APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1992, p. 2271-22790099-2240/92/072271-09$02.00/0Copyright C) 1992, American Society for Microbiology

Application of 13C Nuclear Magnetic Resonance To Elucidatethe Unexpected Biosynthesis of Erythritol by

Leuconostoc oenos

MARIA VEIGA-DA-CUNHA,l PAULA FIRME,12 M. VITORIA SAN ROMAO, 1.3AND HELENA SANTOS14*

Centro de Tecnologia Quimica e Biol6gica, Rua da Quinta Grande 6, Apartado 127, 2780 Oeiras, 1* EstacdoNacional de Tecnologia dos Produtos Agrdrios, Lisbon,2 Estaqdo Vitivinicola Nacional, Instituto Nacional de

Investigaq4o Agrdria, 2575 Runa, and Departamento de Quimica, Faculdade de Ciencias e Tecnologia,Universidade Nova de Lisboa, Quinta da Torre, 2825 Monte da Caparica 4 Portugal

Received 24 February 1992/Accepted 2 May 1992

Natural-abundance "3C nuclear magnetic resonance ("3C-NMR) revealed the production of erythritol andglycerol by nongrowing cells of Leuconostoc oenos metabolizing glucose. The ratio of erythritol to glycerol wasstrongly influenced by the aeration conditions of the medium. The elucidation of the metabolic pathwayresponsible for erythritol production was achieved by 13C-NMR and 'H-NMR spectroscopy using specifically3C-labelled D-glucose. The 1H-NMR spectrum of the cell supernatant resulting from the metabolism of[2-'3C]glucose showed that only 75% of the glucose supplied was metabolized heterofermentatively and that theremaining 25% was channelled to the production of erythritol. The synthesis of this polyol resulted from thereduction of the C4 moiety of the intermediate fructose 6-phosphate. Oxygen has an inhibitory effect on theproduction of erythritol by L. oenos. Preaeration of a suspension of nongrowing cells ofL. oenos resulted in 30%oless erythritol and in 70%o more glycerol formed during the anaerobic metabolism of glucose. The anaerobicproduction of erythritol from glucose was also found in growing cultures ofL. oenos, although to a smaller extent.

The usage of bacterial starter cultures to initiate malolacticfermentation in wines is becoming widespread, especially innewer wine regions (16). Among the strains able to convertmalic acid to lactic acid, Leuconostoc oenos is often favoredfor the preparation of the successful commercial cultures (2,3, 8, 17, 26). In the natural wine fermentations, after a lagphase, the lactic acid bacteria surviving the alcoholic fer-mentation commence multiplication to conduct the malolac-tic fermentation. Almost invariably, L. oenos is the mainspecies that develops here (35).However, the malolactic reaction is not the only metabolic

process performed by L. oenos in wine. Together with malicacid, this strain uses other carbon substrates (e.g., residualsugars and citrate) that are likely to have subtle influences onthe sensory qualities of wine (9). Yet the metabolism ofcarbohydrates by wine lactic acid bacteria (especially L.oenos) is an area of wine biochemistry that so far has notreceived much attention and is still poorly understood.The metabolism of glucose in heterolactic bacteria is

described (15, 31) as a fermentation initiated by the oxidationof glucose 6-phosphate to gluconate 6-phosphate followed bydecarboxylation and splitting of the resulting pentose5-phosphate into a C-2 and a C-3 moiety. Therefore, equi-molar amounts of C02, lactate, and acetate or ethanol are

expected from glucose. However, this pathway of glucosefermentation has not been fully confirmed in L. oenos (11).

In vivo 13C nuclear magnetic resonance (13C-NMR) spec-troscopy can provide precise information on the variationsof metabolite levels and fluxes through biochemical path-ways in a noninvasive way (19, 21, 28). In the present work,'3C-NMR was used to elucidate the metabolic pathway forthe metabolism of glucose under different conditions of

* Corresponding author.

aeration with isotopically enriched substrate. This revealedthe production of unexpectedly high levels of glycerol anderythritol by L. oenos GM, a commercially available bacte-rial starter culture used to initiate malolactic fermentation inwines (17).

MATERIALS AND METHODS

Organism and growth conditions. L. oenos GM is commer-cially available from Microlife Techniques, Sarasota, Fla.The culture was kept at -20°C after revival from thelyophilized form and was resuspended in sterile FT 80growth medium (5) with added 20% glycerol.For the NMR experiments, the bacteria were grown in FT

80 medium (pH 4.8) without MnSO4 and Tween 80. Theglucose and fructose used (each 5.0 g- liter-') were auto-claved separately and added before inoculation of the cul-ture. For each NMR experiment, the cells were grown staticin five 2-liter glass bottles for 3 days at 25°C (3% inoculum).For the growth experiments, the same FT 80 medium was

used but the sugar added was glucose only (7.0 g liter-').L. oenos GM was cultured in a 1.8-liter Biolab ModularMinifermentor (B. Braun Diessel Biotech, Melsungen, Ger-many) inoculated as described above. The pH of the culturewas maintained automatically at 4.8, and the temperaturewas set at 25°C. The cultures were stirred at 250 rpm.Silicone antifoaming agent (Union Carbide Chemicals andPlastics S.A., Versoix, Switzerland) was added to preventexcessive foaming. Anaerobic conditions were maintainedby continuous flushing of N2 gas through the culture. The gaswas homogeneously dispersed in the medium by using an

adequate glass air disperser associated with the constantstirring of the medium. Growth was monitored by measuringthe A600-Sample preparation for the NMR experiments. Fresh cell

2271

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2272 VEIGA-DA-CUNHA ET AL.

suspensions of L. oenos GM were prepared for each exper-iment. The cells were harvested during early exponentialgrowth (A600, approximately 0.15; 0.08 mg [dry weight].ml-'). The culture was centrifuged for 15 min at 2,000 x g at4°C, washed twice with 0.2 M potassium phosphate (pH 4.8),suspended in the same solution to a final volume of approx-imately 3.5 ml, and transferred to a 10-mm NMR tube. H20was added to a final concentration of approximately 5% (byvol) to provide a lock signal. The dry weight of the resultingcell suspension was 0.12 to 0.15 g of cells.NMR spectroscopy. '3C-NMR spectra were recorded at

30°C with a Bruker AMX-300 spectrometer operating at75.47 MHz. For the in vivo experiments using labelledglucose, 13C-NMR spectra (1.75 min each) were consecu-tively acquired following the addition of 25 ,umol of glucose.A pulse of 450 and a repetition time of 0.95 s were used.Proton decoupling was continuously applied by using theWALTZ (wideband alternating phase lower-power tech-nique for zero residue splitting) sequence. Efficient mixingand supply of 02, N2, C02, or air to the cell suspension wereachieved with a simple air lift system (29). Once the sub-strate was exhausted, the cells were removed by centrifuga-tion (20 min at 18,000 x E) and the liquid supernatant was

kept for further '3C- and H-NMR analyses. The '3C-NMRspectra of the cell supernatants were run with a 45° pulse,13-s repetition time, and 32,768 (32 K) acquisition datumpoints. Proton broad band decoupling was applied during theacquisition time only (0.85 s).1H-NMR spectra were recorded at 25°C with a Bruker

AMX-500 spectrometer operating at 500.13 MHz with H20presaturation and using a 45° pulse and a repetition time of9.5 s.Resonance assignments were made by adding the pure

compounds to the cell supernatants.Quantification of the fermentation substrates and end prod-

ucts. The fermentation end products were measured in thecell supernatants. Glycerol was measured by using theappropriate Test Combination Kits of Boehringer MannheimInc. Acetate was measured enzymatically with a SkalarSegmented Flow Analyser (Skalar Analytical B.V., Breda,The Netherlands). For quantitative measurements of eryth-ritol and glyceraldehyde, 13C-NMR spectra of the cell super-natants were acquired as described above. The acquisitionconditions used allowed full relaxation of the resonancescorresponding to the CHOH and CH2OH groups of glycerol,erythritol, and glyceraldehyde. Erythritol and glyceralde-hyde were then measured by comparing the intensities of theresonances corresponding to their CH2OH groups with thoseof glycerol. The quantification of the remaining metabolites,lactate, ethanol, and alanine, was done by comparing theintensities of the resonances of their methyl groups with thatof the methyl group of acetate in the 1H-NMR spectra.For the growth experiments, acetate and glycerol were

measured enzymatically as described above. Glucose, L-(+)-malate, and L-(+)- and D-(-)-lactate were also measuredenzymatically with the Skalar Segmented Flow Analyser.Ethanol was estimated by headspace chromatography usingan HP 5890 Series II gas chromatograph equipped with a

hydrogen flame ionization detector and a fused-silica capil-lary PERMABOND FFAP-DF-0.25 column (25 m by 0.32mm). Erythritol was measured by gas chromatography usingan HR 5300 Mega Series gas chromatograph equipped with a

hydrogen flame ionization detector and an OV-1 fused-silicacapillary column (30 m by 0.32 mm); the carrier gas used wasH2. Erythritol was first converted to its trimethylsilyl deriv-ative (30), inositol being used as an internal standard.

Chemicals. D-[6-'3C]Glucose was purchased from Cam-bridge Isotope Laboratories, Cambridge, Mass. Both D-[1-13C]glucose and D-[2-13C]glucose were supplied by CamproBenelux, Veenendaal, The Netherlands. All three com-pounds were 99% isotopically enriched. The enzymes for theenzymatic methods were obtained from Boehringer Mann-heim Inc. All other chemicals were of reagent grade.

RESULTS

Products from glucose, fructose, and ribose metabolism ofnongrowing cells as determined by '3C-NMR analysis. Themetabolism of glucose by nongrowing cells of L. oenosGM was investigated by in vivo 13C-NMR analysis. Theconsumption of glucose and the metabolites formed weremonitored under anaerobic (CO2 or N2) and aerobic (02 orair) conditions. Consecutive spectra showed that glucosewas consumed faster anaerobically (on average, 3.5,umol. min-' g-1 of cells [dry weight]) than aerobically(1.6 ,umol min'- g-1 cells [dry weight]) with no significantdifference between each of the two gas phases used.

In addition to the expected lactate, ethanol, and acetate,L. oenos was found to produce erythritol and glycerol as twomajor fermentation products (Fig. 1). In the absence of 02,the concentration of erythritol formed was three times higherthan that of glycerol and six times more acetate than ethanolwas detected. When 02 was present, the production ofglycerol increased threefold and erythritol was not detected.There were less ethanol and lactate produced, and thealready high level of acetate formed anaerobically was1.3-fold higher. These results were repeated several times(Fig. 2), showing that under anaerobic conditions the metab-olism of glucose was independent of the presence of N2 orCO2 in the gas phase and that under aerobic conditions the02 present in air was sufficient to cause the changes ob-served. The end products and the carbon balance (Table 1)of two typical anaerobic and aerobic fermentations showedthat 93 and 85% of the glucose carbon, respectively, wererecovered. Since the amount of CO2 formed could not bedirectly measured, it was calculated once the pathway ofglucose metabolism was established.When L. oenos fermented either fructose or ribose, nei-

ther erythritol nor glycerol was produced. Anaerobically,most of the fructose was metabolized to mannitol, the otherend products being acetate, lactate, and small amounts ofethanol. Aerobically, fructose was only partly consumed,and in this case acetate was the major metabolite formed.The mannitol produced decreased, and lactate and ethanolwere absent. During the metabolism of ribose, lactate andacetate were the only two end products formed anaerobi-cally, and if air was used instead of N2, most of the lactateformed was replaced by acetate (data not shown).

Elucidation of the pathway of erythritol production by using'3C-labelled glucose (13C-NMR). If glucose were metabolizedexclusively via the heterofermentative pathway, the isotopi-cally enriched carbon from [2-13C]glucose should all berecovered in the methyl groups of acetate and/or ethanol (seeFig. 4). This was not observed (Fig. 3B), and instead, the 13Cspectra of the cell supernatants after the metabolism of[2-13C]glucose under N2 atmosphere revealed the presenceof labelled carbon not only on the CH3 groups of acetate andethanol but also in the COOH group of acetate and in theCH20H group of ethanol. The hypothesis that not all of theglucose was metabolized heterofermentatively was con-firmed when the above experiment was repeated using[1-13C]glucose. In this case (Fig. 3A), not all of the isotopi-

APPL. ENVIRON. MICROBIOL.

13C-NMR OF GLUCOSE METABOLISM IN L. OENOS 2273

-

a,o

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40

a

a

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C)

70 65PPM

4)400co

ILLI_ 1""L. a .16..IL A

180 90 80 70 60 50 40 30 20PPM PPM

FIG. 1. Natural-abundance 13C-NMR spectra of the end products formed during the anaerobic (A) and aerobic (B) metabolism of glucoseby nongrowing cells of L. oenos GM. Glucose (100 iLmol) was metabolized as described in Materials and Methods. Once all the glucose hadbeen used, the cell suspension was centrifuged and the products in the liquid supernatant (pH 4.9) were analyzed by 13C-NMR spectroscopy.

cally enriched carbon from glucose was lost as '3Co2, andthe labelled end products detected were '3CH3COOH and13CH3CH20H.These two experiments showed that part of the glucose

fermented was not decarboxylated and instead was divertedto an alternative acetate-ethanol-forming pathway. To verifythat erythritol was also formed as a result of this diversion ofC-6 molecules from the usual heterofermentation, the sameexperiment was repeated under aerobic and anaerobic con-ditions using [6-'3C]glucose. Anaerobically (Fig. 3C), theresonances found were assigned to the CH3 of lactate andthe CH20H groups of erythritol and glycerol. The spectra

obtained after the aerobic metabolism of glucose (Fig. 3D)confirmed the decrease in the production of erythritol andlactate (Fig. 1) and furthermore revealed the presence ofacetate labelled on the CH3 group. Figure 4 shows thepathway proposed for glucose metabolism by L. oenos thatexplains the observations described above.

Elucidation of the pathway of erythritol production by using13C-labelled glucose ('H-NMR). 1H-NMR spectra of theliquid supematants resulting from the metabolism of selec-tively labelled glucose and previously analyzed by 13C-NMR(Fig. 3) are shown in Fig. 5. These experiments gaveinformation on the isotopic enrichment of each of the proto-

VOL. 58, 1992

40

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2274 VEIGA-DA-CUNHA ET AL.

Products formed(pmoles)

300

200

100

0

CO2 N2 02 Air

|Ethanol __ co0

Lactate LI Erythritol

Acetate Glycerol

[/ss Alanine Glyceraldehyde

FIG. 2. Mass balance of the glucose metabolites obtained afteranaerobic or aerobic utilization of glucose by nongrowing cells of L.oenos GM. The end products were determined as described inMaterials and Methods.

nated carbons in the end products. The 'H-NMR spectra(Fig. 5) show a singlet at 1.93 ppm assigned to the methylgroup of unlabelled acetate. 1 CH3COOH gives rise to adoublet centered at the same position and with a couplingconstant of 130 Hz (Fig. 5A and B), due to coupling betweenthe methyl carbon and the attached protons. CH313COOHgives rise to another doublet with a smaller coupling con-stant (6.6 Hz; Fig. SB), due to coupling between the methylprotons and the neighbor carboxylate carbon. By comparingthe integrated areas of the two doublets it is possible todetermine the carbon flux between the heterofermentative

TABLE 1. Products from aerobic and anaerobic metabolism ofglucose by nongrowing cells of L. oenos GM

,Lmol of product formed fromEnd product 100 pLmol of glucose ina:detected

N2 Air

Acetate 66 87Lactate 49 13Ethanol 10 1CO2 63 133Erythritol 37 0Glycerol 12 37Glyceraldehyde 0 11Alanine 3 6

a Each experiment was repeated at least three times. The glucose supplied(100 xLmol) was totally consumed under both N2 and air. The end productswere determined as described in Materials and Methods.

and the erythritol-forming pathways (Fig. 4). Therefore,under anaerobic conditions, nongrowing cells of L. oenosGM metabolized heterofermentatively 75% of the 25 ,umol ofglucose supplied and the remaining 25% was channelled tothe production of erythritol.The ratio of the three isotopomers of acetate (Fig. SB) was

found to be 10:8:3 (CH3COOH to 13CH3COOH toCH313COOH), showing that approximately 50% of the ace-tate formed resulted from the metabolism of intracellularlystored compounds. The hypothesis that part of the unla-belled acetate could result from a decarboxylation of pyru-vate was not confirmed when 13CH3COOH production wasfound to be absent after the anaerobic metabolism of[6-13C]glucose (Fig. 3C).The other two 1H-NMR spectra (Fig. SA and C) from the

products formed during the metabolism of glucose isotopi-cally labelled on C-1 and C-6, respectively, agree with theresults obtained with the 13C-NMR spectra (Fig. 3A and C).Figure SA confirms the production of labelled acetate on themethyl group (via the erythritol-forming pathway) from[1-13C]glucose. Figure SC shows the central doublet (5 =1.32 ppm) and the two lateral doublets that are due, respec-tively, to the unlabelled and labelled methyl groups of lactateproduced from [6-13C]glucose. In this case labelled acetatewas not formed.

Effect of 02 on the metabolism of glucose. Oxygen affectedthe metabolism of glucose by nongrowing cells of L. oenosGM by decreasing erythritol, ethanol, and lactate levels aswell as increasing glycerol, glyceraldehyde, and acetatelevels (Table 1; Fig. 1). The inhibitory and stimulatoryeffects of oxygen, respectively, on erythritol and glycerolproduction were tested by measuring both polyols in theliquid supernatants of two cell suspensions that had metab-olized glucose anaerobically. Before the glucose was sup-plied, each of the two cell suspensions was flushed for 2 h at30°C with N2 or 02. The cells that remained in anaerobiosisbefore and during glucose metabolism formed 15 ,umol ofglycerol and 19 ,umol of erythritol from 100 ,umol of glucose.However, if the suspension was aerated before glucosemetabolism, the production of erythritol decreased to 13,umol and that of glycerol increased to 33 ,umol.Production of erythritol by anaerobically growing cultures.

The anaerobic production of erythritol by nongrowing cells(Table 1) was also demonstrated when growing cultures of L.oenos metabolized glucose under an N2 or CO2 atmosphere(Table 2). Growth stopped after 25 days of incubation at25°C. At this stage, the increase in biomass had ended eventhough 30% of the glucose supplied (37 mM) remained in themedium. However, the L-(+)-malate available (49 mM) wasentirely converted to the same isomer of lactate during thefirst 12 to 15 days of growth.From the metabolism of the 22.6 mM glucose used, L.

oenos formed ethanol (16 mM), acetate (5 mM), D-(-)-lactate (19 mM), glycerol (0.5 mM), and erythritol (1.9 mM).Therefore, after 25 days of growth in anaerobic conditionsthis organism channelled 8.5% of the glucose metabolized toform erythritol.

DISCUSSION

The production of erythritol and/or glycerol by microor-ganisms is usually associated with fungi (10) or yeasts (4, 12,14). These eukaryotic microorganisms are described asaccumulating polyols or their derivatives in response toincreased external osmotic pressure (7, 22). Therefore, theoccurrence of these low-molecular-weight soluble sugar al-

APPL. ENVIRON. MICROBIOL.

VOL. 58, 1992

Ery- C1,4

13C-NMR OF GLUCOSE METABOLISM IN L. OENOS 2275

DI

Gly- C1,3

Lac-C3

C

Ac-C1

I I pI

180I I I

175 70 65PPM

Ac-C2

Eth-C2

_,- sIL60 25 20

FIG. 3. '3C-NMR spectra of the products formed anaerobically from [1-13C]glucose (A), [2-13C]glucose (B), and [6-13C]glucose (C) andaerobically from [6-13C]glucose (D) by nongrowing cells of L. oenos GM. Glucose (25 ,umol) was metabolized as described in Materials andMethods. Once all the glucose had been used, the cell suspension was centrifuged and the liquid supernatant (pH 4.9) was analyzed by13C-NMR spectroscopy. Ac-C1 and Ac-C2, C-1 and C-2 of acetate; Eth-C2, C-2 of ethanol; Lac-C3, C-3 of lactate; Gly-C1,3, C-1 and C-3 ofglycerol; Ery-C1,4, C-1 and C-4 of erythritol.

cohols has been described as the way that salt-tolerantorganisms exclude the stress solute and achieve intracellularadjustment by a selective accumulation of solutes morecompatible with cell enzymes (32).

However, in L. oenos the mechanism responsible forpolyol production during the metabolism of glucose is notdue to a salt shock response. Erythritol and glycerol, whichare produced during the metabolism of glucose, are absent

185

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2276 VEIGA-DA-CUNHA ET AL.

6CH20H01

2

GLUCOSE

1 r ATP

Glucose 6-P Fructose 6-P

2 9 2NADH+H

IC02

2CH20HCOCHOHCHOH61CH2OPO3H2

Xylulose 5-P

3COH NAD H3P04 3CH20HH IHHCHOH 1 CHOHCHOH 10 CHOH

61 61CH2OPO3H2 CH20H

Erythrose 4-P Erythritol

9

1~~ATP

CH3 'CH3COOP03H2 2COOH

Acetate5 2NAD+

OH31 3

2CH20HEthanol

2CH3 ATPI kCOOH * -

4Acetate

OCH COH NAD+ H3POI I

. COOPO3H2 CHOH

CH2OPO3H25 42NAD 6 NADH+H+

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2CH3CH20H COOH CO2 NADH

Ethanol CO661CH3

Pyruvate

14 CH20H NADH+H CH20HI I

s- CHOH . CHOH61 61CH20H COH

Glycerol Glyceraldehyde

l+H+ ATP

COOPO3H2 2J COOH61 61CH3 CH3

Acetate

7 NAD

COOHCHOH6COH3

LactateFIG. 4. Pathways of glucose metabolism in L. oenos GM in aerobic and anaerobic conditions. 1, hexokinase; 2, glucose 6-phosphate

dehydrogenase, 6-phosphogluconate dehydrogenase, and ribulose 5-phosphate 3-epimerase; 3, phosphoketolase; 4, acetate kinase; 5,phosphotranscetylase, acetaldehyde dehydrogenase, and alcohol dehydrogenase; 6, enzymes of the Embden-Meyerhof-Parnas pathway; 7,lactate dehydrogenase; 8, glycerol dehydrogenase, glycerol 3-phosphate phosphotransferase; 9, phosphoketolase; 10, erythritol dehydroge-nase and erythrose 4-phosphate phosphotransferase.

during fructose or ribose fermentation, even though thebuffer used always had the same salt concentration. Further-more, the sugar alcohols formed were mostly excretedinstead of being accumulated inside the cell, as they shouldhave been if their role was to stabilize intracellular enzymes.Therefore, in L. oenos, the biosynthesis of erythritol and

glycerol during glucose metabolism must have a differentcause.The production of glycerol by L. oenos has been previ-

ously described (27), and its pathway is similar to the onedescribed for the yeast species (1). Glycerol is synthesizedby reduction of the intermediate metabolite glyceraldehyde

APPL. ENVIRON. MICROBIOL.

1.

V3C-NMR OF GLUCOSE METABOLISM IN L. OENOS 2277

CH313C00

COO

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O COOH

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131

alanineH valine

COOH ;| ethanol A

A S _B

Ai A

I I II

2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8PPM

FIG. 5. 1H-NMR spectra of the products formed anaerobically from [1-_3C]glucose (A), [2-'3C]glucose (B), and [6-'3C]glucose (C) bynongrowing cells of L. oenos GM. Glucose (25 ,umol) was metabolized as described in Materials and Methods. Once all the glucose had beenused, the cell suspension was centrifuged and the liquid supernatant (pH 4.9) was analyzed by 1H-NMR spectroscopy after a fivefold dilutionin 2H,O.

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2278 VEIGA-DA-CUNHA ET AL.

TABLE 2. Production of erythritol by anaerobically growingcells of L. oenos GM

Incubation time Growth Glucose used Erythritol formed(days) (60) (mM) (mM)

0 0.008 0.0 0.001 0.028 0.2 0.004 0.096 1.0 0.055 0.115 2.0 0.087 0.191 4.1 0.33

11 0.277 13.1 0.4415 0.294 18.8 0.6318 0.278 19.4 0.8021 0.283 20.8 1.2025 0.280 22.6 1.90

3-phosphate to glycerol 3-phosphate by a glycerol 3-phos-phate dehydrogenase, followed by dephosphorylation of theglycerol 3-phosphate to glycerol. During this process NADH+ H+ is recycled into NAD+ (Fig. 4).The production of erythritol has to our knowledge been

reported only once for the wine lactic acid bacteria (25), butno attempt was made to elucidate the biochemical pathwayresponsible for production of this polyol. The erythritolfound in yeast and fungus species is synthesized via thepentose 5-phosphate pathway. For example, in the hyphalcells of Aspergillus nidulans the synthesis of erythritol viathe pentose 5-phosphate pathway is favored by growth underglycolytic rather than gluconeogenic conditions (10). Underthese conditions, the high carbon fluxes through both thepentose 5-phosphate pathway and glycolysis generate suffi-cient reduction capacity to lead to the overflow of thevarious polyols. In L. oenos, our results reveal a differentpathway for the synthesis of erythritol as well as show thatnongrowing cells do not metabolize a significant amount ofglucose via the pentose 5-phosphate pathway. As suggestedfrom Fig. 4, we have explained the production of erythritolas resulting from the reduction and dephosphorylation of aC-4 moiety by an erythritol dehydrogenase and a phos-photransferaselike enzyme, the C-4 compound, resultingitself from the splitting of a C-6 intermediate metabolite by aphosphoketolase. This C-6 compound is most likely fructose6-phosphate originating from the isomerization of glucose6-phosphate, and the phosphoketolase is either the same ora different enzyme from the one catalyzing the splitting ofxylulose 5-phosphate into glyceraldehyde 3-phosphate andacetyl phosphate (Fig. 4). The remaining C-2 moiety isresponsible for the observed production of CH313COOHfrom [2-13C]glucose (Fig. 3B) and of 13CH3COOH and/or13CH3CH20H from [1-' C]glucose (Fig. 3A).The data obtained with [1-13C]glucose show that at least

part of the glucose is metabolized in a nondecarboxylativemanner, via a pathway other than the heterofermentativeand the pentose phosphate routes. CO2 fixation, whichwould also explain the incorporation of label into the CH3groups of ethanol and acetate, was never reported in lacticacid bacteria and is extremely unlikely since glucose wasmetabolized under vigorous N2 bubbling which removes theCO2 formed from the cell suspension. The results from themetabolism of [2-13C]glucose (Fig. 3B and SB) rule out anysignificant participation of the pentose 5-phosphate pathwayin the metabolism of glucose and the production of erythri-tol. In fact, if after decarboxylation of the intermediate[2-'3C]6-phosphogluconate part of the resulting [1-_3C]ribu-lose S-phosphate were to be used via the pentose 5-phos-

phate pathway, this would result in a redistribution of thelabel to the C-1 and C-3 of glucose 6-phosphate. From this[1,3-13C]glucose 6-phosphate, the percentage metabolizedheterofermentatively would also result in CH313COOHand/or CH313CH2OH. However, the remaining glucose thatwas shown to be metabolized via the nondecarboxylative(erythritol-forming) pathway should give rise to erythritollabelled on one of the CH2OH groups. Moreover, this[1,3-13C]glucose 6-phosphate, when channelled into the pen-tose 5-phosphate route, would also lead to the production oferythritol labelled also on one of the CH20H groups. Nev-ertheless, it is clear from the results shown in Fig. 3B that no[1-'3C]erythritol is formed from the metabolism of[2-13C]glucose and therefore that the pentose 5-phosphatepathway is not responsible for erythritol production in L.oenos.The shift from ethanol to acetate during aerobic metabo-

lism of L. oenos is explained by the way the alternativeroutes of acetyl phosphate metabolism are organized to takeadvantage of the presence and absence of 02 (6). The lowerlevels of lactate observed aerobically are explained by (i) theincrease in glycerol, limiting the amount of intermediateglyceraldehyde 3-phosphate that is converted to pyruvate,and (ii) the partial substitution of lactate by acetate, sparingthe cell NADH + H+. Similar mechanisms have beendescribed in other homolactic (18, 23, 33) and heterolactic(20, 34) lactic acid bacteria.However, the decline in erythritol and rise in glycerol

observed aerobically are to our knowledge unknown fea-tures of the metabolism of glucose by L. oenos. Thismetabolic shift can be explained in two ways. (i) The activityof the enzymes from the erythritol-forming system is irre-versibly affected by aeration, explaining why L. oenos formsless erythritol and more glycerol if prior to the anaerobicmetabolism of glucose the cell suspension is flushed with 02for 2 h at 30°C. A similar oxygen sensitivity by the enzymesfrom the ethanol-forming system in Leuconostoc mesen-teroides was reported to prevent this organism from metab-olizing glucose anaerobically when the cell suspension hadbeen preaerated (13). (ii) The production of erythritol isdirectly related to an accumulation of the intermediateglucose 6-phosphate in anaerobic conditions when NADH +H+ reoxidation is difficult. In this case, a nonspecific pen-tose phosphoketolase reacting with fructose 6-phosphatecould be responsible for the synthesis of erythrose 4-phos-phate and acetyl phosphate. Further research on the en-zymes involved in the production of erythritol as well as onthe shift from erythritol to glycerol metabolism when thecells are exposed to oxygen is currently on the way.The erythritol produced by growing cells (230 mg. liter-')

after 25 days of incubation at 25°C is in agreement with therange of values found for the concentration of erythritol intable wines (90 to 700 mg- liter-). The presence of eryth-ritol in table wines is often associated with mold-infectedgrapes (24); however, on the basis of the results describedhere, it is possible that at least part of the erythritol found inwine results from the metabolism of L. oenos.

ACKNOWLEDGMENTSWe are grateful to A. V. Xavier for critical reading of the

manuscript as well as for constant encouragement and to E. VanSchaftingen for helpful suggestions.

This work was supported by Junta Nacional de Investigac,oCientifica e Tecnol6gica and by the Biotechnology (BRIDGE)Program, contract BIOT-CT91-0263, of the Commission of theEuropean Communities.

APPL. ENVIRON. MICROBIOL.

13OC-NMR OF GLUCOSE METABOLISM IN L. OENOS 2279

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