Article

Metabolic-Flux Profiling of the Yeasts Saccharomyces cerevisiae and Pichia stipitis

Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland.
Eukaryotic Cell (Impact Factor: 3.18). 03/2003; 2(1):170-80. DOI: 10.1128/EC.2.1.170-180.2003
Source: PubMed
ABSTRACT
The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance.
The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway
for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant
in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts.
The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central
carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the
PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.

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EUKARYOTIC CELL, Feb. 2003, p. 170–180 Vol. 2, No. 1
1535-9778/03/$08.000 DOI: 10.1128/EC.2.1.170–180.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Metabolic-Flux Profiling of the Yeasts Saccharomyces cerevisiae
and Pichia stipitis
Jocelyne Fiaux,
1
Z. Petek C¸akar,
2
Marco Sonderegger,
2
Kurt Wu¨thrich,
1
Thomas Szyperski,
3
* and Uwe Sauer
2
Institute of Molecular Biology and Biophysics
1
and Institute of Biotechnology,
2
ETH Zu¨rich, CH-8093 Zu¨rich,
Switzerland, and Department of Chemistry, State University of New York,
Buffalo, New York 14260
3
Received 13 August 2002/Accepted 28 October 2002
The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in
batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic reso-
nance. The concomitantly characterized network of active metabolic pathways was compared to those identified
in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the
non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P.
stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the
predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat
cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme
activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The
present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of
central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we
demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the
Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.
The two yeasts Saccharomyces cerevisiae and Pichia stipitis
exhibit fundamentally different modes of metabolic regulation
in glucose-containing media. At high extracellular concentra-
tions of glucose, one observes simultaneous fermentation and
respiration (respiro-fermentative metabolism) in S. cerevisiae
at high growth rates even under fully aerobic conditions (11,
34, 53). In Crabtree-positive yeasts, such as S. cerevisiae, ele-
vated glucose concentrations induce the carbon catabolite re-
pression response (16), resulting in low levels of transcription
of genes involved in respiration and in the tricarboxylic acid
(TCA) cycle. In contrast, the Crabtree-negative P. stipitis ex-
hibits predominantly respirative metabolism even at high glu-
cose concentrations (33). These obvious differences in meta-
bolic regulation between the two yeasts provide motivation for
investigation of potential differences in the central carbon
pathways. Stable isotope labeling experiments appear to be a
promising approach, since it reveals in vivo activity of pathways
and reactions (10, 43). Biosynthetically directed fractional
(BDF)
13
C labeling of amino acids can provide comprehensive
insight into central carbon metabolism, yielding a network of
active pathways and quantification of intracellular flux ratios
(36, 37, 44, 46); its use in the present study promises to expand
on the results of earlier labeling studies in yeast that focused
on intermediates or products of individual pathways or reac-
tions (14, 17, 20, 22, 41, 57).
BDF
13
C labeling is achieved by growing cells on mixtures of
unlabeled and uniformly
13
C-labeled [U-
13
C
6
]glucose (40, 44).
Using two-dimensional (2D) nuclear magnetic resonance
(NMR) spectroscopy, the multiplet fine structures due to
13
C-
13
C scalar coupling between adjacent carbons in proteinogenic
amino acids are analyzed in hydrolysates from such fractionally
labeled cells. Decomposition of the fine structures into their
multiplet components allows calculation of the relative abun-
dance of contiguous carbon fragments originating from a single
source molecule of glucose (44). Such intact carbon fragments
are then balanced within a metabolic network to identify the
network of active biosynthetic pathways and to determine
metabolic-flux ratios (44). Although it was initially conceived
for studies of bacterial metabolism, metabolic-flux ratio
(METAFoR) analysis has recently been applied also for the
analysis of eukaryotic metabolism (27), where the spatial sep-
aration of metabolic subnetworks in cytosol and mitochondria
as well as the intercompartmental transport fluxes of pyruvate
(PYR), acetyl coenzyme A (ACoA), and oxaloacetate (OAA)
were taken into consideration.
Here we use METAFoR analysis by NMR for metabolic
profiling of S. cerevisiae (27) and the much less well character-
ized xylose-fermenting yeast P. stipitis in both glucose-limited
chemostat and batch culture. The present investigation focuses
on the metabolic impact of glucose repression (the Crabtree
effect) and pentose phosphate (PP) pathway operation. To
gain further insight into the PP pathway, we further investi-
gated phosphoglucose isomerase (Pgi)-deficient S. cerevisiae,
which catabolizes glucose exclusively via the PP pathway (3).
MATERIALS AND METHODS
Strains and growth conditions. The laboratory yeast strains S. cerevisiae
CEN.PK 113.7D (MATaMAL2-8
c
SUC2) and P. stipitis CBS6054 were used for
* Corresponding author. Mailing address: Department of Chemis-
try, State University of New York, Buffalo, NY 14260. Phone: (716)
645-6800, ext. 2245. Fax: (716) 645-7338. E-mail: szyperski@chem
.buffalo.edu.
† Present address: Department of Molecular Biology and Genetics,
Istanbul Technical University, TR-80626 Maslak, Istanbul, Turkey.
170
Page 1
all labeling experiments. The former was obtained from P. Ko¨tter (Institute of
Microbiology, Johann Wolfgang Goethe-University, Frankfurt, Germany) and
the latter was from B. Hahn-Ha¨gerdal (Department of Applied Microbiology,
Lund University, Lund, Sweden). The strains ENY.WA-1A (MAT ura3-52
leu2-3 trp1-289 his3-1 MAL2-8c MAL3 SUC3) and EBY44 (ENY.WA-1A
pgi1-1::URA3) (kindly provided by E. Boles, Institut fu¨r Mikrobiologie, Hein-
rich Heine Universita¨t, Du¨sseldorf, Germany) were used for UdhA overexpres-
sion experiments. The soluble Escherichia coli transhydrogenase UdhA (4) was
cloned as a BamHI-HindIII fragment from an E. coli overexpression vector (7)
under the control of the constitutive, truncated HXT7 promoter of p425HXT7
(LEU2) (21) (provided by E. Boles). The resulting construct, p425-udhA, was
transformed in S. cerevisiae ENY.WA-1A and in its Pgi-decient derivative.
Batch cultures of 100 ml were grown in 1-liter bafed shake asks on a rotary
shaker at 30°C and 300 rpm in yeast minimal medium, which contained 0.5%
(wt/vol) glucose and 6.7 g of yeast nitrogen base without amino acids (Difco) per
liter. Uracil (50 mg/liter), tryptophan (50 mg/liter), histidine (50 mg/liter), and
leucine (250 mg/liter; to avoid hidden Leu limitations [6]) were supplemented
where necessary.
Glucose-limited chemostat cultures were grown in a 1.5-liter bioreactor (Bio-
engineering, Wald, Switzerland) at 30°C with a working volume of 1 liter. The
lter-sterilized chemostat medium contained the following components (per liter
of distilled water): glucose, 3.6 g; (NH
4
)
2
SO
4
, 5 g; MgSO
4
7H
2
O, 0.5 g;
KH
2
PO
4
, 3 g; 1 ml of vitamin solution (biotin, 0.05 mg/liter; calcium pantothe
-
nate, 1 mg/liter; nicotinic acid, 1 mg/liter; inositol, 25 mg/liter; thiamine-HCl, 1
mg/liter; pyridoxine-HCl, 1 mg/liter; para-aminobenzoic acid, 0.2 mg/liter); and 1
ml of trace element solution (EDTA, 15 mg/liter; ZnSO
4
7H
2
O, 4.5 mg/liter;
CoCl
2
6H
2
O, 0.3 mg/liter; MnCl
2
4H
2
O, 1 mg/liter; CuSO
4
5H
2
O, 0.3 mg/
liter; CaCl
2
2H
2
O, 4.5 mg/liter; FeSO
4
7H
2
O, 3 mg/liter; NaMoO
4
2H
2
O; 0.4
mg/liter; H
3
BO
3
, 1 mg/liter; KI, 0.1 mg/liter) (51). To prevent foaming, 2 ml of
polypropylene glycol 2000 was added (1:10 diluted in H
2
O) per liter of medium.
Anaerobic cultures were supplemented with lter-sterilized Tween 80-ergosterol
solution (1.25 ml/liter) that contained8gofergosterol and 336 g of Tween 80 per
liter of ethanol. The pH was maintained at 5.5 by the addition of 3 M KOH, and
the fermentation volume was kept constant by using a weight-controlled pump.
Aerobic conditions were achieved with a constant airow of 0.5 liters/min and an
agitation speed of 1,200 rpm. To establish anaerobic conditions, air was substi-
tuted with nitrogen at the same ow rate and the agitation speed was reduced to
600 rpm.
Analytical procedures. Cell growth was monitored spectrophotometrically by
determining the optical density of the cultures at 600 nm. Cellular dry weight
(cdw) was determined from six parallel 10-ml culture aliquots that were centri-
fuged for 20 min in preweighed glass tubes at 3,000 g, washed once with water,
and dried at 90°C for 24 h to a constant weight. Concentrations of glucose and
fermentation products in the culture broth were determined with commercial
enzymatic kits (Beckman) or by high-performance liquid chromatography. Phys-
iological parameters were calculated as described previously (37).
13
C labeling experiments. In batch culture, BDF
13
C-labeling of cellular amino
acids was achieved by growth on a 5-g/liter glucose mixture consisting of 90%
(wt/wt) unlabeled and 10% (wt/wt) uniformly labeled [U-
13
C
6
]glucose (degree of
13
C labeling 99% [wt/wt]; Martek Biosciences Corp., Columbia, Md.). These
cultures were inoculated with less than 1% (vol/vol) of a mid-exponential-phase
culture in minimal medium so that the presence of unlabeled biomass could be
neglected. Labeled biomass aliquots were taken from cultures in the mid-expo-
nential phase at an optical density at 600 nm of 1 (37).
Chemostat cultures in physiological steady state were BDF
13
C-labeled by
substituting unlabeled glucose in the feed medium with the above-mentioned
mixture containing 10% (wt/wt) [U-
13
C
6
]glucose. Labeled biomass aliquots were
withdrawn after about one culture volume change so that about 60% of the
biomass was fractionally
13
C-labeled.
Biomass aliquots of about 100 mg (cdw) were harvested from all experiments
by centrifugation of the culture broth at 3,000 g and 4°C for 10 min, washed
once with 20 mM Tris-HCl (pH 7.6), centrifuged again at the previous settings,
and resuspended in 3 ml of 20 mM Tris-HCl (pH 7.6). After addition of 6 ml of
6 M HCl, hydrolysis was performed in sealed glass tubes at 110°C for 24 h, and
the solutions were ltered through 0.2-m-pore-size lters (Millex-GP; Milli-
pore), lyophilized, and dissolved in 700 l of 0.1 M
2
HCl in
2
H
2
O for the NMR
measurements.
NMR spectroscopy and data analysis. 2D proton-detected heteronuclear sin-
gle-quantum
13
C-
1
H correlation NMR spectroscopy (COSY) was employed to
detect aliphatic and aromatic resonances of amino acids at a
1
H resonance
frequency of 500 MHz on a Bruker DRX500 spectrometer as described previ-
ously (37, 44). The overall degree of
13
C labeling was determined by integrating
resolved
13
C satellites in 1D
1
H NMR spectra (27, 44). The thus-obtained degree
of
13
C labeling was in close agreement with the value calculated from the
composition of the minimal medium, and was further conrmed by analysis of
the
13
C scalar coupling ne structure of Leu- (44).
R. Glasers program FCAL (version 2.3.0) (46) was used for integration of
13
C-
13
C scalar coupling ne structures observed for the resonances of 48 carbon
positions in the amino acids. The relative abundances, f, of intact carbon frag-
ments originating from a single source molecule of glucose were calculated from
the relative intensities, I, of the multiplets in the
13
C-
13
C scalar coupling ne (27,
43, 44). These f values (see the appendix for supplementary material) provide
information on the metabolic origin of the amino acid precursor molecules in the
central metabolism. Tracing intact labeled carbon fragments in these metabolites
leads to identication of the active metabolic pathways and to quantication of
the ratios of uxes converging to one particular substrate (45). In cases where
only two reactions contribute to one metabolite pool and where one of the
contributions is assessed from the NMR data, the remaining fraction of the total
pool can be attributed to the competing reaction(s).
Biochemical reaction network for S. cerevisiae and P. stipitis. The biochemical
reaction network considered for S. cerevisiae growing on glucose in batch culture
was recently described (27). Eukaryotic compartmentation into mitochondrial
and cytosolic subsystems was included in the model by considering distinct pools
of PYR, OAA, and ACoA in both compartments (Fig. 1). Firstly, the transport
of PYR into the mitochondria is driven by the proton motive force and was thus
considered to be unidirectional (15). Secondly, although likewise proton driven,
the transport of OAA into the mitochondria is reversible (27, 32), and intercom-
partmental exchange between the two OAA pools may additionally occur via
shuttle transport mechanisms of TCA cycle intermediates (1, 26, 31). Hence,
OAA transport was a priori considered to be bi-directional. Thirdly, mitochon-
drial transport of ACoA was considered reversible because it is mediated by
facilitated diffusion via the so-called carnitine shuttle (48). Although much less is
known about central carbon metabolism of P. stipitis compared to that of bakers
FIG. 1. Biochemical reaction network for yeast central carbon me-
tabolism. The arrows indicate reaction directionality. Letters in bold-
face type indicate metabolites for which the
13
C-labeling pattern can be
accessed through METAFoR analysis. Abbreviations: G6P, glucose-6-
phosphate; F6P, fructose-6-phosphate; P5P, pentose-5-phosphates;
E4P, erythrose-4-phosphate; S7P, seduheptulose-7-phosphate; G3P,
glyceraldehyde-3-phosphate; PGA, 3-phosphoglycerate; ICT, isocitrate;
OGA, oxoglutarate; SUC, succinate; MAL, malate; GOX, glyoxylate.
V
OL. 2, 2003 METABOLIC-FLUX PROFILING OF YEASTS 171
Page 2
yeast, we found no evidence in the literature that the metabolic network of S.
cerevisiae needs to be modied for P. stipitis. In fact, the extensive body of
13
C-labeling data obtained for P. stipitis in the present study was in agreement
with the network proposed for S. cerevisiae.
RESULTS
Central metabolism during growth in batch culture.
METAFoR analysis provides direct information on the biosyn-
thetic pathways for amino acids (23), which are required to
infer the
13
C-labeling patterns of metabolic intermediates from
those detected in the amino acids. For P. stipitis we obtain the
result that amino acids are synthesized along the same path-
ways as in S. cerevisiae. As a continuation of our previous
investigation of S. cerevisiae (27), we rst compared the results
of this earlier study with the ux ratios in central carbon me-
tabolism of exponentially growing P. stipitis in aerobic batch
culture. In these batch cultures with excess glucose concentra-
tions, the maximum specic growth rates were 0.4 and 0.3 h
1
for S. cerevisiae and P. stipitis, respectively. Using BDF
13
C-
labeling, [
13
C,
1
H]-COSY, and METAFoR analysis, we quan
-
tied for several intracellular metabolites the fraction of the
total pool that was derived from the specied substrates (Table
1). In turn, this quanties the ratio of the uxes that converge
to a particular metabolite (Fig. 2). In cases where only two
reactions contribute to one metabolite pool and where one of
contributions is assessed from the NMR data, the remaining
fraction of the total pool can be attributed to the competing
reaction. Five key differences between the two yeast species in
batch culture could be identied.
First, the upper bound on the fraction of phosphoenol-
pyruvate (PEP) molecules derived through at least one trans-
ketolase reaction was widely different (Table 1). This value
provides information about the relative contributions of the
PP pathway and the glucose catabolism via glycolysis to the
generation of trioses. Only an upper bound for the relative
catabolic activity of the PP pathway can be obtained because
glycolytically derived G3P may be exchanged with interme-
diates of the nonoxidative PP pathway via the transketolase
reaction. This exchange reaction generates the same intact
fragments in PEP as glucose catabolism through the PP
pathway. In P. stipitis, the upper bound on the relative PP
pathway ux is less than 57% (Table 1), which is well in the
range of values typically seen in prokaryotic cultures (15, 36,
37). In S. cerevisiae, in contrast, PEP synthesis from pentoses
was not observed, which demonstrates that glucose catabo-
lism proceeds almost exclusively through glycolysis (Fig. 2)
(27). This key difference in glucose catabolism between the
two yeast species becomes readily apparent by visual inspec-
tion of the
13
C-
13
C scalar coupling ne structures of Phe C
,
which reects the labeling pattern of PEP (Fig. 3A). The
doublet component with the smaller coupling constant (Fig.
3A) corresponds to PEP molecules in which the C
1
carbon
and the C
2
-C
3
fragment originate from different glucose
source molecules, and its presence thus provides evidence
for the contribution of transketolase to glucose catabolism.
Despite the absence of glucose catabolism through the PP
pathway in the batch culture of S. cerevisiae (27), evidence
FIG. 2. Metabolic-ux ratios in S. cerevisiae (top values in the in-
sets) and P. stipitis (bottom values) during batch growth with glucose as
the sole carbon source. Connected ratios of uxes that converge to a
particular metabolite are presented in identical geometrical shapes.
For abbreviations see the legend to Fig. 1.
TABLE 1. Origins of metabolic intermediates during aerobic
exponential growth of P. stipitis and S. cerevisiae batch cultures
f
Metabolite
% Fraction of total pool
(mean 2.5 SD)
P. stipitis S. cerevisiae
a
Cytosolic
PEP derived through at least one TK
b
(ub)
57 904
P5P from glucose (lb) 43 232 2
P5P from G3P and S7P (TK reaction) 57 268 2
P5P from E4P (TK and TA
c
reactions)
35 210 2
E4P from F6P (lb) 9 615 4
PEP from OAA
cyt
(PEP carboxykinase
reaction)
5ND
d
OAA
cyt
from PYR
cyt
41 3
e
88100
e
OAA
cyt
reversibly converted to FUM at
least once (cytosolic or
intercompartmental exchange)
24 10 04
Mitochondrial
PYR
mit
from MAL
62530
OAA
mit
from PEP (anaplerosis)
36 276 4
OAA
mit
reversibly converted to FUM
at least once (in the TCA cycle)
58 12 35 4
a
Data taken from Maaheimo et al. (27).
b
TK, transketolase.
c
TA, transaldolase.
d
ND, not detectable because the fragment needed for tracing this activity is
absent.
e
Values calculated assuming absence of cytosolic OAA-to-FUM conversion.
f
The experimental error was estimated from the analysis of redundant
13
C-
13
C
scalar coupling ne structures and the signal-to-noise ratio of the [
13
C,
1
H]-
COSY spectra employing the Gaussian law of error propagation. In certain
cases, the NMR data permit only the determination of upper (ub) or lower (lb)
bounds on the origin of metabolites. Abbreviations are explained in the legend
to Fig. 1.
172 FIAUX ET AL. EUKARYOT.CELL
Page 3
was obtained for signicant anabolic ux via the oxidative
PP pathway to the formation of the biomass precursor P5P:
more than 32% of the P5P molecules originate directly from
glucose (Table 1) (27). This anabolic activity of the PP
pathway is directly apparent in the cross-sections of His C
,
where an additional doublet splitting of the doublet of dou-
blets (Fig. 3B) documents the presence of intact C5-frag-
ments from glucose in the pool of pentoses (43, 44). Con-
sistent with the putative increase of PP activity in P. stipitis,
the minimal fraction of P5P derived from glucose is 43%
(Table 1).
Second, the fraction of OAA
mit
(where the superscript
indicates mitochondrial localization of the metabolite pool)
that is derived from PEP via the anaplerotic PYR carboxy-
lase reaction differs by a factor of 2 in the two yeasts:
anaplerosis contributes 70% in S. cerevisiae but only 36% in
P. stipitis to OAA
mit
synthesis (Table 1). This value species
the relative ux to OAA
mit
required to satisfy the biosyn
-
thetic demands of the TCA cycle. The competing ux from
ACoA
mit
to OAA
mit
is used for complete oxidation of car
-
bon substrates to CO
2
within the TCA cycle. In yeast, PYR
carboxylase is localized exclusively in the cytosol (49). Fol-
lowing (27), anaplerosis is calculated from the fraction of
OAA
mit
derived from PEP (Fig. 1), since the PYR
cyt
(where
the superscript indicates cytosolic localization of the metab-
olite pool) pool is not directly assessable by BDF labeling
because PYR-derived amino acids are synthesized from the
PYR
mit
pool. We conclude that in P. stipitis the TCA cycle
operates predominantly for respiration (Fig. 2). The
13
C-
13
C
scalar coupling ne structures of Glu C
provide direct
evidence for this salient difference (Fig. 4A, left panel). The
higher contribution of the anaplerotic reaction to glutamate
synthesis in S. cerevisiae is reected by the higher intensity
of the doublet with the smaller coupling constant and the
doublet of doublets arising predominantly from carboxyla-
tion of triply
13
C-labeled PYR (44).
Third, while synthesis of OAA
cyt
, which is required for cy
-
tosolic biosynthesis of the Asp amino acid family and anaple-
rosis of the TCA cycle, originates nearly exclusively from PEP
in S. cerevisiae (27), less than half of OAA
cyt
originates from
PEP in P. stipitis (Table 1; Fig. 2). This becomes readily ap-
parent when comparing the
13
C-
13
C scalar coupling ne struc
-
tures of Asp C
(Fig. 4B, left panel). Specically, the large
doublet of doublets in the S. cerevisiae cross section demon-
strates that a high proportion of intact C3-fragments arise from
carboxylation of PYR. On the other hand, the doublet d in Fig.
4B documents that most of the C
2
-C
3
bonds in OAA
cyt
of P.
stipitis were newly formed. This is characteristic for OAA
cyt
synthesized either from oxoglutarate in the TCA cycle or
through the glyoxylate pathway.
Fourth, the fraction of cytosolic or mitochondrial OAA
that was once reversibly converted to fumarate (FUM) is
much higher for P. stipitis than for S. cerevisiae (Table 1).
For OAA
mit
, this conversion occurs via malate dehydroge
-
nase and fumarase in the TCA cycle. The increased ex-
change ux might relate to a predominantly respirative me-
tabolism in P. stipitis. The reversible conversion of OAA
cyt
,
however, may occur either in the cytosol via the glyoxylate
pathway enzyme MAL dehydrogenase in combination with
the cytosolic isoform of fumarase (28) or by intercompart-
mental exchange of OAA molecules (Fig. 1). The small
extent of reversible conversion of OAA
cyt
to FUM in S.
cerevisiae thus demonstrates the absence of glyoxylate shunt
activity and indicates that the ux of OAA
cyt
to OAA
mit
is
unidirectional (27). In P. stipitis, however, about 20% of the
OAA
cyt
molecules are converted to FUM at least once, and
thus one or both of the two above-mentioned ux scenarios
must be invoked.
Fifth, although malic enzyme contributes signicantly to mi-
tochondrial PYR biosynthesis in batch cultures of S. cerevisiae
(27), malic enzyme activity is low in P. stipitis (Table 1). The
manifestation of this reaction in the labeling pattern of PYR
mit
would be consistent with mitochondrial localization of the
malic enzyme not only in S. cerevisiae (2, 27) but in both yeasts.
FIG. 3.
13
C-
13
C scalar coupling ne structures along
1
(
13
C) of a
2D [
13
C,
1
H]-COSY spectrum at the resonance frequency of Phe C
(A) and His C
(B). The spectra were recorded with amino acids
obtained from exponentially growing aerobic S. cerevisiae (left panels)
and P. stipitis (right panels) batch cultures. The corresponding carbon
atom in the precursor metabolites is given in parentheses. Carbon
fragments that can be inferred primarily from a given multiplet com-
ponents are depicted below the left cross-section. The arrow (A) in-
dicates the multiplet component used to trace the generation of PEP
from the PP pathway (see text). Preserved bonds from the source
molecule are shown in bold. The relative abundances of the various
carbon fragments calculated from integration of the ne structures (44,
46) are indicated below each spectrum. Abbreviations: s, singlet; d and d*,
doublets with different coupling constants; dd, doublet of doublets.
V
OL. 2, 2003 METABOLIC-FLUX PROFILING OF YEASTS 173
Page 4
Central metabolism during growth in glucose-limited che-
mostat cultures. While catabolic uxes are maximal during
exponential growth in batch cultures, growth and metabolic
uxes are reduced in chemostat cultures by the limited avail-
ability of glucose. Flux patterns obtained from these differ-
ent types of growth conditions have been shown to differ
signicantly in most microbes (15, 18, 37). To investigate
ux responses to glucose limitation under conditions when
no Crabtree effect is active, we cultivated both yeasts in
aerobic chemostat cultures at a dilution rate of 0.1 h
1
. P.
stipitis grew somewhat more efciently, as evidenced by bio-
mass yields per g of glucose consumed (means standard
deviations) of 0.45 0.03 g and 0.38 0.02 g (cdw) for P.
stipitis and S. cerevisiae, respectively. For both yeasts the
growth was predominantly respirative, with little ethanol
formation (data not shown). When the S. cerevisiae culture
was switched to anaerobic conditions (note that P. stipitis
does not grow under such conditions), metabolism became
entirely fermentative, with a strongly reduced biomass yield
(mean standard deviation) of 0.09 0.00 g (cdw) per g of
glucose and ethanol as the primary metabolic product. Glyc-
erol, acetate, succinate, and PYR were also found in appre-
ciable amounts in the anaerobic culture (data not shown).
METAFoR analyses were performed with hydrolyzed bio-
mass samples that were harvested from these chemostat
cultures in physiological steady-state.
In aerobic chemostats, the upper bound of PEP molecules
that were derived through at least one transketolase reaction
was higher in P. stipitis than in S. cerevisiae (Table 2), but for S.
cerevisiae in both aerobic and anaerobic growth it was much
FIG. 4.
13
C-
13
C scalar coupling ne structures along
1
(
13
C)ofa2D[
13
C,
1
H]-COSY spectrum at the resonance frequency of Glu C
(A) and
Asp C
(B). The spectra were recorded with amino acids obtained from batch and chemostat cultures of S. cerevisiae and P. stipitis. The
corresponding carbon atom in the precursor metabolites is given in parentheses. As indicated in the left-most cross-sections, all these multiplets
consist of a singlet (s) representing the
12
C
13
C
12
C
isotopomer, a doublet (d) originating from
12
C
13
C
13
C
, a second doublet (d*) for
13
C
13
C
12
C
, and a doublet of doublets (dd) arising from
13
C
13
C
13
C
.
TABLE 2. Origins of metabolic intermediates during growth of
P. stipitis and S. cerevisiae in glucose-limited chemostat
cultures at a dilution rate of 0.1 h
1c
Metabolite
% Fraction of total pool
(mean SD)
P. stipitis
(aerobic)
S. cerevisiae
Aerobic Anaerobic
Cytosolic
PEP derived through at least
one TK (ub)
61 11 40 88 5
P5P from glucose (lb) 28 241 211 2
P5P from G3P and S7P (TK
reaction)
72 259 289 2
P5P from E4P (TK and TA
reactions)
43 233 215 2
E4P from F6P (lb) 27 56 644 4
PEP from OAA
cyt
(PEP
carboxykinase reaction)
032 8ND
a
OAA
cyt
from PYR
cyt
24 3
b
62 4
b
40 10
b
OAA
cyt
reversibly converted to
FUM at least once (cytosolic
or intercompartmental
exchange)
47 16 0837 7
Mitochondrial
PYR
mit
from MAL
7 13 10
OAA
mit
from PEP
(anaplerosis)
32 231 298 2
OAA
mit
reversibly converted
to FUM at least once (in
the TCA cycle)
58 14 56 14 43 4
a
Not detectable because of degeneracy of the f values.
b
Values calculated assuming absence of cytosolic OAA-to-FUM conversion
(27).
c
See Table 1 footnotes and legend to Fig. 1 for abbreviations.
174 FIAUX ET AL. EUKARYOT.CELL
Page 5
higher than previously described for batch cultures of (27)
(compare Tables 1 and 2). Thus, the difference in catabolic PP
pathway usage is much less pronounced under glucose-limited
growth conditions than in batch cultures because the chemo-
stat cultures grew at only about one third of the maximum
specic growth rate. Under anaerobic conditions, the fraction
of PEP derived through at least one transketolase reaction is
signicantly reduced (Table 2), which indicates reduced cata-
bolic use of the PP pathway, as was shown previously also for
anaerobic batch cultures (27).
During aerobic chemostat growth, both yeasts exhibit a com-
parable contribution of about 30% from the anaplerotic reac-
tion to the synthesis of OAA
mit
(OAA
mit
from PEP in Table
2), while the remaining OAA
mit
originates from the TCA cycle.
This ux partitioning is very similar to the value obtained for
the P. stipitis batch culture, but it is in contrast to the values
obtained for the S. cerevisiae batch culture (Table 1). Thus, P.
stipitis appears to exhibit predominantly respirative metabo-
lism in both glucose-limited chemostat and batch culture,
whereas S. cerevisiae metabolism is respirative in the chemostat
but respiro-fermentative in the batch culture, which is consis-
tent with the physiological data. In anaerobic chemostat cul-
ture, the TCA cycle operates through two branches, which both
serve exclusively anabolic functions (Table 2). As described above
for anaplerosis, these ux responses are readily apparent by in-
spection of the Glu C
cross-sections (Fig. 4A, right panel).
In contrast to the S. cerevisiae batch culture (Table 1),
OAA
cyt
is not derived exclusively from PYR
cyt
in the che
-
mostat cultures (Table 2). As discussed above for the batch
cultures, this observation could be explained either by sig-
nicant exchange between the OAA
cyt
and OAA
mit
pools,
by high activity of the glyoxylate pathway, or by a combina-
tion of these two factors. Consistently, a signicant fraction
of OAA
cyt
was reversibly converted at least once to FUM in
aerobic P. stipitis and anaerobic S. cerevisiae cultures (Table
2). Furthermore, the data indicate that a small fraction of
PYR
mit
originates from MAL (Tables 1 and 2), thus dem
-
onstrating activity of the malic enzyme in S. cerevisiae and P.
stipitis chemostat cultures. The relative contribution of
malic enzyme to the synthesis of PYR
mit
in S. cerevisiae is,
however, signicantly reduced in the chemostat culture
when compared with the batch culture (27) (Tables 1 and 2).
The malic enzyme activity in aerobic and anaerobic S. cer-
evisiae cultures is comparable (Table 2).
Transhydrogenase expression compensates for Pgi knock-
out in S. cerevisiae. The ux ratios in batch culture might
suggest that S. cerevisiae has relatively low catabolic capacity
of the PP pathway, which would be consistent with the
apparent difference in pentose utilization by the two yeasts
considered here (20, 25, 39, 52). To address this hypothesis,
we used a Pgi-decient S. cerevisiae strain which catabolizes
glucose exclusively via the PP pathway. Since S. cerevisiae
does not contain a transhydrogenase that could transfer
electrons from NADPH to NAD
(47), this mutant cannot
reoxidize PP pathway-produced NADPH and, consequently,
it cannot grow on glucose as the sole carbon source (3). To
enable recycling of NADPH, we overexpressed the soluble
transhydrogenase UdhA from E. coli (4, 7) in this Pgi mu-
tant. As expected, the resulting Pgi
UdhA
strain grows
aerobically on glucose with a specic growth rate of 0.15
h
1
, which is half the rate observed for the control strain
with a functional Pgi. Hence, S. cerevisiae is fully capable of
exclusive glucose catabolism via the PP pathway at a rela-
tively high metabolic rate, provided that concomitantly pro-
duced NADPH is recycled.
DISCUSSION
Here, we report a metabolic network analysis of P. stipitis
central carbon metabolism, which shows that S. cerevisiae
network topology (27) allows consistent data interpretation
also for P. stipitis. The following key factors were identied
for the ux distributions: (i) There is signicant in vivo
activity of the nonoxidative PP pathway. (ii) There is evi-
dence for PYR carboxylase activity in the anaplerotic reac-
tion. (iii) There must be intercompartmental exchange of
OAA if one assumes that the glyoxylate pathway is inactive.
(iv) There is evidence for malic enzyme activity in the mi-
tochondria, as was also reported for S. cerevisiae (2, 27).
Furthermore, there was no evidence in the aerobic regime
for activity of the gluconeogenic PEP carboxykinase in ei-
ther of the two yeasts, which is consistent with the reported
glucose repression of the PEP carboxykinase-encoding gene
in S. cerevisiae (13, 35, 42). The present data thus suggest
that a similar regulation operates in P. stipitis, and that the
pathways for amino acid biosynthesis in P. stipitis are iden-
tical to those used in S. cerevisiae.
Overall, comparative ux proling by METAFoR analysis
revealed distinctly different metabolic regimes for cultures
growing either in batch or in glucose-limited chemostat
mode. Metabolic differences between the two yeast species
were minor in glucose-limited chemostats, where cultures
exhibited a predominantly respirative mode of metabolism,
but were signicant in batch culture. First, in batch culture,
S. cerevisiae exhibited only 24% cyclic (respiratory) TCA
cycle ux (from oxoglutarate) compared to 76% anaplerotic ux
(from OAA
cyt
) into the OAA
mit
pool of the TCA cycle, but the
inverse ratio for these two uxes was found for P. stipitis (Fig.
2), as was also reported for other yeasts (29). This ux ratio is
TABLE 3. Comparison of the ux ratios in S. cerevisiae batch and
chemostat cultures determined by metabolic ux balancing (18)
and by METAFoR analysis (Tables 1 and 2)
Pathway and
metabolite
% Fraction of total pool (mean SD)
Batch culture Chemostat culture
Flux
balancing
METAFoR
analysis
Flux
balancing
METAFoR
analysis
Glucose catabolism
through the PP pathway
(PEP derived through at
least one TK reaction)
14
a
0452
a
40 8
Anaplerosis
OAA
mit
from PEP
100 76 442 31 2
OAA
cyt
from PYR
cyt
82 88100 60 62 4
Malic enzyme (PYR
mit
from MAL)
22842 5 213
a
Note that Gombert et al. estimated the split ratio of glycolysis to PP pathway.
To compare this value to the value of PEP via TK, one needs to subtract the
biosynthetic withdrawal of PP pathway intermediates.
VOL. 2, 2003 METABOLIC-FLUX PROFILING OF YEASTS 175
Page 6
consistent with the generally held view that sugar catabolism in
aerobic batch cultures is respiro-fermentative in S. cerevisiae
(53), and predominantly respirative in Crabtree-negative
yeasts such as P. stipitis (33). Second, during respiro-fermen-
tative metabolism of S. cerevisiae in batch culture, cytosolic
OAA originates exclusively from PYR and is transported uni-
directionally into the mitochondria (27). The predominantly
respirative metabolism in all other aerobic cultures, however,
generates the OAA
cyt
pool containing signicant fractions of
molecules that do not originate from PYR
cyt
but come either
from intercompartmental exchange of TCA cycle intermedi-
ates or from the cytosolic glyoxylate pathway. Third, although
some in vivo activity of the mitochondrial malic enzyme is
detected in all cultures, the highest relative contribution to
PYR
mit
synthesis was observed for respiro-fermentative S. cer-
evisiae in the batch culture. Fourth, our results reveal a major
difference in the contribution of the PP pathway to glucose
catabolism in batch cultures of P. stipitis and S. cerevisiae. The
fraction of PEP molecules derived through at least one trans-
ketolase provides the sum of the net ux through the PP
pathway and the reversible exchange of trioses with interme-
diates of the nonoxidative PP pathway. This value is close to
zero in the S. cerevisiae batch culture (27), indicating absence
of signicant in vivo activity of the nonoxidative PP pathway
branch in S. cerevisiae batch culture, which is thus different
from P. stipitis batch culture (Table 1; Fig. 2).
From this exceptionally low catabolic PP pathway ux in S.
cerevisiae one might conclude that the in vivo activity of this
pathway limits catabolism of pentoses in recombinant S. cer-
evisiae (20, 25, 52), but this appears to be incompatible with the
comparatively rapid growth of Pgi-decient, transhydrogenase-
overexpressing S. cerevisiae. Since it depends on exclusive glu-
cose catabolism via the PP pathway, this strain grows at a
specic rate that is half of that observed with the control. As a
reference, a corresponding E. coli strain grew at about one
third of the wild type rate (7). Thus, sufcient PP pathway
capacity is potentially available in S. cerevisiae, although it
might not be fully activated during pentose catabolism. The
conclusion that the nonoxidative pathway does not principally
limited pentose catabolism in recombinant S. cerevisiae agrees
well with the results of a recent study to simultaneous overex-
press all four pathway enzymes (24). Our results, however, do
not exclude the possibility that the rate of pentose catabolism
is affected by the activity of one or more PP pathway enzymes.
Like in E. coli, our results reveal that UdhA catalyzes the
electron transfer from NADPH to NAD
, as was observed
previously during overexpression of another soluble transhy-
drogenase in S. cerevisiae (30).
Using a different methodology, Gombert et al. recently
reported on network identication and ux quantication in
the same S. cerevisiae strain that was used here (18), leading
to a mutual identity of the metabolic network models in both
cases. To facilitate direct comparison between the results of
both studies, we calculated several metabolic-ux ratios
from the net ux data of Gombert et al. (18) (Table 3). The
ux ratios obtained with the two different approaches are in
good agreement; i.e., both reveal increased glucose catabo-
lism via the PP pathway and reduced anaplerosis in glucose-
limited chemostat culture when compared to batch culture.
Both cultures show some malic enzyme activity and very
little if any glycine cleavage to CO
2
(data not shown). In
particular, the consistent ux distributions obtained for glu-
cose-limited S. cerevisiae by these two and other methods
support that both techniques provide reliable results (8, 9, 18).
Three apparent differences between the results obtained
with the method used by Gombert et al. and with our ap-
proach concern primarily the batch growth of S. cerevisiae.
First, Gombert et al. report exclusive operation of the TCA
cycle as a two-branched pathway in batch culture that does
not contribute to glucose catabolism, while we found previ-
ously that cyclic TCA cycle uxes contribute about 24% of
the total ux to OAA
mit
(27). Since the absolute ux to
OAA
mit
is relatively low in batch culture, this minor net
TCA cycle ux may have been missed by the ux estimation
procedure due to its small contribution to the global target
function of the isotopomer balancing. Second, our results
suggest a signicant contribution of malic enzyme to PYR
mit
synthesis in batch culture (27), where this activity is not
reported by Gombert et al. (18). Similar to the above case,
absolute uxes to PYR
mit
are low under these respiro-fer-
mentative conditions, so that the higher relative contribu-
tion of malic enzyme to PYR
mit
synthesis not necessarily
reect a higher absolute ux. Third, our data show that the
catabolic ux through the PP pathway in batch cultures of S.
cerevisiae contributes less than 4% to PEP biosynthesis (27),
compared to an estimate of 14% calculated from the data of
Gombert et al. (Table 3). Both estimates are consistent with
the calculated minimal ux into the PP pathway of 2% of the
total glucose consumption for NADPH formation with am-
monia as the sole nitrogen source (5, 47).
Global interpretation of
13
C-labeling and physiological
data on the basis of isotopomer balancing (12, 38, 50, 56) as
used by Gombert et al. extracts the maximum information
from labeling experiments, but local features of the ux
distribution may be inaccurately reected as a result of the
global error minimization function used. METAFoR analy-
sis differs from this approach by providing a strictly local
analysis of
13
C-labeling data, which is independent of abso-
lute uxes and physiological data (15). Additionally, varia-
tions in the growth conditions used (i.e., 0.5 units pH dif-
ference, different composition of the minimal medium, and
bioreactor versus shake ask cultivation) may have led to
slightly different ux distributions. A possible dependence
of catabolic PP pathway uxes on environmental conditions
may also explain, at least in part, previously reported dif-
ferences in PP pathway ux estimates for S. cerevisiae on the
basis of isotope labeling studies (17, 18, 27, 54; P. M. Bru-
inenberg, G. W. Waslander, J. P. van Dijken, and W. A.
Scheffers, unpublished data [presented at the Physiological
and genetic modulation of product formation, Como,
1986]). Overall, the combined use of global isotopomer bal-
ancing and local METAFoR analysis promises to further enhance
the reliability of future ux determinations (10, 15, 55).
ACKNOWLEDGMENTS
We thank Jay Bailey for his continuous support and Eckhard Boles
for sharing the Pgi mutant and the HXT promoter with us.
Financial support for this work was partly obtained from the Swiss
Priority Program in Biotechnology.
176 FIAUX ET AL. EUKARYOT.CELL
Page 7
TABLE A1. Relative abundances of intact C2 and C3 fragments in the biosynthesis of fractionally
13
C-labeled amino acids for chemostat
cultivations of S. cerevisiae and P. stipitis
a
Carbon
position
Chemostat
Relative abundance
S. cerevisiae P. stipitis
f
(1)
f
(2)
f
(2)
f
(3)
f
(1)
f
(2)
f
(2)
f
(3)
Ala C Aerobic 0.04 0.11 0.04 0.81 0.01 0.15 0.03 0.81
Anaerobic 0.00 0.07 0.00 0.94
Ala C Aerobic 0.08 0.92 0.06 0.94
Anaerobic 0.01 0.99
Arg C Aerobic 0.66 0.32 0.00 0.02 0.63 0.00 0.33 0.04
Anaerobic 0.07 0.93 0.00 0.00
Arg C Aerobic 0.14 0.86 0.09 0.91
Anaerobic 0.26 0.74
Asp C Aerobic 0.14 0.10 0.19 0.57 0.24 0.14 0.42 0.20
Anaerobic 0.01 0.22 0.01 0.76
Asp C Aerobic 0.17 0.65 0.16 0.02 0.26 0.25 0.41 0.08
Anaerobic 0.01 0.79 0.00 0.20
Glu C Aerobic 0.28 0.22 0.42 0.08 0.24 0.22 0.45 0.09
Anaerobic 0.02 0.75 0.03 0.20
Glu C Aerobic 0.69 0.31 0.00 0.68 0.32 0.00
Anaerobic 0.00 1.00 0.00
Glu C Aerobic 0.08 0.00 0.92 0.00 0.02 0.00 0.98 0.00
Anaerobic 0.14 0.00 0.86 0.00
Gly C Aerobic 0.19 0.81 0.18 0.82
Anaerobic 0.10 0.90
His C Aerobic 0.04 0.02 0.04 0.90 0.03 0.00 0.02 0.95
Anaerobic 0.02 0.01 0.00 0.97
His C Aerobic 0.10 0.49 0.00 0.41 0.12 0.60 0.00 0.28
Anaerobic 0.07 0.82 0.00 0.11
His C
2
Aerobic 0.32 0.68 0.43 0.57
Anaerobic 0.15 0.85
Ile C Aerobic 0.25 0.00 0.75 0.00 0.37 0.01 0.62 0.00
Anaerobic 0.23 0.00 0.77 0.00
Ile C
2
Aerobic 0.12 0.88 0.06 0.94
Anaerobic 0.03 0.97
Ile C
1
Aerobic 0.82 0.18 0.00 0.52 0.48 0.00
Anaerobic 0.82 0.18 0.00
Ile C
1
Aerobic 0.84 0.16 0.53 0.47
Anaerobic 0.91 0.09
Leu C Aerobic 0.10 0.00 0.90 0.00 0.05 0.00 0.95 0.00
Anaerobic 0.11 0.00 0.89 0.00
Leu C Aerobic 0.95 0.01 0.04 1.00 0.00 0.00
Anaerobic 1.00 0.00 0.00
Leu C
1
Aerobic 0.24 0.76 0.17 0.83
Anaerobic 0.13 0.87
Leu C
2
Aerobic 1.00 0.00 1.00 0.00
Anaerobic 1.00 0.00
Lys C Aerobic 0.07 0.05 0.85 0.03 0.06 0.03 0.91 0.00
Anaerobic 0.18 0.00 0.82 0.00
Lys C Aerobic 0.70 0.30 0.00 0.70 0.29 0.01
Anaerobic 0.03 0.97 0.00
Continued on following page
177
Page 8
TABLE A1Continued
Carbon
position
Chemostat
Relative abundance
S. cerevisiae P. stipitis
f
(1)
f
(2)
f
(2)
f
(3)
f
(1)
f
(2)
f
(2)
f
(3)
Lys C Aerobic 0.66 0.34 0.00 0.68 0.32 0.00
Anaerobic 0.04 0.96 0.00
Lys C Aerobic 0.11 0.79 0.10 0.05 0.95 0.00
Anaerobic 0.22 0.77 0.01
Lys Cε Aerobic 0.14 0.86 0.07 0.93
Anaerobic 0.24 0.76
Met C Aerobic 0.14 0.12 0.19 0.55 0.20 0.22 0.32 0.26
Anaerobic 0.03 0.20 0.02 0.75
Phe C Aerobic 0.04 0.09 0.01 0.86 0.04 0.10 0.00 0.86
Anaerobic 0.03 0.02 0.00 0.95
Phe C Aerobic 0.03 0.97 0.00 0.00 0.02 0.98 0.00 0.00
Anaerobic 0.02 0.98 0.00 0.00
Pro C Aerobic 0.12 0.88 0.09 0.91
Anaerobic 0.22 0.78
Pro C Aerobic 0.30 0.22 0.43 0.05 0.27 0.28 0.33 0.12
Anaerobic 0.07 0.75 0.00 0.18
Pro C Aerobic 0.66 0.34 0.00 0.67 0.33 0.00
Anaerobic 0.02 0.18 0.80
Pro C Aerobic 0.09 0.88 0.03 0.06 0.91 0.03
Anaerobic 0.20 0.76 0.04
Ser C Aerobic 0.06 0.07 0.30 0.57 0.05 0.08 0.23 0.64
Anaerobic 0.02 0.02 0.34 0.62
Ser C Aerobic 0.39 0.61 0.30 0.70
Anaerobic 0.39 0.61
Thr C Aerobic 0.15 0.11 0.18 0.56 0.25 0.14 0.41 0.20
Anaerobic 0.02 0.23 0.02 0.73
Thr C Aerobic 0.17 0.41 0.42 0.27 0.68 0.05
Anaerobic 0.04 0.82 0.14
Thr C2 Aerobic 0.83 0.17 0.50 0.50
Anaerobic 0.83 0.17
Tyr C Aerobic 0.05 0.10 0.00 0.85 0.04 0.12 0.00 0.84
Anaerobic 0.03 0.03 0.00 0.94
Tyr C Aerobic 0.034 0.96 0.00 0.00 0.05 0.95 0.00 0.00
Anaerobic 0.03 0.97 0.00 0.00
Tyr C
1
Aerobic 0.05 0.95 0.00 0.04 0.96 0.00
Anaerobic 0.04 0.96 0.00
Tyr Cε
1
Aerobic 0.31 0.08 0.18 0.43 0.24 0.05 0.16 0.55
Anaerobic 0.29 0.00 0.27 0.44
Val C Aerobic 0.14 0.00 0.83 0.03 0.12 0.01 0.83 0.04
Anaerobic 0.10 0.00 0.90 0.00
Val C
1
Aerobic 0.11 0.89 0.06 0.94
Anaerobic 0.02 0.98
Val C
2
Aerobic 0.97 0.03 0.96 0.04
Anaerobic 0.96 0.04
a
The fractions f
(i)
, named according to reference 44 and corresponding to the relative amount of C
i
intact fragments, were calculated from the relative intensities
of the
13
C multiplet components using the probabilistic equations described by Szyperski (44). Note that in reference 44 f
(2a)
and f
(2b)
correspond to f
(2)
and f
(2)
,
respectively.
178
Page 9
APPENDIX
The supplementary material mentioned in Materials and Methods is
presented in Tables A1 and A2.
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