Appl Microbiol Biotechnol(1993) 38:776-783
App//ed
Xylose fermentation by Saccharomyces cerevisiae
Peter K6tter*, Michael Ciriacy
Institut ftir Mikrobiologie, Heinrich-Heine-Universit/~t,Universit/itsstrasse 1, W-4000 Dtisseldorf 1, Federal Republic of Germany
Received: 12 August 1992/Accepted: 8 October 1992
Abstract. We have performed a comparative study of
xylose utilization in Saccharomyces cerevisiae transfor-
mants expressing two key enzymes in xylose metabol-
ism, xylose reductase (XR) and xylitol dehydrogenase
(XDH), and in a prototypic xylose-utilizing yeast, Pichia
stipitis. In the absence of respiration (see text), baker's
yeast cells convert half of the xylose to xylitol and etha-
nol, whereas P. stipitis cells display rather a homofer-
mentative conversion of xylose to ethanol. Xylitol pro-
duction by baker's yeast is interpreted as a result of the
dual cofactor dependence of the XR and the generation
of NADPH by the pentose phosphate pathway. Further
limitations of xylose utilization in S. cerevisiae cells are
very likely caused by an insufficient capacity of the non-
oxidative pentose phosphate pathway, as indicated by
accumulation of sedoheptulose-7-phosphate and the ab-
sence of fructose-l,6-bisphosphate and pyruvate accu-
mulation. By contrast, uptake at high substrate concen-
trations probably does not limit xylose conversion in S.
cerevisiae X Y L 1 / X Y L 2 transformants.
Introduction
D-Xylose is the major product of the hydrolysis of hem-
icellulose from many plant materials, in which xylose
can represent up to 34% (Wong et al. 1988; Schneider
1989). The microbial conversion of this renewable car-
bon source to ethanol has been regarded to be of eco-
nomical interest (Jeffries 1985). Xylose can be fer-
mented to ethanol by many bacteria, yeasts and fungi
(Skoog and Hahn-H~igerdal 1988), although by-product
formation or slow xylose conversion limits economical
application for ethanol production (Neirinck et al. 1982;
Slininger et al. 1985). In general, yeasts convert xylose
to xylulose in a two-step reaction mediated by oxidore-
* Present address: Institut ftir Mikrobiologie, Johann-Wolfgang-
Goethe-Universit~it, Theodor-Stern-Kai 7, W-6000 Frankfurt a.
M., FRG
Correspondence to: M. Ciriacy
Microbiology
Biotechnology
© Springer-Verlag 1993
ductases. Firstly, xylose is reduced to xylitol by an
NADPH/NADH-Iinked xylose reductase (XR), fol-
lowed by oxidation of xylitol to xylulose by a NAD-
linked xylitol dehydrogenase (XDH). By contrast, iso-
merisation o f xylose in bacteria is carried out by a
unique enzyme xylose isomerase. In yeasts as well as in
bacteria, xylulose is subsequently phosphorylated by xy-
lulokinase to form xylulose-5-phosphate, which is
thought to be channelled into the pentose phosphate
pathway (Gong et al. 1981; Jeffries 1983).
Baker's yeast, Saccharomyces cerevisiae, normally
used in ethanol production from hexoses is unable to
utilize xylose (Barnett 1976). However, this yeast can
slowly metabolize xylulose, the ketoisomer of xylose
(Wang and Schneider 1980). Thus, construction of a xy-
lose-utilizing S. cerevisiae strain should be feasible by
introducing the xylose isomerisation step from a xylose-
utilizing microorganism into baker's yeast. So far, ap-
proaches to establish the xylose-utilizing pathway in S.
cerevisiae using the xylose isomerase gene from various
bacterial sources have not been successful (Wilhelm
1986; Sarthy et al. 1987; Amore et al. 1989). We have
recently isolated the X F L 1 and X F L 2 genes coding for
XR and XDH proteins from Pichia stipitis, a yeast able
to ferment xylose under anaerobic conditions (Bruinen-
berg et al. 1984; Dellweg et al. 1984). S. cerevisiae cells
transformed with these genes are able to grow aerobical-
ly on xylose as sole carbon source. These transformants
use xylose at a considerably slower rate than glucose.
Furthermore, xylose utilization was almost entirely oxi-
dative, as indicated by the low ethanol yield (K6tter et
al. 1990).
We report here on xylose utilization and product for-
mation in S. cerevisiae X Y L 1 / X Y L 2 transformants un-
der various conditions. In order to elucidate the slow xy-
lose utilization and ethanol production we compared the
intracellular metabolite concentrations of S. cerevisiae
transformants and P. stipitis cells grown on glucose or
xylose. From these data it becomes apparent that xylose
conversion to ethanol in S. cerevisiae is limited by cofac-
tor imbalance and by an insufficient capacity of xylulose
conversion by the pentose phosphate shunt.

Materials and methods
Yeast strains. S. cerevisiae strain PUA6-9 (MA Tc~ his3 leu2) was
constructed in this laboratory (Porep 1987). P. stipitis CBS5774
was obtained from the Centraalbureau voor Schimmelcultures,
Yeast Division, Delft, The Netherlands.
Media. Yeast strains were grown at 30°C in 0.67% Difco yeast
nitrogen base (YNB) without amino acids and supplemented with
appropriate amino acids and nucleobases if not otherwise indi-
cated. Media were supplemented with either 2% xylose or 2°70glu-
cose.
Plasmid. Plasmid pRD1 derived from plasmid YEpl3 (Broach
1983) contains both the XYL1 and XYL2 genes from P. stipitis
(KOtter et al. 1990; Amore et al. 1991). Yeast transformation was
performed as described by Dohmen et al. (1989).
Xylosefermentation. S. cerevisiae cells were pre-grown to mid-ex-
ponential phase in 2% xylose medium. Cells were harvested by
centrifugation and washed twice with 2% xylose medium. For fer-
mentation studies cells were resuspended in 1/10 the volume of the
same medium. The cell titre in all experiments was about 4x 108
cells/ml.
Assay methods. Enzymatic analyses of xylose and extracellular
metabolites were performed according to standard methods (xy-
lose: Halliwell and Lovelady (1981); ethanol, xylitol, glycerol and
acetate: Bergmeyer (1984).
Sugar uptake measurements. Xylose uptake was measured with
modifications as described by Bisson and Fraenkel (1983). Uptake
was initiated by addition of 13C-labelled sugar (3.7-14.8 x 103 Bq/
mmol) to yeast ceils at a final concentration of 5 mg/ml (wet
weight). After incubation for 5 s at 30° C, uptake was stopped by
addition of 10 ml ice-cold water. Cells were collected on glass-
fibre filters (Whatman GF/C, Maidstone, UK), and washed on
the filter with 10 ml ice-cold water. The filters were subsequently
placed in scintillation liquid (Quicksafe A, Zinsser Analytik,
Frankfurt/M, FRG) and incorporated radioactivity was deter-
mined in a Beckman scintillation counter.
Determination of intraeellular metabolites. Yeast cells of log-
phase cultures were harvested by passing rapidly 50-ml samples
through a membrane filter (pore size 5 gin, Millipore, Eschborn,
FRG) and immediate incubation in 5 ml boiling ethanol (Ciriacy
and Breitenbach 1979). After 2 min, ethanol was removed under
vacuum at 30° C. The dried samples were resuspended in 4 ml imi-
dazole buffer (50mM, pH 7.0) and cell debris was removed by
centrifugation. The extracts were stored at -70°C until used.
Levels of sugar phosphates were determined as described by Berg-
meyer (1984). Dry weight of cells was determined with appropriate
culture aliquots collected and dried on membrane filters.
Results
Xylose fermentation
As previously shown, xylose fermentation under aerobic
conditions in S. cerevisiae cells transformed with the P.
stipitis X Y L 1 and X Y L 2 genes is characterized by rather
slow and incomplete sugar consumption. Oxidative me-
tabolism of xylose was indicated by low ethanol produc-
tion (KOtter et al. 1990). In order to understand in more
detail the xylose metabolism in S. cerevisiae transfor-
mants and to increase ethanol productivity, fermenta-
tion studies were done in the presence of the respiratory
777
inhibitor, antimycin A. Under this condition the cyto-
plasmic redox balance is maintained by coupling the oxi-
dation of glycolytic N A D H to the reduction of acetalde-
hyde to ethanol.
In the absence of respiration, S. cerevisiae (pRD1)
transformants did not grow on xylose medium. This was
unexpected since S. cerevisiae PUAs trains used in these
experiments are able to grow in the absence of respira-
tion on xylulose, the ketoisomer of xylose. This differ-
ence in growth behaviour on xylose and xylulose, re-
spectively, in the presence of antimycin A is very likely
due to the coenzyme specificity of the enzymes asso-
ciated with the conversion of xylose to xylulose. Since a
transhydrogenase activity is apparently absent in yeasts
(Bruinenberg et al. 1983, 1985; Lagunas and Gancedo
1973) the redox equivalents N A D + / N A D H and
N A D P ÷ / N A D P H , respectively, must be balanced. The
dual cofactor dependence of XR could lead to a deficit
in NAD since N A D H generated by the X D H reaction
cannot completely be reoxidized by XR. As a conse-
quence, the surplus N A D H could limit metabolic activi-
ty and could explain the growth defect of S. cerevisiae
transformants on xylose medium in the absence of respi-
ration. To verify this hypothesis, fermentation studies
were done in the presence of antimycin A.
Comparison of xylose fermentation studies of S. cer-
evisiae strain PUA6-9 transformed with plasmid pRD1
grown either in the presence or absence of antimycin A
are shown in Fig. 1A and B. In the presence of the res-
piratory inhibitor (Fig. 1A) we observed nearly complete
xylose utilization with S. cerevisiae transformants. After
about 60 h, 1.9% xylose were metabolized to the major
fermentation products, xylitol and ethanol. About 50%
of the utilized xylose was found as xylitol in the culture
supernatant. The maximal ethanol concentration ob-
served was about 68 mM, i.e. about 34% of the theoreti-
cal yield from complete xylose conversion to ethanol. In
the absence of antimycin A (Fig. 1B), the xylose con-
sumption rate was about twofold higher and most of the
xylose (about 55%) was utilized oxidatively. As ob-
served in the absence of respiration, ethanol and xylitol
were the major fermentation products.
In a parallel set of experiments we examined xyl0se
utilization of P. stipitis cells in the presence or absence
of antimycin A (Fig. 1C and D). P. stipitis cells showed
a 3.5-fold higher rate of xylose utilization than S. cerevi-
siae independent of the presence of antimycin A: 2°7o xy-
lose was completely metabolized after 10h under the
conditions used. Ethanol was the major fermentation
product, and only very small amounts of xylitol accu-
mulated. In the culture without antimycin A the maxi-
mal concentration of ethanol was 56 mM; inhibition of
respiration resulted in a two-fold higher ethanol yield.
Surprisingly, we observed in the P. stipitis cultures a
rapid disappearance of ethanol after xylose depletion
even in the presence of antimycin A. This could suggest
that mitochondrial electron transport in P. stipitis is in
part insensitive to antimycin A. As a result, part of the
xylose carbon would be oxidised leading to a deficit in
fermentation products. This was indeed observed: in P.
stipitis cultures with antimycin A the yield of fermenta-
778

S. cerevisiae PUA6-9 (pRD~I)

160 70 160- 70
,u
Ethanol ~ : = = * ~ : ~ 2 2 Z ~
1,0 60 ~ 140 : 60
.~
Xylose
120- . ~ 120"
50 50
® 100- ~ loo-
40 40
Ethanol
~" ~o' ~ ~ ~o-
30
~ ~ Xyfitol 30
~ ~
E ~0' . 8o-
20 2O
40' ~ 40"
N E

~0 / 10 ~ 20- O ",0 lO
-I I
Glycerol
o ~ ~ ' , ..... ~ ~ 0 O~ " ~ - '"' i ~ ~ ~ ~' 0
20 40 60 80 100 20 40 60 80 100
& Timelh} T~me(h)

P. stipitis CBS5774
t60 160
OD 6 0 O n t o ~
OD 60Ohm ~ 140 ' 140
;

~1o~, ~....
5
L 120 120 '.~
,~
100 '100 *~
E 10
~ ~ ~"
o~ 8O w 80 ,;i,

0 6O 8 60 _~
•
40 -40 =
E
20 ~0

1~- ~ ,,m = ~3 ~ ~3 ~ ~ ~
I
0 -; , ~ o
0 5 10 15 20 25 3O o 5 lO 15 2o 25 3o
Time(h) d Time (h)

Fig. l a - d . Batch fermentation of xylose by Saccharomyces cerevi- m~ncd wffhom (b, d), o~ £n ~hc p~c~cnc¢ o~ ~nfimyci~ ~ (a, c),
siae PUA6-9 transformed with plasmid pRD1 (a, b) and by Pichia ~m~myc£n ~ was added to a fin~] conccmr~fion o~ 2 ~ / m ] . ~ach
stipitis CBS5774 (e, d). Initial cell densities were uniformly 4 x l0 s v a l u e ~cp~cscms the m c a ~ o~ t w o d e t e r m i n a t i o n s . ~ o [ c t h i n scales
cells/ml. Xylose utilization and metabolite formation were deter- ~o~ ~. cemvisiGe and P. sdpitis ~ c diffc~cm; O D , o p f i c ~ ] d c ~ s f f y

tion products was only 52% of the maximal theoretical
yield for complete fermentative xylose conversion.
Carbon balance of xylose utilization
From the fermentation experiments shown above, a car-
bon balance for xylose utilization in the absence of res-
piration was calculated for S. cerevisiae PUA6-9 (pRD1)
transformants (Table 1). On the basis of the amount of
utilized xylose, about 90°7o of the carbon was recovered
in the fermentation products, ethanol and xylitol. As-
suming that the two reactions involved in xylose/xylu-
lose isomerization use the same cofactor, the theoretical
yield of ethanol in the absence of respiration is given by
the equation:
'6 xylose --, 10 ethanol + 10 CO2
The yield of 0.51 g ethanol/g xylose is equivalent to
that for the conversion of glucose to ethanol by S. cere-
visiae according to Neuberg's first form of fermenta-
tion. Xylose metabolism of S. cerevisiae (pRD1) trans-
formants showed an apparently different stochiometry.
This can be interpreted as a result of the dual cofactor
dependence of the XR (NADH/NADPH) and the de-
pendence of XDH on NAD only. Since about 50% of
the utilized xylose was secreted as xylitol (Fig. 1B, and
Table 1) we conclude that in S. cerevisiae (pRD1) trans-
formants half of the xylose is reduced to xylitol using
NADPH as cofactor. It is plausible to assume that
NADPH is generated by channeling part of the xylose
carbon through the pentose phosphate cycle. Approxi-
mately half of the xylose is converted by NADH-linked
xylose reductase. The NAD generated in this reaction is
subsequently reduced to NADH by XDH thereby bal-
ancing the NAD/NADH ratio. Subsequently, xylulose is
phosphorylated to xylulose-5-phosphate, which is meta-
bolized further by the non-oxidative part of the pentose
(1)
phosphate pathway yielding fructose-6-phosphate and
glyceraldehyde-3-phosphate. Part of the fructose-6-
phosphate is channelled into the oxidative part of the
pentose phosphate cycle. Oxidation to ribulose-5-phos-

Table 1. Carbon balance of Saccharomyces cerevbiae PUA6-9
(pRD1) transformants based on the operation of Neuberg's first
and second forms of fermentation
Metabolite Concentration
(g/l) ~
Xylitol 9.88
CO2b ] .43
Ethanol c 3.08
CO2c'e 2.95
Glycerold 0.28
Acetaldehyde d,~ 0.13
COza'e 0.13
Total amounts of fermentation products 17.88
Xylose metabolized 19.65
H20b 0.58
Total amounts of fermentation educts 20.23
a Metabolite concentrations were determined in culture superna-
tants. Values (duplicate determinations) were taken from the ex-
periments depicted in Fig. 1A at t=76 h
b CO2 formed and H20 used to regenerate NADPH by operation
of the oxidative pentose phosphate pathway were deduced from
xylitol formed
Neuberg's first form of fermentation
Neuberg's second form of fermentation
e Acetaldehyde and C Q were deduced from metabolites formed
in equimolar amounts (glycerol and ethanol, respectively)
phate yields 2 N A D P H per fructose-6-phosphate. In the
absence of respiration this cofactor moiety is used in the
NADPH-linked xylose reduction resulting in xylitol se-
cretion. Thus, xylitol secretion prevents accumulation of
N A D H , which cannot be reoxidized in the absence of
respiration. Xylose utilization of S. eerevisiae transfor-
mants in the absence of respiration can thus be de-
scribed by the equation
12xylose + 3 H 2 0 ~ 6 xylitol + 9 ethanol + 12 CO2 (2)
or 0.23 g ethanol/g xylose.
On the basis of this scheme we calculated the net pro-
duction of ATP. Xylose fermentation in the presence of
antimycin A results in 0.75 tool A T P / m o l xylose. By
contrast, xylose fermentation on the basis of Eq. 1
would result in 1.66 tool A T P / m o l xylose, which is equi-
valent to 2 tool A T P / m o l glucose during glucose fer-
mentation. Tentatively, the absence of cell growth on
xylose may be explained both by the slow fermentation
of xylose and by inefficient A T P generation due to loss
of half of the xylose carbon in the form of secreted xyli-
tol.
Intracellular metabolite concentrations
In order to characterize differences in pentose and
hexose metabolism we compared the intracellular meta-
bolite concentrations of S. cerevisiae (pRD1) transfor-
mants and P. stipitis grown on glucose and xylose, re-
spectively (Fig. 2). This would allow some conclusions
about rate-limiting steps in xylose utilization by S. cere-
779
visiae transformants. With regard to the fermentation
studies described above differences in metabolite con-
centrations were determined also in the absence of respi-
ration. Concentrations of intracellular metabolites ob-
tained with S. cerevisiae transformants grown on glu-
cose were in close agreement with those reported by oth-
ers (Gancedo and Gancedo 1973; Ciriacy and Breiten-
bach 1979). Typically, fructose-l,6-bisphosphate and
pyruvate accumulated. Expectedly, sugar phosphate ac-
cumulation was not significantly affected by inhibition
of respiration. Metabolite concentrations were generally
lower in xylose-grown S. cerevisiae cells than in glucose-
grown cells, with the exception of sedoheptulose-7-phos-
phate. The concentration of this intermediate of the
pentose phosphate pathway was about tenfold higher in
cells grown on xylose than on glucose. Levels of xylu-
lose-5-phosphate and 6-phosphogluconate in xylose-
grown cells were also significantly increased. Addition
of antimycin A to xylose-grown S. cerevisiae transfor-
mants caused a significant decrease in the levels of sedo-
heptulose-7-phosphate and 6-phosphogluconate. All
other sugar phosphates were unaffected by the inhibi-
tion of respiration and remained at low levels.
In contrast to S. cerevisiae transformants, intracellu-
lar metabolite concentrations in P. stipitis were general-
ly lower both on glucose and xylose. P. stipitis cells did
not show the typical accumulation of fructose-l,6-bis-
phosphate and pyruvate observed with S. cerevisiae dur-
ing glucose fermentation. Fructose-l,6-bisphosphate
and pyruvate concentrations on glucose were lower com-
pared with S. cerevisiae. This is in good agreement with
the observation that P. stipitis metabolizes glucose and
xylose, respectively, almost entirely oxidatively under
aerobic conditions. Also, intermediates of the pentose
phosphate pathway did not accumulate during xylose
utilization by P. stipitis. In the presence of antimycin A
the level of pyruvate increased dramatically (about six-
fold in xylose-grown cells, cf. Fig. 2D). This accumula-
tion of pyruvate coincides with the enhanced production
of ethanol under these conditions (cf. Fig. 1C and D,
and unpublished data).
Xylose uptake
Limitations in xylose utilization by S. cerevisiae X Y L 1 /
X Y L 2 transformants, compared to cells fermenting glu-
cose, could result from xylose transport. In S. cerevi-
siae, glucose is transported into the cell by a carrier-me-
diated process (Cirillo 1961). Basically, two separate sys-
tems have been described, a glucose-repressible high-af-
finity (Krn=l.5mM) and a constitutive low-affinity
(Km=20mM) transport system (Bisson and Fraenkel
1984; Bisson 1988; Lang and Cirillo 1987; Ramos et al.
1988). In xylose uptake experiments, a biphasic Eadie-
Hofstee plot was obtained, indicating that transport sys-
tems differing in their affinities for xylose are present
(Fig. 3). For the high-affinity system, a Km for xylose of
about 190mM was calculated. The low-affinity system
has an apparent Km of about 1.5 M. These values agree
with previous reports (Serrano and de la Fuente 1974;
780
S. cerevisiae PUA6-9 (pRD1)
15
~ 10
.~
~.
-
0
a~ Xu5P S7P E4P 6PG Q6P F6P F16P GAP DAP 2PG PEP PYR
P. stipitis CBS5774
15-
~ ~o-
-o
Xu5P S7P E4P 6PQ G6P F6P F16P GAP DAP 2PQ PEP PYR
Fig. 2a-d. Intracellular metabolite concentrations of S. cerevisiae
strain PUA6-9 transformed with plasmid pRD1 (a, b) and P. sti-
pitis (c, d). Yeast cells were grown in 2% (w/v) glucose (a, c) or
2% (w/v) xylose (b, d) and harvested during exponential growth
(4x 107 cells/ml). Intracellular metabolites were extracted from
cells either grown in the absence of antimycin A (m) or incubated
for 30 min after addition of the respiratory inhibitor (final concen-
tration 2 gg/ml, 1~): Xu5P, xylulose-5-phosphate; S7P, sedohep-
Busturia and Lagunas 1986). In glucose transport exper-
iments carried out under the same conditions, Km values
of about 1.5 mM and 35 mM for the high- and low-affin-
ity systems, respectively, were determined (S. Ozcan,
this laboratory; personal communication) in agreement
with those reported by Bisson and Fraenkel (1983). This
would mean that the monosaccharide transport system
in S. cerevisiae has nearly a 200-fold lower affinity for
xylose than for glucose. Under the conditions of our fer-
mentation studies (the initial xylose concentration was
133 raM) the high-affinity carrier may be responsible for
the xylose uptake. However, even the initial xylose con-
centration was clearly below the Km value of the high-
affinity uptake system. The maximal rate of xylose
transport deduced from Fig. 3 is about 240 nmol/min
per milligram of cells for the high-affinity system. For
initial concentrations of xylose ranging from about
144 mM to 100 raM, as used in xylose fermentation (Fig.
1A), the actual rate of xylose uptake was calculated
from Fig. 3. Under these conditions the velocity of xy-
lose transport was about 100 nmol/min per milligram of
cells. The calculated transport rate was about 30-fold
b XuSP ~7P E4P 6PG G6P F6P F16P GAP DAP 2PG PEP PYR
,
d XuSP S7P E4P 6PQ G6P F6P F16P GAP DAP 2PG PEP PYR
tulose-7-phosphate; E4P, erythrose-4-phosphate; 6PG, 6-phos-
phogluconate; G6P, glucose-6-phosphate; F6P, fructose-6-phos-
phate; F16P, fructose-l,6-bisphosphate; GAP, glycerinaldehyde-
3-phosphate; DAP, dihydroxyacetone phosphate; 2PG, 2-phos-
phoglycerate; PEP, phospho(enol)pyruvate; PYR, pyruvate. Cal-
culations were based on four determinations each. Standard errors
were <0.1-0.4 nmol/mg dry mass
higher than the observed rate of xylose consumption
(Fig. 1A and B). Thus, at first glance the capacity of the
xylose uptake system does not seem to be a limiting fac-
tor in xylose utilization.
Discussion
We have shown that simultaneous expression of the P.
stipitis X Y L 1 and X Y L 2 genes in baker's yeast allows
growth on xylose as sole carbon source. Xylose utiliza-
tion in S. cerevisiae is characterized by high xylitol secre-
tion, which is even increased in the presence of the respi-
ratory inhibitor antimycin A. We interpret this finding
by assuming a considerable flow of xylose carbon
through the oxidative pentose phosphate pathway. This
assumption takes into account that XR can effectively
use N A D P H as a cofactor instead of NAD H. As a cor-
ollary, oxidation of xylitol to xylulose by X D H is lim-
ited by the availability of NAD. Consistent with this as-
sumption is the finding that xylitol accumulation is in-
creased in the absence of respiration. These findings are

800
600
+ 6 Xylulose
E 400
.E
÷ ®
-6
>
1 +
÷
+
0 I , , , ' , " " ~
0.2 0.4 0.6 0.8 1 1.2
V/[S] nmol/min mM rag(wet wt)
Fig. 3. Eadie-Hofstee plot of xylose transport in S. cerevisiae
xyll/xyl2 transformants. Cells were grown in 2°7o(w/v) xylose and
harvested during exponential growth. Initial transport rates were
measured after incubation for 5-s intervals using D-[U-14C] xylose
at concentrations ranging from 1 mM to 1 M. The values for two
independent culture samples are shown: V, velocity; [S], xylose
concentration
summarized in a putative scheme of xylose metabolism
in S. cerevisiae (Fig. 4). From this it is also obvious that
net A T P production is significantly reduced to less than
half the theoretical value. It is reasonable to assume that
this low A T P yield together with the relatively slow xy-
lose metabolism causes the growth arrest on xylose ob-
served in the absence of respiration. Xylitol production
from xylose in S. cerevisiae has recently been shown in
X Y L 1 transformants (Tran-Dinh et al. 1991). In this
case however the accumulation of xylitol is due to the
absence of the X D H reaction. Xylose/xylitol conversion
with a remarkably high yield has also been demon-
strated recently in the yeast Candida guillierimondii
(Meyrial et al. 1991). It is not unlikely that this natural-
ly-occurring xylitol production is regulated in a similar
way as in S. cerevisiae.
One might argue that the dual cofactor dependence
of the XR is the sole reason for xylitol production. This
is obviously not correct since expression of XR in P. sti-
pitis is not accompanied by xylitol formation (cf. Fig.
1C, D; Dellweg et al. 1990; Skoog and Hahn-H~tgerdal
1990; Prior et al. 1989) regardless of the growth condi-
tions. Model simulations predict that in P. stipitis under
aerobic conditions N A D P H is the preferred cofactor for
XR, whereas under oxygen deficiency xylose is reduced
by N A D H (Dellweg et al. 1990; Rizzi et al. 1988; Dell-
weg et al. 1989). The absence of xylitol accumulation
under oxidative conditions could be interpreted as a re-
sult of xylitol oxidation. However, this model does not
explain the method of N A D P H generation. Notably, an
investigation of (1-13C) xylose metabolism in P. stipitis
781
[12 Xylose[
' I0 ×ylitol]
o .,o---IQ ° .Ao
OP
6 Xylitol
®r °x'#'/e
3 Xu5P
6 ADP
6 Xu5P
, 3 F6P
2 GAP 1 F6P 1 GAP 2 F6P
I I
1.4 1 , I
3 GAP 3 F6P
1
Fig. 4. Scheme of xylose utilization and mechanism of cofactor
regeneration in S. cerevisiae xyll/xyl2 transformants in the ab-
sence of respiration: EMP, Embden-Meyerhof-Parnas pathway;
1, xylose reductase; 2, xylitol dehydrogenase; 3, xylulose kinase;
4, ribulose-5-phosphate epimerase, ribose-5-phosphate isomerase,
transaldolase and transketolase; 5, glucose-6-phosphate dehydro-
genase, 6-phosphogluconate dehydrogenase. Abbreviations for su-
gar phosphates as in Fig. 2
by nuclear magnetic resonance spectroscopy revealed
that only very little xylose carbon is channelled through
the oxidative part of the pentose phosphate cycle (Lig-
thelm et al. 1988). From these observations we speculate
that xylitol secretion in S. cerevisiae is rather a corollary
of N A D P H availability. We are currently testing this
hypothesis by using appropriate S. cerevisiae mutants
with a defect in the oxidative pentose phosphate path-
way, which is, according to this working hypothesis, the
major source of N A D P H .
Another limiting factor of xylose fermentation by S.
cerevisiae may be the capacity of the non-oxidative pen-
tose phosphate pathway, as indicated by a significant
accumulation of sedoheptulose-7-phosphate concentra-
tions during xylose fermentation, which was not ob-
served in glucose-fermenting cells. Accumulation of se-
doheptulose-7-phosphate was observed during fermenta-
tion of xylulose (Ciriacy and Porep 1986; Senac and
Hahn-H~gerdal 1990). This may suggest that the trans-
aldolase-transketolase reactions are insufficient. Trans-
aldolase catalyses the reaction of sedoheptulose-7-phos-
phate with glycerinaldehyde-3-phosphate yielding fruc-
tose-6-phosphate and erythrose-4-phosphate. Since glyc-
782
erinaldehyde-3-phosphate is also a key intermediate o f
glycolysis, its rapid glycolytic conversion could lead to a
depletion o f this intermediate f r o m the pentose phos-
phate pathway. Furthermore, the slow xylose consump-
tion by S. cerevisiae coincides with the low intracellular
concentration o f fructose-l,6-bisphosphate, and in par-
ticular, o f pyruvate. N o r m a l l y these two intermediates
o f glycolysis accumulate in b a k e r ' s yeast to significant
amounts during growth on glucose (Gancedo and Gan-
cedo 1973). It has been shown that the intracellular con-
centration o f pyruvate is directly connected with the de-
gree o f fermentation. High pyruvate concentrations are
required for efficient ethanol p r o d u c t i o n under aerobic
conditions. This p h e n o m e n o n has been explained by the
different affinities for pyruvate o f pyruvate dehydrogen-
ase and pyruvate decarboxylase. The Km value for pyru-
vate dehydrogenase is approximately ten times lower
than for pyruvate decarboxylase (Holzer and G o e d d e
1957). It has been concluded that ethanol f o r m a t i o n is
due to an overflow reaction at the pyruvate level when
the oxidative p a t h w a y is saturated. With regard to xy-
lose utilization in b a k e r ' s yeast, slow xylose metabolism
would prevent a significant pyruvate a c c u m u l a t i o n and
consequently efficient ethanol f o r m a t i o n under aerobic
conditions.
Xylose transport in S. cerevisiae takes place by facili-
tated diffusion (Cirillo 1961). In S. cerevisiae X Y L 1 /
X Y L 2 transformants, xylose transport does not seem to
be a limiting reaction because the observed rate o f xy-
lose c o n s u m p t i o n was about 30-fold lower than the ac-
tual rate o f xylose uptake, at least at high substrate con-
centrations. At lower extracellular xylose concentrations
however, transport could become a critical step because
o f the remarkably low substrate affinity o f the X D H
[68-97 mM, depending on the cofactor (Rizzi et al.
1988)]. In contrast to S. cerevisiae, P. stipitis takes up
xylose by active p r o t o n symport (Does and Bisson 1989;
Kilian and van U d e n 1988). This system allows intracel-
lular accumulation o f xylose and could be a prerequisite
for efficient xylose metabolism.
Acknowledgements. We greatly appreciate the excellent technical
assistance of J. Lepique. This work was supported by Bundes-
ministerium fiir Forschung und Technologie grant 0316700/B2 to
M.C.
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