, with concomitantrapid intracellular accumulation of glucose and protons leadingto cell death and lysis (41). Release of glucose upon exposureto excess maltose has also been observed in S. cerevisiae mutantsdefective in glucose catabolite repression (5, 11, 26).

The aim of the present study was to investigate the mechanismresponsible for glucose release during maltose fermentationby S. cerevisiae. Special attention was paid to a possible roleof the HXT-encoded glucose transporters in mediating glucoseefflux.

MATERIALS AND METHODS

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Materials and Methods

Strains and maintenance.
The strains used in this study (Table 1) were grown to the stationaryphase in shake flask cultures on synthetic medium (55) adjustedto pH 6.0 and containing 2% (wt/vol) glucose. After additionof sterile glycerol (30%, vol/vol), 2-ml aliquots were storedin sterile vials at -80°C. These frozen stock cultures wereused to inoculate precultures for chemostat cultivation.

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TABLE 1. S. cerevisiae strains used in this study

Media.
Synthetic medium containing mineral salts and vitamins was preparedand sterilized as described previously (55). To meet the auxotrophicrequirements of strains CEN.PK.2-1C and EBY.VW.4000, media forcultivation of these strains were supplemented with uracil (113mg · liter^-1), L-histidine (45 mg · liter^-1),L-leucine (180 mg · liter^-1), and L-tryptophan (27 mg· liter^-1). Auxotrophic requirements were calculatedas described by Oura (38), and the required concentrations weremultiplied by 2 to prevent limitation by nutrients other thanthe sugar substrate. For chemostat cultivation, the glucoseor maltose concentration in reservoir media was 7.5 g ·liter^-1 (0.25 mol of C · liter^-1). Media for maltose-limitedcultivation of strains CEN.PK.2-1C and EBY.VW.4000 were alsosupplemented with ethanol (5% on a total carbon basis) to eliminatethe persistent metabolic oscillations (6, 28) in maltose-limitedchemostat cultures of these strains.

Chemostat cultivation.
Aerobic chemostat cultivation was performed at a dilution rateof 0.10 h^-1, at pH 5.0, and at 30°C in laboratory fermentors(Applikon, Schiedam, The Netherlands) as described previously(51). Steady-state data are data for cultures without detectableoscillations. A maximum culture age of 170 h (25 generations)was used to minimize selection of mutants.

Off-gas analysis.
The exhaust gas was cooled in a condenser (2°C) and driedwith a Perma Pure dryer (type PD-625-12P). O₂ and CO₂ concentrationswere determined with a Rosemount NGA2000 analyzer. The exhaustgas flow rate was determined and specific rates of CO₂ productionand O₂ consumption were calculated as described previously (54,60).

Determination of culture dry weights.
Culture dry weights were determined by filtration of sampleswith nitrocellulose filters and drying in a microwave oven asdescribed previously (51).

Extracellular metabolite analysis.
Glucose, maltose, ethanol, glycerol, acetate, and pyruvate concentrationsin the supernatants of chemostat cultures were determined byhigh-performance liquid chromatography (HPLC) analysis by usingan HPX-87H Aminex ion-exchange column (300 by 7.8 mm; Bio-Rad)at 60°C. The column was eluted with 5 mM sulfuric acid ata flow rate of 0.6 ml · min^-1. Pyruvate and acetate weredetected at 214 nm with a Waters 441 UV meter coupled to a Waters741 data module. Glucose, maltose, ethanol, and glycerol weredetected with an ERMA type ERC-7515A refractive index detectorcoupled to a Hewlett-Packard type 3390A integrator. Glucoseand maltose in reservoir media were also analyzed by HPLC.

Anaerobic fermentation assays.
Samples containing exactly 200 mg (dry weight) of biomass wereharvested from a steady-state chemostat culture by centrifugation(5,000 x g, 3 min) and were resuspended in 10 ml of fivefold-concentratedsynthetic medium (pH 5.6). Subsequently, the cell suspensionswere introduced into a thermostat-controlled (30°C) vessel.The volume of each suspension was adjusted to 40 ml with demineralizedwater. After 10 min of incubation, 10 ml of a maltose solution(100 g · liter^-1) was added, and samples (two 1-ml samples)were taken at appropriate time intervals for 2 h. The 10-mlheadspace was continuously flushed with water-saturated carbondioxide at a flow rate of approximately 30 ml · min^-1.Sugar concentrations and metabolite levels in the supernatantswere determined by HPLC analysis. The ethanol concentrationin the supernatant was determined by a colorimetric assay (56)by using partially purified alcohol oxidase from Hansenula polymorpha(a kind gift from Bird Engineering, Rotterdam, The Netherlands).At the end of the experiments (after 2 h) some growth had takenplace (data not shown). Consequently, the levels of carbon recoverywere only ca. 90% if growth of biomass was not taken into account.

Intracellular metabolite measurements.
Biomass samples (4 ml of a 4-g [dry weight]/liter suspension)were taken from an anaerobic fermentation assay mixture andimmediately quenched with 20 ml of 60% methanol at -40°C.After the cells were washed twice with cold 60% methanol, intracellularmetabolites were extracted by resuspending the cell pelletsin 5 ml of boiling 75% ethanol and incubating them for 3 minat 80°C (19). Cell debris and intracellular metaboliteswere dried at room temperature with a vacuum evaporator (typeAES1010 Savant Automatic Environmental SpeedVac system). Finally,0.5 ml of demineralized water was added to each preparation.The resulting suspension was stored at -20°C. Before metaboliteanalysis the suspension was centrifuged. The ATP content wasdetermined by a commercial bioluminometric luciferase assay(catalog no. 1699695; Roche, Almere, The Netherlands). Glucose6-phosphate was measured by monitoring its conversion by NADP⁺-dependentglucose-6-phosphate dehydrogenase (obtained from Roche). Thereaction was carried out in the presence of 0.04 mM NADP⁺ and0.05 U of glucose-6-phosphate dehydrogenase. Microtiter plateswith total volumes of 100 and 60 µl were used for analysisof ATP and quantification of NADPH formation, respectively,in the glucose 6-phosphate measurements. A Mediators PHL luminometer(Mediators Diagnostic Systems, Vienna, Austria) was used toanalyze the bioluminescence, and a Perkin-Elmer HTS 7000 Plusbioassay reader was used to quantify NADPH formation. Glucosecontents were determined with a commercial kit (catalog no.716251; Boehringer) based on conversion of glucose via hexokinaseand NADP⁺-dependent glucose-6-phosphate-dehydrogenase. Glucoseassays were performed with an Amersham Pharmacia Biotech NovaspecIIspectrophotometer by using 1-ml cuvettes. Intracellular maltosecontents were determined by HPLC analysis by using an AminexHPX-87K column (300 by 7.8 mm; Bio-Rad) at 85°C. The columnwas eluted with demineralized water at a flow rate of 0.5 ml· min^-1. Maltose was detected with a TSP type RI-150refractive index detector. Intracellular metabolite concentrationswere calculated based on the assumption that 1 g of biomasshas a cellular volume of 2 ml.

Fluorescent staining for yeast viability.
A commercial LIVE/DEAD yeast viability kit (L-7009; MolecularProbes, Leiden, The Netherlands) was used to estimate the fractionsof dead cells in samples obtained from anaerobic fermentationassays. FUN-1 and Calcofluor White M2R cell stain were addedto yeast cell suspensions (10⁶ to 10⁷ cells/ml) at final concentrationsof 5 to 20 and 25 µM, respectively. After staining, thesuspensions were mixed thoroughly and incubated in the darkat 30°C for 30 min. Five microliters of a stained yeastsuspension was trapped between a coverslip and an object slideand analyzed with a fluorescence microscope (Zeiss Axioplan2 Imaging, Weesp, The Netherlands) by using appropriate filtersets (fluorescein isothiocyanate, Zeiss 09 450-490 FT510 LP515;and 4',6'-diamidino-2-phenylindole [DAPI], Zeiss 02 G365 FT395LP420).

Maltase activity assay.
As a check for extracellular maltase activity, a standard anaerobicfermentation assay was performed. At 0, 30, and 60 min, a sample(2 ml) was centrifuged. Each supernatant (1 ml) was incubatedat 30°C. Samples were taken at different times and analyzedfor glucose by using the UV method (Boehringer kit no. 716251).A 10% (wt/vol) maltose solution in water was used as a negativecontrol.

Protein concentration determination.
Protein concentrations in the supernatants of anaerobic fermentationassay mixtures and in cell extracts were estimated by the methodof Lowry et al. (31). Dried bovine serum albumin (fatty acidfree; obtained from Sigma, Zwijndrecht, The Netherlands) wasused as a standard.

Determination of viable counts.
Viable counts of S. cerevisiae CEN.PK113-7D were determinedon 2% (wt/vol) YPD agar plates. This complex medium contained(per liter) 10 g of yeast extract (Difco, Detroit, Mich.), 20g of peptone from casein (Merck, Darmstadt, Germany), 20 g ofD-glucose, and 20 g of agar (Difco). After appropriate dilutionof the culture and plating (which yielded 50 to 400 coloniesper plate), colonies were counted following 48 h of incubationat 30°C. At least 1,000 colonies were counted to calculateviable counts.

RESULTS

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Results

Release of glucose during maltose fermentation.
When anaerobic maltose fermentation was studied with S. cerevisiaeCEN.PK113-7D pregrown in aerobic, maltose-limited chemostatcultures, substantial amounts of glucose were produced in additionto fermentation products like ethanol and glycerol (Fig. 2A).The highest rate of glucose production (dglucose/dt) took placeduring the first 45 min after maltose addition. During thisperiod, ca. 0.6 mol of glucose was released for each 1 mol ofmaltose consumed (Fig. 2B), corresponding to 30% of the maltosecarbon. After 45 min, the amount of glucose released graduallydecreased, and after ca. 2 h no further net production of glucoseoccurred (Fig. 2). When similar experiments were performed withcells pregrown in aerobic, glucose-limited chemostat cultures,the initial maltose consumption rates were low. No glucose releasewas observed during the slow induction of maltose-fermentingcapacity (data not shown).

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FIG. 2. Anaerobic fermentation of maltose by cells harvested from an aerobic, maltose-limited chemostat culture of S. cerevisiae CEN.PK113-7D and subsequently exposed to excess maltose. The values are averages ± mean deviations for three experiments with cells from independent chemostat cultures. (A) Extracellular metabolite concentrations as determined by pulse assays of three independent cultures. Symbols: ${circ}$ , maltose; •, glucose; ${square}$ , ethanol. (B) Amount of glucose released per mole of maltose consumed during the anaerobic maltose fermentation experiment, calculated by dividing the slopes of the glucose and maltose curves in panel A.

To investigate whether glucose release also occurs in otherwild-type S. cerevisiae strain backgrounds, anaerobic maltosefermentation experiments were performed with maltose-grown culturesof the industrial baker's yeast strain S. cerevisiae DS28911(51, 52) and the laboratory strain S. cerevisiae CBS8066. Qualitatively,these two strains exhibited product formation profiles thatwere very similar to that of the CEN.PK113-7D strain (data notshown). The maximum glucose concentrations observed under standardizedconditions were 9, 4, and 12 mM for strains CEN.PK.113-7D, DS28911,and CBS8066, respectively.

These results indicate that glucose release during maltose fermentationis a general phenomenon in S. cerevisiae cultures but that theamount of glucose released is strain dependent.

Glucose release is not caused by cell lysis.
Postma et al. (41) observed cell death and release of proteinsafter exposure of aerobic, maltose-limited chemostat culturesof S. cerevisiae CBS8066 to excess maltose. To investigate whetherthe glucose release observed during anaerobic incubation ofmaltose-grown S. cerevisiae CEN.PK113-7D with excess maltose(Fig. 2) was due to cell lysis, possibly accompanied by therelease of maltase into the extracellular medium, several controlexperiments were performed.

Plating on complex medium did not reveal a marked decrease inviable counts during anaerobic maltose fermentation (Fig. 3A).During the first 1 h of the experiments, during which glucoserelease was most pronounced (Fig. 2), the viable counts werereduced by only ca. 4% (Fig. 3A). These results were corroboratedby a fluorescent live-dead staining technique, which indicatedthat throughout the pulse experiments virtually all cells remainedmetabolically active (data not shown). As a further indicatorof possible cell lysis, protein concentrations were analyzedin culture supernatants (29). No significant increase in theextracellular protein concentration was observed during thepulse (Fig. 3A). More specifically, the possibility that theextracellular glucose encountered during anaerobic maltose fermentationwas due to an extracellular maltase was investigated. Incubationof supernatant samples taken during the maltose fermentationexperiments did not reveal any extracellular maltase activity(Fig. 3B).

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FIG. 3. Controls for cell integrity and viability during anaerobic fermentation of maltose by cells harvested from aerobic, maltose-limited chemostat cultures of S. cerevisiae CEN.PK113-7D. (A) Extracellular protein concentrations and fraction of viable cells as determined from plate counts. The values are averages ± mean deviations for two experiments performed with cells from independent chemostat cultures. (B) Extracellular maltase activity, plotted as glucose concentration during incubation of supernatant samples taken at different times during anaerobic fermentation experiments. Symbols: ${circ}$ , zero-time supernatant; •, 30-min supernatant; ${square}$ , 60-min supernatant; ${blacksquare}$ , maltose control (100 g · liter^-1), revealing contamination of commercially available maltose with glucose. The values are averages ± mean deviations for two experiments performed with cells from independent chemostat cultures.

Absence of glucose release during maltose fermentation in an hxt null strain.
To investigate whether glucose release during anaerobic maltosefermentation was mediated by plasma membrane glucose transporters(Fig. 1), anaerobic maltose fermentation experiments were performedwith the hxt null strain S. cerevisiae EBY.VW.400. In this strain,all members of the HXT gene family have been deleted, whicheliminates glucose uptake via facilitated diffusion (58). S.cerevisiae EBY.VW.4000 is a member of the CEN.PK strain family,but in contrast to the prototrophic CEN.PK113-7D strain, itcarries four auxotrophic markers. Duplicate control experimentsperformed with cells from independent maltose-grown chemostatcultures of the isogenic, auxotrophic reference strain CEN.PK2-1Cyielded the same glucose and ethanol profiles as the experimentsperformed with the prototrophic CEN.PK113-7D strain (data notshown). In contrast, hardly any extracellular glucose (maximumconcentration, 1.6 ± 0.1 mM) was found during anaerobicmaltose fermentation experiments performed with the hxt nullstrain (Fig. 4). The concentrations of other extracellular metabolites(ethanol, glycerol, and acetate) were comparable to those inthe reference strains (Fig. 2A and 4).

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FIG. 4. Anaerobic fermentation of maltose by cells harvested from aerobic, maltose-limited chemostat cultures of S. cerevisiae EBY.VW.4000 ( ${Delta}$ hxt1 through -17 ${Delta}$ gal2 ${Delta}$ agt1 ${Delta}$ YDL247w ${Delta}$ YJR160c) (A) and S. cerevisiae CEN.PK2-1C (reference strain) (B). Extracellular concentrations of maltose ( ${circ}$ ), glucose (•), and ethanol ( ${square}$ ) are shown. The values are averages ± mean deviations for two experiments performed with cells from independent chemostat cultures.

The strongly reduced glucose release by the hxt null strainsuggests that one or more HXT-encoded hexose transporters areinvolved in glucose efflux. To investigate whether the absenceof functional hexose transporters led to intracellular accumulationof glucose, intracellular metabolite assays were performed duringanaerobic maltose fermentation by S. cerevisiae EBY.VW.4000.In this experiment, the intracellular glucose levels increasedto 41.4 ± 1.3 mM during anaerobic maltose fermentationby the hxt null strain (Fig. 5A). The intracellular concentrationsof glucose 6-phosphate and ATP were 1.8 ± 0.1 and 1.0± 0.1 mM, respectively (Fig. 5B). High concentrationsof intracellular maltose (ca. 130 mM) accumulated during anaerobicmaltose fermentation, suggesting that maltase activity was saturatedunder the experimental conditions.

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FIG. 5. Intracellular metabolite levels during anaerobic maltose fermentation by maltose-pregrown cells of S. cerevisiae EBY.VW.4000 ( ${Delta}$ hxt1 through -17 ${Delta}$ gal2 ${Delta}$ agt1 ${Delta}$ YDL247w ${Delta}$ YJR160c). (A) Symbols: ${circ}$ , maltose; •, glucose. (B) Symbols: ${square}$ , glucose 6-phosphate; ${blacksquare}$ , ATP. Most of the values are averages ± mean deviations for two experiments performed with cells from independent chemostat cultures; the only exceptions are the maltose values, which are values from a single experiment.

DISCUSSION

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Discussion

The results presented in this paper demonstrate that one ormore HXT-encoded glucose transporters are involved in glucoseefflux during exposure of S. cerevisiae to excess maltose. Thereversibility of glucose transport has been demonstrated inprevious studies with kinase-less mutants (14, 15). However,to our knowledge the present study provides the first experimentalproof that Hxt transporters are involved in glucose export.There is no reason to assume that the ability to export glucoseis confined to one or a few members of the Hxt family. However,as has been demonstrated for glucose uptake via the individualHxt transporters, it is likely that the kinetic properties ofthe Hxt-encoded transporters for glucose efflux are different(44, 57).

The simultaneous uptake of maltose and efflux of glucose resultin reduced ATP yields from maltose dissimilation. When the protonssymported with maltose are expelled via the plasma membraneATPase complex, the combination of maltose uptake via protonsymport and glucose efflux via facilitated diffusion resultsin a net hydrolysis of ATP (Fig. 1). The physiological responseof S. cerevisiae to excess maltose reported here is less dramaticthan that reported in a previous study (41), in which exposureto excess maltose resulted in a loss of viability and cell lysis.The reason for this difference is not known but may be relatedto the use of a different strain background. Furthermore, theexperimental conditions were different; in contrast to the dataobtained in the present study, the data reported by Postma etal. (41) were obtained with aerobic, respiring cultures.

The detrimental effects of an imperfect balance between maltoseuptake and glucose dissimilation are likely to be relevant forthe development of maltose-constitutive S. cerevisiae strainsfor baker's yeast production and brewing. For example, constitutiveoverproduction of maltose permease and maltase, which has beenproposed as a means to increase fermentative capacity with maltoseas the substrate, is likely to result in an imbalance betweenmaltose uptake and glycolysis. This complication may also occurin other cases where disaccharides are transported via protonsymport and hydrolyzed intracellularly. A relevant example isthe metabolic engineering of S. cerevisiae for lactose fermentationby constitutive expression of a lactose-proton symporter andintracellular beta-galactosidase (1, 47, 48).

To prevent the dissipation of metabolic energy that resultsfrom noncoordinated uptake and hydrolysis of disaccharides,two regulatory mechanisms can be envisaged. One possible mechanismwould involve downregulation of disaccharide hydrolysis (forinstance, by glucose inhibition of the disaccharide hydrolase).Although maltase in S. cerevisiae is not known to be regulatedvia glucose inhibition or glucose catabolite inactivation, transcriptionof the MALx2 genes is subject to glucose repression (16, 17).

The second possibility is downregulation of maltose uptake tomatch the uptake rate to the glycolytic activity of the cells(for example, by glucose repression of the synthesis of maltosepermease or glucose-induced inactivation of the maltose carriers).Albeit with different time scales, both mechanisms should leadto a situation where the level of maltose permease is adaptedto the capacity of glucose metabolism. The intricate mechanismsdescribed for glucose repression and glucose inactivation ofthe S. cerevisiae maltose permease are generally explained byconsidering glucose the preferred carbon source (32, 62). Sucha preferred status of glucose can be explained by the lowerATP yield from maltose due to an energy requirement for maltoseuptake (49, 59). The present study offers an alternative, additionalexplanation: these control systems may have evolved to preventATP dissipation via simultaneous energy-dependent maltose uptakeand glucose efflux.

ACKNOWLEDGMENTS

We thank Hans van Dijken for many stimulating discussions andEckhard Boles for providing the hexose-transport-negative strain.

This work was financially supported by the Dutch Ministry ofEconomic Affairs via the EET program.

FOOTNOTES

* Corresponding author. Mailing address: Kluyver Laboratory of Biotechnology, Julianalaan 67, 2628 BC Delft, The Netherlands. Phone: 31 15 278 3214. Fax: 31 15 213 3141. E-mail: J.T.Pronk{at}TNW.TUDelft.NL.

REFERENCES

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