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Nonstatistical 13C Distribution during Carbon Transfer fromGlucose to Ethanol during Fermentation Is Determined bythe Catabolic Pathway Exploited*

Received for publication, October 24, 2014, and in revised form, December 22, 2014 Published, JBC Papers in Press, December 23, 2014, DOI 10.1074/jbc.M114.621441

Kevin Bayle, Serge Akoka, Gerald S. Remaud, and Richard J. Robins1

From the Elucidation of Biosynthesis by Isotopic Spectrometry Group, CEISAM, UMR 6230, CNRS-University of Nantes, BP 99208,F-44322 Nantes, France

Background: Different fermentation pathways should lead to distinctive isotope patterns in the products.Results: Three pathways for glucose catabolism show discrete isotope patterns in ethanol.Conclusion: Catabolism by different enzyme sequences leads to differential isotope redistribution patterns.Significance: Learning how isotope fractionation occurs in nature is crucial for interpreting fractionation during biochemicaland physical processes for traceability.

During the anaerobic fermentation of glucose to ethanol, thethree micro-organisms Saccharomyces cerevisiae, Zymomonasmobilis, and Leuconostoc mesenteroides exploit, respectively,the Embden-Meyerhof-Parnas, the Entner-Doudoroff, and thereductive pentose phosphate pathways. Thus, the atoms incor-porated into ethanol do not have the same affiliation to theatomic positions in glucose. The isotopic fractionation occur-ring in each pathway at both the methylene and methyl positionsof ethanol has been investigated by isotopic quantitative 13CNMR spectrometry with the aim of observing whether an iso-tope redistribution characteristic of the enzymes active in eachpathway can be measured. First, it is found that each pathwayhas a unique isotope redistribution signature. Second, for themethylene group, a significant apparent kinetic isotope effect isonly found in the reductive pentose phosphate pathway. Third,the apparent kinetic isotope effects related to the methyl groupare more pronounced than for the methylene group. These find-ings can (i) be related to known kinetic isotope effects of some ofthe enzymes concerned and (ii) give indicators as to which stepsin the pathways are likely to be influencing the final isotopiccomposition in the ethanol.

Efforts to understand the causes of isotopic fractionation2 inthe 13C/12C ratio during metabolism are hampered by a lack ofdata on the intramolecular 13C composition of the compoundsinvolved. In particular, it is valuable to understand how differ-ent routes to the same product can influence isotope redistri-bution patterns. The anaerobic fermentation of glucose to eth-anol can be carried out by a number of different pathways in

which, although the substrate consumed and product accumu-lated are the same, the intermediate compounds and enzymesinvolved are not. It follows, therefore, that because isotopicfractionation is associated with reaction mechanism, differentpathways should lead to different isotope patterns in the finalproduct. The other principal factor that will determine the iso-topic composition of a product is the position-specific isotopiccomposition of the substrate.

A combination of these two phenomena, position-specificisotope ratios in the substrate and position-specific fraction-ation during metabolism, determines therefore the final iso-topic composition in the product(s) of fermentation. In previ-ous studies, we have shown by isotopic quantitative 2H NMRspectrometry that the isotopic fractionation in 2H is distinctivedepending on whether the Embden-Meyerhof-Parnas (EMP)3

pathway or the reductive pentose phosphate (RPP) pathway isused and that the hydrogen atoms in the product can be linkedto the positions in the substrate (1, 2).

Until recently, however, a similar approach to obtain 13C/12Cisotopic ratios was not possible. To exploit 13C NMR for thestudy of intramolecular 13C distributions, several additionaldifficulties had to be overcome. First, the isotopic variation of13C in natural compounds has a range �10-fold less than 2H(�50‰ and 500‰, respectively, on the �-scale). As a result,isotopic 13C NMR requires a 10-fold higher precision. Second,for the effective use of quantitative 13C NMR at natural abun-dance, uniform proton decoupling of 13C-1H interactions isrequired. This was achieved by the use of adiabatic decoupling(3). Third, slow relaxation times can lead to long acquisitiontimes and associated potential instability, a difficulty largelysolved by the use of relaxation agents (4). Now, this techniquecan be used for quantification of individual isotopomers byexploiting (i) the separation of the resonance signals from thedifferent carbon positions caused by their degree of shieldingand (ii) the direct relationship of the peak area to the amount of

* This work was supported in part by funds from the French Research Ministry(to K. B.) and by funding from the ISOTO-POL project through FrenchNational Research Agency Project ANR-11-CESA-009.

1 To whom correspondence should be addressed: Elucidation of Biosynthesisby Isotopic Spectrometry Group, CEISAM, UMR 6230, CNRS-University ofNantes, 2 rue de la Houssiniere, BP 92208, Nantes 44322, France. Tel.: 332-5112-5701; Fax: 332-5112-5712; E-mail: [email protected].

2 Isotopic fractionation is the selection of one isotopomer versus another dur-ing a physical or (bio)chemical process which leads to a nonstatistical dis-tribution of isotopes in the population of isotopomers within the finalproduct and the residual substrate.

3 The abbreviations used are: EMP, Embden-Meyerhof-Parnas; G6P, glucose-6-phosphate; GAP, glyceraldehyde-3-phosphate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; ED, Entner-Doudoroff; RPP, reductive pentose phos-phate pathway; KIE, kinetic isotope effect.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 7, pp. 4118 –4128, February 13, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

4118 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 7 • FEBRUARY 13, 2015

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13C resonating at a given frequency (5). Acquisition conditionsneed to be carefully defined and rigorously controlled to obtainrepeatable spectra of sufficient quality for the required preci-sion. Parameters such as concentration of sample, temperature,stability of the lock need to be the same, and spectra with asignal to noise ratio above 750 are recorded. The NOE has to beeliminated, and the efficiency of the 1H decoupling sequencemust be uniform over the whole range of 1H chemical shifts (12ppm). Studies (3, 5–7) on the optimization of an NMR meth-odology for accurate and precise measurements of 13C/12C iso-tope ratios have shed light on how the decoupling conditions of1H in 13C single-pulse NMR experiments strongly affect theprecision of measured peak surface areas. A major breakthroughwas achieved with the development of an optimized adiabatic 1Hdecoupling sequence (3). Provided this is respected, individual 13Cisotopomers can be observed and the absolute intramoleculardistributions of 13C can be determined (8). A curve fitting basedon a total line shape analyses is used to obtain the area undereach peak, which provides the reduced molar fraction fi/Fi of13C at each carbon position from which the �13Ci (‰) can becalculated (see “Experimental Procedures” and Ref. 9 for defi-nitions). The extent to which individual positions are eitherenriched or depleted in 13C relative to the statistical mean indi-cates metabolically induced isotope fractionation.

The determination of KIEs, calculated from isotope fraction-ation, is recognized as an important way to obtain mechanisticinformation about enzymes, because they are determined bythe rigidity of the bonds within the substrate and the transitionstate structure. They may be normal (KIE � 1) or inverse(KIE � 1), leading respectively to impoverishment or enrich-ment in the pertinent positions of the product. The magnitudeof the isotope effect is, in general, greater for primary KIEs, inwhich a bond to the atom under consideration is broken. How-ever, significant secondary isotope effects also occur: for exam-ple, a change in hybridization sp2-sp3 theoretically causes a nor-mal KIE, whereas a change sp2-sp3 generates an inverse KIE(10). When a metabolic pathway consisting of a cascade ofenzymes is studied, the observed fractionation will be thesummed effect of the KIEs of the enzymes involved. Because theenzymes are probably not active under fully saturating condi-tions, as each product of an enzymatic reaction is the substratefor the next reaction, kcat is not accessible (11). Nevertheless, aneffect on Vmax/Km can be obtained and an apparent KIE(appKIE) potentially characteristic of the overall pathway underconsideration.

That photosynthesis, wherein the assimilation of carbondioxide by plants can involve three types of metabolism: C3, C4and CAM, leads to different patterns of 13C distribution in theaccumulated hexoses has been clearly demonstrated, initiallyby indirect analysis following fragmentation (12) and morerecently with iq-13C NMR (13, 14). This shows that the pho-toassimilates are composed of mixtures of isotopomers that aredetermined by the physicochemical processes (15) and theenzymes (16) involved in the particular metabolism exploited.These mixtures of isotopomers are determined by the presenceof isotope effects in the Calvin cycle and indicate the role ofcertain enzymes in the 13C isotope discrimination. For exam-ple, the aldol condensation of glyceraldehyde-3-phosphate

(GAP) with dihydroxyacetone phosphate by fructose-1,6-bis-phosphate aldolase (EC 4.1.2.13) to form fructose 1,6-bisphos-phate could be identified as the origin of the relative 13C enrich-ment at the C-3 and C-4 positions of glucose (17).

However, very much less is known of the 13C fractionationimplicit in the pathways utilizing glucose, the postphotosyn-thetic isotope fractionation. Three well characterized pathwaysof microbial metabolism: the Embden-Meyerhof-Parnas, thereductive pentose phosphate, and the Entner-Doudoroff (ED),convert glucose to ethanol and are exploited by a variety ofmicro-organisms to carry out the anaerobic fermentation ofglucose. The first two are also active in plants.

Of these three pathways, only the EMP has been closelyexamined for position-specific 13C fractionation in ethanol bio-synthesis (13, 14, 18), whereas some data from the RPP are alsoavailable (12). These studies clearly demonstrated that fermen-tation by the EMP route leads to 13C/12C isotopic ratios for themethyl (CH3) and methylene (CH2) carbon positions that differdepending on the source glucose. However, in these studies theaim was complete degradation of glucose, so no information onKIEs during fermentation was obtained.

To better understand the origins of 13C isotope fractionationduring fermentation, we have fermented glucose to ethanolusing the three common pathways: EMP, ED, and RPP. Ethanolis a convenient analyte for this type of study because it is readilyproduced in large quantities by easy to cultivate micro-organ-isms and can be obtained in pure form by distillation. Key fea-tures of the three target pathways are illustrated in Fig. 1A, andthe origin of the carbons incorporated into ethanol is shown inFig. 1B.

The anaerobic oxidative catabolism of glucose to pyruvatevia the EMP pathway is exploited by a large range of organisms,including the yeast Saccharomyces cerevisiae, producing 2 molof each of ethanol and CO2 per mol of glucose (Fig. 1B). Withinthe present context, the critical feature is the homolytic split-ting of the six-carbon unit by fructose 1,6-bisphosphate aldol-ase (EC 4.1.2.13) to form two three-carbon units, which isom-erize and then follow the same path from GAP to pyruvate,thence, by decarboxylation, to ethanol. The ED pathway, whichis unique to prokaryotes, is less frequently exploited for anaer-obic oxidative catabolism, the bacterium Zymomonas mobilisbeing an example of an organism that uses it in a strictly fer-mentative sense. The ED pathway also produces 2 mol of eachof ethanol and CO2 per mole of glucose and also involves thehomolytic splitting of the six-carbon unit into two three-carbonunits. However, the key enzyme here is 2-dehydro-3-deoxy-phosphogluconate aldolase (EC 4.1.2.14), which produces 1mol of pyruvate and 1 mol of GAP. As a result, the pattern ofallocation of carbon to ethanol is modified (Fig. 1B). The RPP,in contrast to these two pathways, involves the heterolyticcleavage of the six-carbon unit into CO2, a two-carbon and athree-carbon unit (Fig. 1B). Although the three-carbon unit isagain GAP and is metabolized to pyruvate as in the EMP and EDpathways, it is not converted to ethanol but to lactic acid. Thetwo-carbon unit, acetyl-phosphate, is converted directly to eth-anol without the intervention of pyruvate. This pathway is usedprimarily by heterolactic bacteria, including species of Lactoba-cillus and Leuconostoc.

13C Isotopic Relationship in Glucose Fermentation

FEBRUARY 13, 2015 • VOLUME 290 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4119


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Early studies of 13C fractionation concluded “that glucosedoes not have large differences in the isotope ratios in the indi-vidual carbon atoms and that the Embden-Meyerhof reactionsdo not have a large carbon isotope effect” (19). However, at thattime (1961) isotope measurement by mass spectrometry gaveonly a value for �13Cg (‰), the global or mean isotope content,and the technique is poorly adapted to the determination of theintramolecular distribution of isotopes. Subsequently, mea-surement of �13Ci (‰), the position specific isotope distribu-tion, has proved the first part of the above conclusion to be

incorrect (12, 18): as yet, evidence is still to be presented thatglycolysis proceeds without position-specific isotopic discrim-ination. To probe this, we have carried out an analysis of �13Ci(‰) in ethanol from the EMP pathway. In addition, we haveexamined whether fermentation by alternative pathways bywhich the glucose is converted to ethanol by reactions involvingalternative mechanisms will indicate distinct 13C appKIEs.

EXPERIMENTAL PROCEDURES

Materials—D-Glucose (batch 071M014552V), L-ascorbic acid,KH2PO4, and (NH4)2SO4, were obtained from Sigma-Aldrich;K2HPO4, MgSO4, sodium glycerophosphate, glycerol, Tris(2,4-pentadionato)chromium-(III) (Cr(Acac)3), and casein meatpeptone were from Merck; tryptone and yeast extract (auto-lytic) was from Biokar; and soya flour peptone (prepared usingpapain) was from Fluka. The same supply of each componentwas used for all fermentations. DMSO-d6 was obtained fromEuriso-top.

Bacterial Cultures—S. cerevisiae was obtained from MartinVialatte Oenologie, and Saccharomyces bayanus was from theOenology Laboratories Group. Both were stored dry at �4 °C.Leuconostoc mesenteroides strain 19D cit� was provided byProfessor Herve Prevost (Unite de Recherche Securite des Ali-ments et Microbiologie, ONIRIS, Nantes, France) and stored at�80 °C in M17 medium (20) with 15% (v/v) glycerol. Z. mobilisZM6 (DSMZ 3580) was provided by Professor Michel Rohmer(Laboratory of the Chemistry and Biochemistry of Micro-or-ganisms, Chemistry Institute, UMR7177, CNRS-University ofStrasbourg, Strasbourg, France) and stored at �80 °C in ZGM(see below for composition) with 15% (v/v) glycerol.

Bacterial Culture Conditions—All fermentations were con-ducted using the same batch of D-glucose. Fermentations wereperformed using a Labfors-5 7.5-liter bioreactor equipped witha stirrer, regulated aeration rate, digital pumps for addingmedium, acid, basic, or antifoam, pH and O2 sensors, automaticcontrol, and environmental monitoring. For each organism,two or three fermentations were carried out anaerobically at30 °C in appropriate medium with gentle mixing (100 rpm).Samples of appropriate volume (1000 to 250 ml) were taken atappropriate times to give between 10 and 75% utilization ofD-glucose. Supernatant was recovered by centrifugation (4250g; 10 min; 4 °C) and kept at �20 °C.

For S. cerevisiae/S. bayanus culture medium D-glucose (300g), (NH4)2SO4 (12 g), K2HPO4 (4.5 g), KH2PO4 (4.5 g), MgSO4(3 g), casein meat peptone (3 g), and soya flour peptone (3 g)were dissolved in 2.8 liters of distilled water, and the pH wasadjusted to 6.8. Culture was initiated by adding S. cerevisiae (6g) and S. bayanus (6 g) suspended in 200 ml of water to theculture vessel.

For L. mesenteroides, 2 liters of M17 medium (20), pH 6.3,was sterilized by autoclaving (121 °C, 20 min) and transferredaseptically to the culture vessel. Just before initiating the fer-mentation, D-glucose (300 g in 1 liter of water sterilized by auto-claving (110 °C for 20 min)) was added aseptically. A preculturewas prepared by suspending bacteria stored at �80 °C in 2 �200 ml of M17 medium and incubating without agitation for24 h. Following harvesting, the cells were washed twice with

FIGURE 1. Summary of the three metabolic pathways evaluated. A, themetabolic routes from glucose to ethanol following the EMP, the ED, and theRPP pathways. B, the affiliation between the carbon positions in glucose andthe carbon positions in ethanol and other fermentation products of thesepathways. Enzymes indicated are: 1, G6P dehydrogenase (EC 1.1.1.49); 2, glu-cose-6-phosphate isomerase (EC 5.3.1.9); 3, phosphogluconate dehydroge-nase (decarboxylating), (EC 1.1.1.44); 4, phosphogluconate dehydratase (EC4.2.1.12); 5, 2-dehydro-3-deoxy-phosphogluconate aldolase (EC 4.1.2.14); 6,fructose-1,6-bisphosphate aldolase (EC 4.1.2.13); 7, triose phosphate isomer-ase (EC 1.2.1.12); 8, phosphoketolase (EC 4.1.2.9); 9, enolase (EC 4.2.1.11); 10,pyruvate kinase EC 2.7.1.40); 11, pyruvate dehydrogenase (EC 1.8.1.4); 12,alcohol dehydrogenase (EC 1.1.1.1). P, phosphate; DHAP, dihydroxyacetonephosphate.




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MP17 (50 ml), resuspended in M17 (50 ml), and transferredaseptically to the fermenter.

For Z. mobilis, fermentation culture medium (NH4)2SO4 (12g), KH2PO4 (3 g), MgSO4 (1.5 g), and yeast extract (15 g) weredissolved in 2 liters of distilled water. The pH was adjusted to6.8, and the culture was sterilized by autoclaving (121 °C, 20min) and transferred aseptically to the culture vessel. An over-night preculture of Z. mobilis was grown in medium (ZGM)composed of yeast extract (5 g/liter), (NH4)2SO4 (1 g/liter),KH2PO4 (1 g/liter), MgSO4 (0.5 g/liter), and glucose (20 g/liter),pH 5.0, was prepared. This and D-glucose (20 –150 g/liter) wereintroduced to the fermenter as described for L. mesenteroides.

Extraction of Ethanol from Fermentation Medium—Ethanolwas recovered from the fermentation medium by distillationusing a Cadiot column equipped with a Teflon turning band.Care was taken to ensure that at least 90% of the ethanol presentwas recovered so as to avoid isotopic fractionation (21).

Quantification of Ethanol—Fermentation medium wasdiluted to give an ethanol concentration in the range 1–10mg/ml. To 1 ml of this, 0.5 ml of ethyl acetate was added. Aftervigorous agitation (3 min), the phases were separated by cen-trifugation (13,500 � g for 1 min), and the ethyl acetate phasewas recovered. Ethanol was quantified by gas chromatographyon an Agilent 7820A gas chromatograph fitted with a PTA-5(30 m � 0.32 mm; film thickness, 0.5 �m) with helium as acarrier gas at a constant flow (1.2 ml/min) and an injector tem-perature of 250 °C. Elution conditions were as follows: 70 °C for4 min, 20 °C/min to 200 °C, and 200 °C for 1.9 min. Detectionwas by flame ionization at 250 °C. Calibration was with knownconcentrations of ethanol (0.67– 8.34 mg/ml) treated exactlylike the samples.

Quantification of D-Glucose—D-Glucose concentration wasdetermined by HPLC on a Lichrosphere 100-NH2 (250 � 46mm, 5 �m) column eluted isocratically with acetonitrile/water(80:20) at 1 ml/min. Detection was by refractive index. Calibra-tion was with pure D-glucose in the range 10 –250 mg/ml.

Isotope Ratio Measurement by Mass Spectrometry—Theglobal value for the whole molecule, �13Cg (‰), is the deviationof the carbon isotopic ratio Rs relative to that of the interna-tional standard Vienna Pee Dee Belemnite, Rv-PDB. It is deter-mined by isotope ratio measurement by mass spectrometry andcalculated from Equation 1.

�13Cg�‰ � � RS

RV�PDB� 1� � 1000 (Eq. 1)

13C NMR Acquisition Conditions—Relaxation agent Cr(A-cac)3 solution (0.1 M) was prepared by dissolving 34.9 mg ofCr(Acac)3 in 1 ml of DMSO-d6 in a 4-ml vial. To this was added600 �l of ethanol and 100 �l of DMSO-d6 to act as lock. Fol-lowing mixing, the solution was left 2– 4 h, filtered to removeundissolved relaxation agent, and transferred to a 5-mm NMRtube. Quantitative 13C NMR spectra were recorded at 100.6MHz using a Bruker 400 NMR spectrometer fitted with a 5-mm1H/13C dual� probe. The temperature of the probe was set at303 K. The offsets for both 13C and 1H were set at the middle ofthe frequency range. Inverse-gated decoupling was applied, andthe repetition delay between each 90° pulse was set at 10�

T1max of ethanol to avoid the NOE and to achieve full relaxation

of the magnetization. The decoupling sequence used adiabaticfull passage pulses with cosine square amplitude modulation(�2

max 17.6 kHz) and offset independent adiabaticity withoptimized frequency sweep (3). Each measurement consisted ofthe average of five independently recorded NMR spectra.

Spectral Data Processing—The positional isotopic distribu-tion in ethanol was obtained from the iq-13C NMR spectrumessentially as described previously (9, 22). To obtain Si, the areaunder the 13C signal for the C-atom in position i (in this case themethyl and methylene positions), curve fitting based on a totalline shape analyses (deconvolution) is carried out with aLorentzian mathematical model using PerchTM NMR Soft-ware. In this procedure, line shape parameters are optimized interms of intensities, frequencies, line width, and line shape(Gaussian/Lorentzian, phase, asymmetry) by iterative fitted to aminimal residue. All line fitting was performed by the sameoperator.

Each Si has to be corrected to compensate for the slightloss of intensity caused by satellites (13C–13C scalar couplinginteractions) by multiplying by (1 � n � 0.011), where n isthe number of carbon atoms directly attached to the C-atomposition i, and 1.1% ( 0.011) is the average natural 13C-abundance. (For ethanol, n 1 for both the methyl andmethylene positions.)

Stot is the sum of the areas of all 13C signals from themolecule.

Stot � �n

Si (Eq. 2)

The 13C mole fraction fi, the area of the peak corresponding tothe carbon position i divided by the sum of all the carbon sitesof the molecule, is obtained by Equation 3.

fi � Si��n

Si (Eq. 3)

Fi is the statistical mole fraction for a carbon site i, that is themolar fraction for the carbon position i in the theoretical situ-ation where there is a homogeneous 13C distribution within themolecule,

Fi �c

C(Eq. 4)

where c is the number of carbon equivalents in the moleculeresonating at a given frequency, and C is the total number ofcarbon atoms in the molecule (for ethanol Fi 1/2 for both themethyl and the methylene positions).

Hence, the site-specific reduced molar fraction can bedefined as fi/Fi. From the reduced molar fraction �13Ci, the spe-cific isotope composition of the carbon position i is obtainedfrom the isotope composition of the whole molecule (�13Cg)measured by isotope measurement by mass spectrometry asfollows.

Ai (%), the isotopic abundance for carbon position i, isobtained from Equation 5,

Ai � fi/Fi � Ag (Eq. 5)




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where Ag is the isotopic abundance of a whole molecule and isobtained from the following,

Ag � Rg��Rg � 1 (Eq. 6)

with

Rg � ��13Cg

1000� 1� � RV�PDB (Eq. 7)

where RV � PDB 0.0112372. Then

Ri � Ai��1 � Ai (Eq. 8)

hence

�13Ci�‰ � � Ri

RV�PDB� 1� � 1000 (Eq. 9)

Calculation of Isotope Effects—Kinetic isotope effects werecalculated using a modified form of the Biegeleisen equation,

KIE �kL

kH�

ln�1 � feth

ln�1 � �feth � Reth,t

RG,0�� (Eq. 10)

where kL and kH are the rates of overall reaction with light (12C)and heavy (13C) isotopomers, respectively; feth is the fraction ofproduct formed ([ethanol]/[ethanoltheoretical]) determined bygas chromatography for ethanol and HPLC for the initial [glu-cose]; Reth, t is the isotopic ratio for ethanol at time t, deter-mined by iq-13C NMR; and Rg, 0 is the mean isotopic ratio forthe relevant positions in glucose at t 0, determined by iq-13CNMR as described previously (23).

RESULTS

To calculate the appKIE values, the position-specific 13Ci con-tent of the accumulated product, ethanol, was determined. Toobtain the greatest degree of isotopic fractionation betweensubstrate and product, fermentation to a low feth is advanta-

geous. Fermentations in anaerobic conditions adapted to eachmicro-organism were sampled in the range 10 –70% ofadvancement of the reaction, ethanol was recovered by distilla-tion, and the iq-13C NMR spectra were recorded (Fig. 2).

From this, the �13Ci (‰) values were calculated for themethyl and methylene positions (see “Experimental Proce-dures”). The mean for the fi/Fi for each fermentation (5 spectra)and the mean � S.E. values of �13Ci (‰) are given in Table 1.

From the calculated �13Ci (‰) data, the position-specificKIEs were calculated using the modified Biegeleisen equation.These are given in Table 2.

DISCUSSION

Unique Features of the EMP Pathway—The position-specificappKIE observed for the fermentation of D-glucose by S. cerevi-siae/S. bayanus culture is not significant for the CH2 positionand only shows a small normal effect for the CH3. Thus, theprevious proposal that the Embden-Meyerhof reactions do nothave a large carbon isotope effect (19) is substantiated by directmeasurement. However, because the appKIE determined on thefinal product (ethanol) reflects the sum of counteracting nor-mal and inverse KIEs, this does not exclude the possibility offractionation having occurred at several steps in the pathway,which consists of a series of thirteen enzymatic reactions. Inaddition, each value is the mean of the isotope effects on theC-2 � C-5 and C-1 � C-6 positions of D-glucose for the CH2and CH3, respectively.

Five enzymes have the potential to generate an isotopic frac-tionation on positions C-2G and/or C-5G on their route to becomethe CH2 of ethanol (Fig. 3). Two of these enzymes, glucose-6-phos-phate (G6P) isomerase (EC 5.3.1.9) and triose phosphate isomer-ase (EC 1.2.1.12), catalyze steps that are unique to the EMP path-way, whereas three others, enolase (EC 4.2.1.11), pyruvate kinase(EC 2.7.1.40), and the pyruvate dehydrogenase complex (EC1.2.4.1 � EC 2.3.1.12 � EC 1.8.1.4) are common to at least two ofthe three pathways studied (Fig. 1). It should be noted that anyprimary KIE associated with fructose-1,6-bisphosphate aldolase

FIGURE 2. 13C NMR spectrum of ethanol in DMSO-d6 acquired under quantitative conditions. See “Experimental Procedures” for the acquisitionconditions.




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will not manifest itself, because the C-3 and C-4 are both lost asCO2. G6P isomerase causes an sp3-sp2 change in hybridization atthe C-2, a modification commonly associate with a normal sec-ondary kinetic isotope effect (24). That this is manifest is highlyprobable, because glucose isomerase (EC 5.3.1.5) from Strepto-myces murinus has an inverse isotope effect in the direction fruc-tose to glucose (18), which would act to impoverish the C-2 of F6Pderived from G6P, by �15‰ at equilibrium. This depletion ismuch higher than that measured in ethanol, implying that furthersteps compensate. Although triose phosphate isomerase is a

potential candidate, because during the reaction an sp2-sp3 changein hybridization occurs at the C-2 position (Fig. 3A), it can be rea-sonably argued that this is unlikely, because the limiting step of thereaction (proton transfer from His-95 of the active site) does notinvolve a primary 13C isotope effect (25). The C-2 position isinvolved in further reactions, notably enolase, pyruvate kinase, andpyruvate dehydrogenase, which are all susceptible to introducingisotopic fractionation. These enzymes are all located in the part ofthe EMP that is common to other pathways and are discussedbelow.

TABLE 1Data obtained by iq-13C NMR for the 13C distribution in ethanol produced by fermentation of glucoseFor definitions of terms, see “Experimental Procedures.” All fermentations were made using the same batch of D-glucose obtained from Sigma-Aldrich (batch071M014552V).




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Unique Features of the ED Pathway—The fermentation ofD-glucose by Z. mobilis gives values of appKIE similar to thoseobtained for the fermentation of D-glucose by S. cerevisiae/S. bayanus culture (Table 2), with no significant isotope effect atthe CH2 but a larger normal effect for the CH3 of appKIECH3

1.0035 � 0.0002. As with the G6P to F6P transformation, theC-2 undergoes an sp3-sp2 transition in the conversion of6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate(KDPG) by phosphogluconate dehydratase (EC 4.2.1.12).Although the mechanism has been defined (26) and the C-2Gundergoes cleavage of the C-H bond and ketonization (Fig. 4),the lack of any KIE indicates that, as with triose phosphateisomerase, this is not the kinetically limiting step. However, thelack of any KIE supports the suggestion that the G6P to F6Pisomerization in the EMP pathway is indeed responsible for asmall fractionation, because this step is absent in the ED path-way, and the C-2G, unlike the C-5G, does not undergo the stepscommon to the three pathways (Fig. 1).

The normal appKIECH3is considerably greater than for the

EMP pathway. This may reflect the origin of the CH3 being theC-3G � C-6G in this pathway. The C-3G position is potentiallysubjected to a primary KIE during the fission of the C-3G–C-4Gbond by the action of KDPG aldolase, which cleaves KDPG toyield directly pyruvate and GAP. Thus, the C-3G enters the CH3of ethanol, in marked contrast to the EMP pathway. Althoughno 13C KIE studies have been done on the KDPG aldolase, itsmechanism (27) is closely similar to that of fructose 1,6-bis-phosphate aldolase (28), for which a normal KIE of 1.0254 hasbeen determined (17). This value is in consensus with theappKIECH3

found here.Unique Features of the RPP Pathway—The fermentation of

D-glucose by L. mesenteroides shows a distinctly different iso-topic fractionation compared with the EMP or ED pathways.Three features are striking: (i) the CH2 position shows a normalappKIECH2

1.0058 � 0.0007, (ii) the appKIECH3 1.0057 �

0.0011 is of a similar magnitude, and (iii) both values are greaterthan for the other pathways (Table 2). As in the EMP and EDpathways, cleavage of the C-3G–C-4G bond occurs, but cru-cially, it is only the (C-2–C-3)G unit that is converted to ethanol,the (C-4 –C-5–C-6)G yielding D-lactic acid (Fig. 1). It is foundthat this difference has a major influence on the appKIE values.An analysis of the data is simplified, especially for the CH3

of ethanol, as the C2G is only involved in one reaction: phos-phogluconate dehydrogenase (decarboxylating) (EC 1.1.1.44),which simultaneously ketonizes the C-2G position and cleavesthe C-1G–C-2G bond and could therefore involve a primary KIE(Fig. 5). The CH2 position similarly is derived from a cleavagereaction that catalyzed by phosphoketolase (EC 4.1.2.9), whichcleaves the C-3G–C-4G bond and therefore could involve a pri-mary KIE. The C-3G does undergo further reactions, but theonly one susceptible to any isotope fractionation is the finalalcohol dehydrogenase (EC 1.1.1.1/EC 1.1.1.2), in which achange in hybridization sp2-sp3 occurs (see below).

The C-1 of D-glucose is lost during the decarboxylation of6-phosphogluconate to ribulose-5-phosphate catalyzed by6-phosphogluconate dehydrogenase (decarboxylating). Decar-boxylation is characteristically associated with a normal pri-mary KIE (29, 30). The mechanism of 6-phosphogluconatedehydrogenase has been elucidated (31, 32) and a 13C KIE 1.0209 � 0.0005 has been determined for the C-1G duringdecarboxylation by the enzyme from the yeast Candida utilis(31). Although no data are available for the C-2G position, a KIEof a similar magnitude is probable (33), so an appKIE �1.006 iscompatible with the proposed mechanism. It should be notedthat, because reactions are not occurring under Vmax/Km con-ditions, values of appKIE obtained in vivo can be expected to belower than values of KIE obtained under in vitro conditions.

Considering the origin of the CH3 of ethanol, a KIE associ-ated with the cleavage of the C-3G–C-4G bond is probable (34).Although no isotopic data are available for this reaction, itssimilarity to the better studied fructose-1,6-bisphosphate aldol-ase (35) for which a normal KIE of 1.0254 (17) has been deter-mined, making it probable that the appKIECH3

1.0057 is due tothe activity of this enzyme. That this value is smaller thandetermined for the fructose-1,6-phosphate aldolase mightreflect the sp2-sp3 change during the alcohol dehydrogenasestep, which is likely to manifest a small secondary inverseKIE (see below).

Features Common to the EMP and ED Pathways—Both theEMP and ED pathways produce ethanol from GAP via pyruvate(Fig. 1A). Only the reaction by which 2-phosphoglycerate isconverted to phosphoenolpyruvate involves a change in hybrid-ization state of sp3-sp2 for both the C-5G and C-6G and couldtherefore manifest a secondary 13C KIE. This is catalyzed by

TABLE 2Apparent kinetic isotope effects obtained for the methylene (CH2) and methyl (CH3) positions of ethanol produced by the fermentation ofD-glucose

Fermentation no.

appKIE

S. cerevisiae/S. bayanus Z. mobilis L. mesenteroides

feth CH2 CH3 feth CH2 CH3 feth CH2 CH3

% % %1 11.3 1.0000 1.0017 14.3 0.9991 1.0033 24.2 1.0068 1.0059

49.1 0.9992 1.0012 30.1 0.9991 1.0034 58.1 1.0059 1.007355.3 0.9988 1.0012 43.0 0.9985 1.0039

2 9.1 0.9991 1.0018 12.0 0.9997 1.0036 30.4 1.0055 1.004911.6 0.9991 1.0014 17.5 0.9995 1.0036 57.1 1.0049 1.004437.3 0.9997 1.0013 30.1 0.9994 1.0034 73.7 1.0057 1.0060

3 9.4 1.0001 1.0012 8.1 1.0003 1.003552.6 0.999 1.0015 14.9 0.9998 1.003460.2 0.9981 1.0014 26.2 0.9995 1.0038

Mean 0.9992 1.0014 0.9994 1.0035 1.0058 1.0057S.D. 0.0006 0.0002 0.0005 0.0002 0.0007 0.0011




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enolase in an elimination reaction involving a carbanion inter-mediate (36, 37). The slow step of the reaction, the abstractionof the H at the C (i.e. the C-2G or C-5G) is associated with astrong 2H isotope effect: subsequent enolization of the C (i.e.

the C-1G or C-6G) is not. Therefore, it can be concluded thatthis step is unlikely to contribute to the appKIECH3

of ethanol.Furthermore, any normal isotope effect at the C is likely to bereversed by an inverse isotope effect in the subsequent reaction

FIGURE 3. Enzymatic reactions of the Embden-Meyerhof-Parnas pathway indicating possible associated isotopic fractionation sites. A, steps unique tothe EMP pathway potentially affecting fractionation at the C-2 position. B, steps common to the EMP, ED, and RPP pathways, potentially affecting the C-1, C-2,C-5, and C-6 positions. Enzymes involved are numbered as in Fig. 1.




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catalyzed by pyruvate kinase in which an sp2-sp3 change inhybridization occurs.

The only common step in which a primary KIE might occuris pyruvate dehydrogenase (decarboxylating) (EC 1.2.4.1),responsible for the bond cleavage within the pyruvate dehydro-genase complex. Primary 13C KIEs have been shown for bothcarbon positions involved in the decarboxylation, the C (i.e.the C-2G or C-5G of glucose) showing a 13C KIE 1.0213 �0.0017 and 1.0254 � 0.0016 for the enzyme from bacteria (Esch-erichia coli) and yeast (S. cerevisiae) respectively (29). Further-more, a small secondary effect on the C of 1.0031 � 0.0009 wasfound. It can therefore be proposed that this enzyme makes amajor contribution to the values of appKIECH3

obtained of1.0014 � 0.0002 and 1.0035 � 0.0002 for the ethanol fromS. cerevisiae and Z. mobilis fermentations, respectively.

Following on from this proposal is the possibility to estimatethe effect isotopic caused by the KDPG aldolase on C-3G bycomparing the appKIECH3

calculated for the two types of fer-mentation. The appKIECH3

for Z. mobilis is 1.0035, and forS. cerevisiae it is 1.0014. The appKIECH3

observed for Z. mobiliscan be estimated as the average of the appKIECH3

for S. cerevisiaeand the KIE caused by KDPG aldolase, because no other reac-tion is likely to cause fractionation at that position (C-3G) in theupper portions of these two pathways (see Fig. 1 and above). Bycalculation, the deduced KIECH3

for KDPG aldolase on the C-3Gis 1.0021. Although only 10% the magnitude of the primary KIEdetermined for fructose-1,6-bisphosphate aldolase (17), this

once again indicates that reactions are occurring under condi-tions far from Vmax/Km. In addition, these two enzymes differmechanistically (27). It is notable, however, that the valueobtained is of the same magnitude as for the RPP pathway(Table 2), in which a similar cleavage catalyzed by phosphoke-tolase, is involved.

Features Common to All Three Pathways—The only stepcommon to all three pathways in the final step: the reduction ofacetaldehyde to ethanol, during which a change of hybridiza-tion sp2-sp3 occurs that could induce a small secondary inverseKIE. The yeast and liver enzymes gave intrinsic 13C equilibriumisotope effects of 1.0164 and 1.0149, respectively, with benzylalcohol as substrate (38). However, the value was highlydependent on the C-H bond distance in the transition state,which will not be equivalent in acetaldehyde. Only in the case ofL. mesenteroides is the appKIECH2

of the same order. However,because any isotope effect is predicted to be inverse, it appearsthat this step does not influence the final 13C value of the CH2group.

Conclusions—The application of iq-13C NMR to the study offermentation pathways has made possible the detection of weakappKIEs in the formation of ethanol from D-glucose. These iso-tope effects are manifest because the transformation is to anend product that accumulates. Thus, despite the potentialreversibility of a number of the individual steps in each path-way, the overall vector is driven by the metabolic need to regen-erate NAD� in order that metabolism can continue. Hence, the

FIGURE 4. Enzymatic reactions unique to the Enterer-Doudoroff pathway indicating possible associated isotopic fractionation steps. Enzymesinvolved are numbered as in Fig. 1.




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pathway overall is a unidirectional closed system, and any iso-tope fractionation associated with reverse reactions will benegated. Although it is confirmed that glycolysis (EMP) essen-tially proceeds without isotopic discrimination, as previouslysupposed (19), the data support the previous observation thatthe methylene group becomes relatively enriched during fer-mentation and the methyl becomes relatively impoverished(13). As indicated in Table 2, a normal KIE is found for the CH3,which will lead to impoverishment, and a negligible or slightlyinverse KIE is found for the CH2. Furthermore, rather moresignificant KIEs are seen to be present in the ED and RPP routesof D-glucose catabolism.

The relatively small size of the appKIEs observed and theabsence of comprehensive mechanistic data on all the enzymesinvolved limits the extent to which the data can be interpreted.Nevertheless, a number of enzymes can be implicated, notablythose involved in key steps characteristic to each pathway. Littlecan be concluded for the EMP pathway, other than that isotopiceffects are relatively small and fully consistent with a series ofsecondary isotope effects in which normal and reverse effectscounteract, giving an overall negligible effect for the appKIECH2

and a very small normal appKIECH3. For the ED pathway, a net

estimate for the KIE at C-3G caused by KDPG aldolase can bepostulated. This effect is relatively small for a primary KIE,which suggests that isotopic fractionation caused by otherenzymes is also playing a contributory role. The RPP pathwayshows more intense isotopic fractionation at both the methyl

and methylene positions. This fractionation is satisfactorilyexplained by a normal KIE for both positions associated withthe action of phosphoketolase. The data obtained serve to focusattention on these enzymes, for which more details of theirspecific properties need to be obtained.

Acknowledgments—We thank Virginie Silvestre for help with 13CNMR, Anne-Marie Schiphorst and Mathilde Grand for help withisotope measurement by mass spectrometry, and Denis Loquet forassistance with HPLC.

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Kevin Bayle, Serge Akoka, Gérald S. Remaud and Richard J. Robinsduring Fermentation Is Determined by the Catabolic Pathway Exploited

C Distribution during Carbon Transfer from Glucose to Ethanol13Nonstatistical

doi: 10.1074/jbc.M114.621441 originally published online December 23, 20142015, 290:4118-4128.J. Biol. Chem.

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