Oxygen- and Glucose-Dependent Regulation of Central Carbon Metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxicmetabolism of the biocontrol and non-Saccharomyces wine yeastPichia anomala. In aerobic batch culture, P. anomala grows inthe respiratory mode with a high biomass yield (0.59 g [dryweight] of cells g of glucose^–1) and marginal ethanol,glycerol, acetate, and ethyl acetate production. Oxygen limitation,but not glucose pulse, induced fermentation with substantialethanol production and 10-fold-increased ethyl acetate production.Despite low or absent ethanol formation, the activities of pyruvatedecarboxylase and alcohol dehydrogenase were high during aerobicgrowth on glucose or succinate. No activation of these enzymeactivities was observed after a glucose pulse. However, afterthe shift to oxygen limitation, both enzymes were activatedthreefold. Metabolic flux analysis revealed that the tricarboxylicacid pathway operates as a cycle during aerobic batch cultureand as a two-branched pathway under oxygen limitation. Glucosecatabolism through the pentose phosphate pathway was lower duringoxygen limitation than under aerobic growth. Overall, our resultsdemonstrate that P. anomala exhibits a Pasteur effect and nota Crabtree effect, i.e., oxygen availability, but not glucoseconcentration, is the main stimulus for the regulation of thecentral carbon metabolism.

INTRODUCTION

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Introduction

The ascomycetous yeast Pichia (Hansenula) anomala is an efficientbiocontrol agent against mold infections in airtight storedgrain (11, 37), in apples (21), and on grapevines (28). P. anomalahas also been reported to be a non-Saccharomyces wine yeast(31, 44) and can be a spoilage yeast of food (23, 24) and fermenteddrinks (25). Growth and survival in these environments requirerapid adaptation of central carbon and energy metabolism toaerobic or oxygen-limited conditions, i.e., regulation of respirationand fermentation. Moreover, products of the glucose metabolismof P. anomala, in particular the volatile compound ethyl acetate,produced in significant amounts by this yeast (8, 38, 49), areimportant for wine aroma (41).

Respiration and fermentation in the model yeast Saccharomycescerevisiae are regulated by sugar availability (9, 19). A glucosepulse rapidly induces alcoholic fermentation and the activitiesof alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC).This induction is correlated with the accumulation of certainglycolytic metabolites (3) and an enhanced specific growth rate(40, 52). The sugar regulation in S. cerevisiae is an exceptionto most other known yeasts that respond to oxygen availabilityrather than to glucose concentration (51, 54). In the Crabtree-negativeyeasts Kluyveromyces lactis and Pichia stipitis, the inductionof PDC and ADH activity does not depend on the glycolytic fluxbut on the O₂ tension (22, 36). Contrary to the general convictionthat Crabtree-negative yeasts lack glucose induction of thealcoholic fermentation, this has been reported for K. lactis(22). P. anomala is a Crabtree-negative yeast but is able togrow anaerobically (18).

Individual enzyme activities or production rates can provideimportant metabolic insight. However, integrating such dataon isolated components into knowledge about the whole metabolicnetwork is rather complicated. Metabolic flux analysis basedon ¹³C labeling experiments provides a tool to study centralcarbon metabolism as a network (46). The general principle isthat uptake and secretion rates are interpreted within a stoichiometricmodel of metabolism, together with nuclear magnetic resonanceor mass spectrometry (MS) data on the distribution of ¹³C labelfrom a tracer experiment. The calculated fluxes are thus a bestfit to physiological and ¹³C data. Several yeasts were successfullyinvestigated by this method, including S. cerevisiae (20, 26),Saccharomyces kluyveri (32), and P. stipitis (13). Importantfindings derived from flux analysis include the discovery ofquantitative differences between yeasts with respect to theactivity of the pentose phosphate (PP) pathway, the importanceof anaplerosis for the contribution of oxaloacetate to the tricarboxylicacid (TCA) cycle, the activity of the TCA cycle, and the presenceof a mitochondrial malic enzyme.

No data are available on the regulation of the respiratory andfermentative enzymes in P. anomala, nor on the metabolic fluxesor product formation under controlled aerobic and oxygen-limitedconditions. This information is necessary to understand andpredict the growth and metabolic activity of P. anomala in differenthabitats like grain, silage, and various food environments.In addition, regulation of the central energy metabolism needsto be understood for determining and improving the role of P.anomala in biocontrol or wine making. Using flux analysis, quantitativephysiology, and in vitro enzyme activities, we attempt to providea baseline study for central carbon metabolism and its globalregulation in the yeast P. anomala.

MATERIALS AND METHODS

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Materials and Methods

Yeast strain and growth media.
The haploid strain NRRL-Y-366-8 (+; CBS 1984), derived fromthe diploid type strain NRRL-Y-366 (CBS 5759) of P. anomala(BioloMICS Database; http://www.cbs.knaw.nl/databases/index.htm),was chosen for the growth experiments in this study. Definedmedia were used for all cultivations (10, 34) and either glucose(20 g liter^–1), succinate (10 g liter^–1), or ethanol(2 g liter^–1) was used as the sole carbon source. Theinocula were precultivated for 20 to 30 h at 25°C, 150 rpm,in YNB medium (Difco Laboratories, Detroit, Mich.) with thesame carbon source as that for the subsequent batch cultivationand added to the fermentor vessel to an optical density at 600nm (OD₆₀₀) of 0.1 to 0.15. All growth experiments were repeatedat least three times.

Batch cultivation under controlled conditions.
All experiments were performed in a Belach fermentor with a1-liter working volume at 25°C. The pH was automaticallykept constant at 5.0 ± 0.1 by titration with 2 M KOH.The dissolved oxygen tension was measured with an autoclavableO₂ sensor (D100 series OxyProbe; Bradley James Corporation).

In aerobic cultivations the oxygen saturation was kept at 50%± 5% by regulating the stirring velocity between 100and 900 rpm at an aeration rate of 1 liter of air min^–1.In the oxygen shift experiments, the culture was flushed withnitrogen (Air Liquid, Malmö, Sweden) until the dissolvedoxygen tension reached 0%, which was achieved within 1 min.To avoid anaerobic conditions, the culture was aerated with0.3 liters of air min^–1 at a stirring speed of 90 rpm.The dissolved oxygen tension was always 0% after the shift,indicating that the oxygen demand of the yeast cells was higherthan the oxygen transfer rate from the gas phase to the medium,i.e., the cultures were oxygen limited (12).

Experimental setup.
In the oxygen shift experiments, cells were grown aerobicallyuntil the culture reached an OD₆₀₀ of 2 to 3. One sample wastaken from the aerobic culture (t₀) before the oxygen tensionwas shifted from 50 to 0%. Then samples were taken at 0.5, 2,4, 6, 8, and 12 h after the oxygen shift. In the glucose pulseexperiment, cells were grown aerobically with succinate as thesole carbon source until the culture reached an OD₆₀₀ of 2 to3. Samples were taken before glucose addition (t₀) and then0.5, 2, 4, 6, and 8 h after glucose addition (20 g liter^–1).

Growth measurements.
Growth was estimated by measuring the OD₆₀₀. The OD₆₀₀ was correlatedwith the dry weight of cultures growing in aerobic and oxygen-limitedconditions with controlled aeration. Dry weight was determinedgravimetrically from triplicate 10-ml samples of culture supernatantas described earlier (36). One OD unit corresponded to 0.42± 0.03 mg of cells (dry weight) (cdw) ml^–1.

Enzyme measurements.
Enzyme assays were done on fresh cell extracts at 30°C witha Beckman DU 600 spectrophotometer at 340 nm (Beckman Instruments,Inc., Fullerton, Calif.). Samples were taken 0 to 8 h afterglucose pulse and 0 to 12 h after shift to oxygen limitation(see Results). Samples of 50 to 100 ml of the supernatant werewithdrawn, and protein was extracted according to the methodof Ciriacy and Breitenbach (7). Protease inhibitors (Complete;Roche Diagnostics, Mannheim, Germany) were added to the crudeextract buffer (1 tablet, 50 ml^–1). The protein concentrationswere determined by comparison with standard solutions of bovineserum albumin by use of the Bio-Rad Laboratories protein assaybased on the work of Bradford (5).

The activities of ADH (EC 1.1.1.1) (1), PDC (EC 4.1.1.1) (50),and the NAD⁺-dependent aldehyde dehydrogenase (ALD; EC 1.2.1.5)(4) were analyzed. One activity unit corresponds to the amountof enzyme catalyzing the conversion of 1 µmol of substratemin^–1, and specific activities are expressed as unitsmilligram of protein^–1.

Determination of sugar and metabolite concentrations.
Glucose, succinate, and secreted metabolite concentrations weredetermined by high-pressure liquid chromatography analysis.Samples were prepared as previously described (17) and analyzedusing an HC-75 column (305 by 7.8 mm; Hamilton, Reno, Nev.)and an RI detector (Agilent 1100 Series; Agilent Technologies,Waldbronn, Germany; 40°C). The column was eluted at 60°Cwith 5 mM sulfuric acid at a flow rate of 0.6 ml min^–1.The metabolites were identified and quantified by comparisonof peak areas with standard curves obtained by analyzing authenticreference compounds.

Determination of ethyl acetate.
To allow for analysis of the volatile ester ethyl acetate, thegas outflow of the fermentor was led through two serial tubescontaining 4 ml of decane each (99%; Sigma). Oda et al. (33)showed that the organic solvent decane could be used as a hydrophobictrap for volatile esters. Ethyl acetate was quantified witha Hewlett-Packard 6890 gas chromatography column, equipped witha flame ionization detector and a capillary column (HP19091S-833;250 µm by 30 m). The oven temperature was programmed to60°C for 3 min followed by a temperature rise of 20°Cmin^–1 to 250°C, where it was held for 2 min. Samples(1 µl) were injected in splitless mode with hydrogen asthe carrier gas at 40 ml min^–1. The compound was identifiedand quantified by comparison of retention time and peak areasto external standards of known concentrations of ethyl acetate(Sigma).

¹³C labeling experiments.
The labeling experiments were carried out in batch culturesassuming pseudo-steady-state conditions during the exponentialgrowth phase (14, 47). ¹³C labeling of proteinogenic amino acidswas achieved by growth on 5 g of glucose liter^–1 (aerobicgrowth) or 10 g liter^–1 (oxygen-limited growth), suppliedas a mixture of 80% (wt/wt) unlabeled and 20% (wt/wt) uniformlylabeled [U-¹³C]glucose (¹³C, >99%; Martek Biosciences Corp.,Columbia, Md.). The inocula were pregrown under aerobic or oxygen-limitedconditions, respectively, and added to the fermentor vesselto an OD₆₀₀ of 0.01 (less than 1% of final OD) to minimize theinfluence of unlabeled glucose from the inocula. Aliquots wereharvested (three to four per cultivation) by centrifugationwhen the culture reached an OD₆₀₀ of 0.8 to 2 and washed withdistilled water. Cellular protein was hydrolyzed by incubatingresuspended cells in 6 M HCl for 24 h at 105°C. Subsequently,the samples were dried at 85°C. The free amino acids werederivatized at 85°C for 1 h with 25 µl of dimethylformamide and 25 µl of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide(15, 56). Gas chromatography-MS analysis was carried out asreported recently (14).

Metabolic flux ratio analysis.
The gas chromatography-MS data include the mass isotopomer distributionsof the following amino acids: alanine, aspartate, glutamate,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tyrosine, and valine.For each amino acid fragment ${alpha}$ , one mass isotopomer distributionvector (MDV) was assigned,

(1)
withm₀ being the fractional abundance of the lowest mass and m_i_>₀being the abundances of molecules with higher masses. The MDVwere corrected for naturally occurring isotopes as describedpreviously (14), to obtain the exclusive mass isotopomer distributionsof the carbon skeletons, which were used to calculate the aminoacid (MDV_AA) and metabolite (MDV_M) mass distribution vectors.Metabolic flux ratios were calculated from the MDV_M as describedpreviously (2).

¹³C-constrained flux balancing.
Intracellular fluxes were calculated with a flux model thatcomprised all major pathways of yeast central carbon metabolism(see Fig. 4). The resulting stoichiometric model of 27 linearequations is underdetermined, thus having an infinite solutionspace. Hence, additional constraints were required to uniquelysolve the mass balance equations and to estimate the in vivofluxes. For this purpose, a set of linearly independent equationswere obtained from METAFoR analysis to render the system solvable(see also Table 2).

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FIG. 4. Metabolite flux distributions in P. anomala. The numbers represent the percentages of carbon passing through each reaction mixture. The upper numbers in each set represent flux distribution for an aerobic culture, whereas the lower numbers are those of an oxygen-limited culture.

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TABLE 2. Metabolic flux ratios in P. anomala when grown aerobically and under oxygen limitation

The set included the upper bound of PEP through the PP pathway:

(2)
the fraction of mitochondrial oxaloacetate derivedthrough anaplerosis:

(3)
the fractionof PEP originating from cytosolic oxaloacetate:

(4)
and the upper and lower bounds for mitochondrialpyruvate derived through the malic enzyme (from mitochondrialmalate):

(5/6)
The biomass compositionand the resulting precursor requirements were used as describedfor S. cerevisiae (20). The minimization of errors from theextracellular fluxes, the metabolite balances, and the fluxratios was carried out as recently described (15).

RESULTS

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Results

Growth and fermentation profiles during batch cultivation of P. anomala.
To characterize the regulation of the respiratory and fermentativemetabolism, the yeast strain was cultivated in controlled aerobicand oxygen-limited batch cultures. Under aerobic conditions,P. anomala grew exponentially with a growth rate of 0.22 h^–1and a biomass yield of 0.59 g of cdw g of glucose^–1 (Table1). Only low amounts of ethanol, acetate, glycerol, and ethylacetate were detected (Table 1; Fig. 1), indicating a preferentiallyrespiratory metabolism. When the oxygen tension was reducedfrom 50 to 0%, growth was arrested for approximately 0.5 h.Growth resumed within 2 h, with a much lower growth rate thanthat for aerobic growth (Fig. 2 and Table 1). Under our experimentalconditions, we could not conclude whether the growth of theyeast cells was oxygen limited. Such limitation is characterizedby a linear growth curve (12). Indeed, linear regression ofthe growth curve yielded an R² value of 0.99. However, a fitto an exponential function also resulted in an R² value of 0.99,with a growth rate of 0.056 h^–1. The biomass yield wasreduced sixfold to 0.11 g of cdw g of glucose^–1. The productionrates of ethanol, glycerol, and ethyl acetate increased considerably(Table 1; Fig. 1). The ethanol yield was close to the theoreticalmaximum, indicating a preferentially fermentative metabolism.The glucose consumption rate increased more than twofold inresponse to oxygen limitation (Table 1). The ethanol productionrate of 6.2 ± 0.6 mmol g of cdw^–1 h^–1 duringfermentative growth was slightly lower than that in K. lactis(10 mmol g of cdw^–1 h^–1) and P. stipitis (0.38 gg of cdw^–1 h^–1, equal to 8.3 mmol g of cdw^–1h^–1) (22, 48) and significantly lower than the 24 mmolg of cdw^–1 h^–1 that has been described for anaerobicallycultivated S. cerevisiae (42).

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TABLE 1. Specific growth rate, production and consumption rates, and yields^a in P. anomala when grown aerobically, exposed to a shift in oxygen tension or to a glucose pulse^b

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FIG. 1. Semiquantitative analysis (see text) of ethyl acetate production in aerobic (t₀) and oxygen-limited batch cultures. Values are means ± standard deviations (n = 3).

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FIG. 2. Growth of P. anomala in batch cultures grown under aerobic conditions ( ${blacktriangleup}$ ) and after a shift to oxygen limitation ( ${blacksquare}$ ), on glucose as the sole carbon source.

Ethyl acetate production increased 10-fold within 1 h afteroxygen limitation (Fig. 1). The production rate then decreasedbut was still higher than that during aerobic conditions (4to 8 h after the shift; Fig. 1). The contact time between thegas outflow (containing ethyl acetate) and decane in the tubeswas only a few seconds (aeration rate, 0.3 or 1 liter min^–1in 2 x 4 ml of decane). Oda et al. (33) as well as Rojas etal. (45) measured ester concentration in decane that had beenin contact with the yeast cultures for 24 to 48 h. We thereforeused two serial tubes to trap as much ethyl acetate as possiblewithout creating a dilution effect from large decane volumes.During aerobic growth, all ethyl acetate was found in the firsttube, indicating a low and continuous production. In the firstsample after the oxygen shift (0 to 1 h), the concentrationwas almost equally high in both tubes, indicating that the contacttime was too short to trap all ethyl acetate. In the subsequentsamples (4 to 8 h after oxygen shift) the concentration in thesecond tube was 10 to 50% of that in the first. This shows thatthe decane trap method is only semiquantitative at high productionrates and that the increase in ethyl acetate production rateis probably even higher than that shown in Fig. 1.

To investigate if alcoholic fermentation in P. anomala can alsobe induced by glucose, a glucose pulse was applied to culturesgrown on succinate. With succinate as a carbon source, P. anomalagrew linearly. After the pulse, the cultures resumed exponentialgrowth within 6 h (Table 1). The growth rate (0.21 h^–1)and the glucose consumption rate (2.65 mmol g of biomass^–1h^–1) correspond to the values obtained from the aerobicbatch cultures grown on glucose (Table 1). The glucose pulsedid not induce ethanol formation within the time of analysis(8 h), but small amounts of glycerol, arabitol, and acetatewere detected in the growth medium (data not shown). Arabitolwas detected in low amounts under all growth conditions duringlate exponential and stationary phase, independent of glucoseconcentration or oxygen tension (data not shown).

There seems to be no strict glucose repression of the consumptionof ethanol, acetate, or succinate, since these compounds werecoconsumed with glucose when present in the culture (data notshown). The results show that P. anomala is a respiratory yeastthat lacks a Crabtree effect, i.e., having no induction of alcoholicfermentation in response to a high glucose concentration.

Enzyme activities during batch cultivation of P. anomala.
To investigate whether the oxygen-dependent changes in the respiratoryand fermentative metabolism are connected to enzyme regulation,we determined activities of the fermentative key enzymes, PDCand ADH, as well as ALD, an enzyme in the pyruvate bypass, underaerobic (t₀) and oxygen-limited conditions (0.5 to 12 h afterthe shift) in glucose-grown cultures. The three enzymes hadsignificant activities in the aerobic cultures (Fig. 3) withADH having the highest activity (2 U mg of protein^–1)and ALD having the lowest (100-fold lower than that of ADH).When the culture conditions were shifted from aerobic to oxygenlimited, PDC and ADH were activated threefold and reached maximumactivity after 6 h (Fig. 3a and b). ALD was only slightly activatedand reached its highest activity 12 h after the oxygen shift(Fig. 3c). The high enzyme activities of PDC and ADH in aerobicbatch culture might indicate a glucose-dependent induction ofthese fermentative enzymes. To further investigate the roleof glucose in the induction of the fermentative pathways, thesame enzyme activities were measured in cultures before (t₀)and after the addition of a glucose pulse (0.5 to 8 h afterthe pulse). We observed considerable activities for PDC, ADH,and ALD in cultures growing on succinate (Fig. 3). Both ADHand ALD activities were higher than in cells grown aerobicallyon glucose. After glucose addition, the ADH and ALD activitieswere down regulated (Fig. 3b and c). The activity of ADH reachedthe level of the aerobic glucose culture after 8 h (Fig. 3b).The activity of PDC was lower during growth on succinate thanduring growth on glucose and was not affected by the additionof glucose within the 8 h of analysis (Fig. 3a).

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FIG. 3. Specific enzymatic activities of PDC (a), ADH (b), and ALD (c) in crude extracts from cells grown on glucose under initially aerobic conditions (t₀) after a shift to oxygen limitation (•) and from cells grown on succinate (t₀) pulsed with glucose ( ${blacksquare}$ ). The curves show representative results from three independent cultivations.

¹³C metabolic flux analysis in aerobic and oxygen-limited batch cultures.
To monitor the global effects of oxygenation on metabolism,the metabolic flux distributions in P. anomala were determinedby metabolic flux analysis. For this purpose, extracellularfluxes (Table 2) and the known precursor requirements for thedetermined biomass formation were balanced in a stoichiometricmodel. The analytically determined flux ratios (Table 2) wereused as constraints in the calculation to obtain a unique fluxsolution (15).

The intracellular flux distribution changed significantly inresponse to oxygen availability (Table 2; Fig. 4). Under aerobicconditions, about 30% of the glucose-6-phosphate (glucose-6-P)was directly oxidized and entered the PP pathway (Fig. 4), whereasonly 10% of the glucose-6-P was metabolized via the glucose-6-Pdehydrogenase under oxygen limitation. The flux response tothe two aeration regimens was even more drastic at the pyruvatebranching point. Under aerobic conditions, the pyruvate fluxinto the mitochondria was 59%, but under oxygen limitation theflux was only 7% and pyruvate was mainly decarboxylated by PDC.Reducing oxygen availability caused a switch in operation ofthe TCA cycle from a cyclic activity to a two-branched pathwaythat sustained only synthesis of the biomass precursors oxaloacetateand ${alpha}$ -ketoglutarate. Akin to S. cerevisiae and P. stipitis (13),no flux was detected via the gluconeogenic PEP-carboxykinasethat catalyzes the reaction from oxaloacetate to PEP. Finally,low in vivo activities of the mitochondrial malic enzyme weredetected in the aerobic culture (Fig. 4). This contributionto the mitochondrial pyruvate pool was also reported for S.cerevisiae (20).

DISCUSSION

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Discussion

A decrease in oxygen tension was the inducing stimulus for theactivation of fermentation, and the doubling of the glucoseuptake rate shows that P. anomala exhibits a clear Pasteur effect.The activities of fermentative key enzymes PDC and ADH wereinduced threefold by oxygen limitation but not by a glucosepulse. In addition, glucose availability did not induce ethanolformation. This regulation pattern is in sharp contrast to S.cerevisiae, where a glucose pulse activates PDC, the fermentativeADH (ADH1), and ethanol production (53, 55).

Three major differences were observed in the flux distributionsof cultures grown under different aeration regimens. The fluxthrough the PP pathway was lower during oxygen-limited growth;more reduced metabolites, mainly glycerol and ethanol, wereproduced; and the TCA cycle operated as a cycle under aerobicgrowth but as a two-branched pathway under oxygen limitation.Our analysis shows that P. anomala is very similar to S. cerevisiaein the general flux distribution under respiratory and fermentativemetabolism (13, 20). However, the signals that induce respiratoryand fermentative metabolism are completely different. We obtainedrespiratory growth from aerobic batch cultures with a high glucoseconcentration, and fermentation could be induced only by shiftingthe cultures to oxygen limitation. In contrast, S. cerevisiaeshows fermentative behavior when cultivated in aerobic batchcultivation (19). This confirms that oxygen availability isthe main inducer of the fermentative metabolism in P. anomala.Among the respiratory yeasts, P. anomala is similar to K. lactis(22) with only minor fermentation despite the considerable activitiesof PDC and ADH, but different from P. stipitis (36).

The ADH and ALD activities were higher in cells grown on succinatethan in those grown aerobically on glucose. This was unexpected,as no ethanol was formed during these cultivations and no ethanolwas present as a carbon source. Possibly, the ethanol assimilationpathway is controlled by glucose repression but constitutivelyexpressed when no glucose is present. It is also possible thatit fulfills other, as yet unknown functions in the metabolism.Moreover, we also found considerable PDC activity during growthon succinate. We can presently only speculate about the functionof the PDC under these conditions. Flikweert et al. (16) demonstratedthat in S. cerevisiae the PDC is indispensable for growth onglucose. Possibly, in P. anomala this enzyme is also requiredfor growth on gluconeogenetic carbon sources.

The regulation of ethyl acetate production by P. anomala hasnot previously been studied under controlled oxygenation. Thiscompound essentially contributes to the aroma of wine (41).Ethyl acetate was produced during all aeration conditions, butthe production rate increased considerably after a shift tooxygen limitation. This result differs from those of Rojas etal. (45), who demonstrated a stronger ethyl acetate productionfrom a P. anomala wine strain under higher aeration. However,these experiments were done with shake-flask cultures whereoxygen limitation, despite rotation, is likely to occur (6).We recently demonstrated that oxygen limitation rapidly occurredin P. anomala shake-flask cultures even at low cell densities(17); thus, the high values from the wine yeast might be dueto a beginning oxygen limitation. In yeasts, ethyl acetate isproduced from ethanol and acetyl coenzyme A (CoA) by the actionof an alcohol acetyltransferase (EC 2.3.1.84) or from ethanoland acetate by the reversed action of an esterase (EC 3.1.1.1)(27, 30, 39, 43, 58). The contribution of these two enzymesto the production of ethyl acetate differs among several species(27, 57). Proposed reasons for the ethyl acetate productionare that the ester synthesis is (i) a detoxification mechanismto remove the toxic compounds acetate and ethanol from the cellsor (ii) a recirculation pathway for free CoA (CoA-SH), whichis liberated in the condensation reaction between acetyl-CoAand ethanol (29). The molar amount of ethyl acetate is only1% of the ethanol produced, and thus the removal is not significant.However, the removal of acetate may be important under oxygenlimitation, if the cell is more sensitive to intracellular organicacids under these conditions. However, the second hypothesisis more likely since the ester production is induced duringoxygen limitation when the lipid synthesis and the resultingrelease of CoA-SH are reduced. Moreover, the yeast grows inmany different environments and shows considerable antimicrobialactivity (35). We recently found an antifungal activity of ethylacetate during airtight storage of cereal grain (18a). Thus,the volatile compound might contribute to the competitivenessof P. anomala in closed environments, i.e., with limited gasexchange with the surroundings.

This metabolic and physiological study demonstrates that P.anomala is a respiratory yeast. Oxygen is the main regulatorof the central energy metabolism; glucose does not repress respirationand does not even completely repress the utilization of alternativecarbon sources. These findings also show that regulating oxygenavailability may be the main factor in manipulating the activityof P. anomala in its numerous habitats, including biocontroland wine making or as a spoilage yeast in beverages or yoghurt.

ACKNOWLEDGMENTS

This work has been financially supported by The Foundation forStrategic Environmental Research (MISTRA). Lars M. Blank gratefullyacknowledges financial support by the Deutsche Akademie derNaturforscher Leopoldina (BMBF-LPD/8-78).

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, Swedish University of Agricultural Sciences (SLU), P.O. Box 7025, SE-750 07 Uppsala, Sweden. Phone: 46 18 673212. Fax: 46 18 673392. E-mail: Elisabeth.Fredlund{at}mikrob.slu.se.

REFERENCES

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