FEMS Yeast Research 3 (2003) 185^189
www.fems-microbiology.org
Production of ethanol from L-arabinose by Saccharomyces cerevisiae
containing a fungal L-arabinose pathway
Peter Richard , Ritva Verho, Mikko Putkonen, John Londesborough, Merja Penttila«
VTT Biotechnology, P.O. Box 1500, Tietotie 2, Espoo 02044 VTT, Finland
Received 31 May 2002; received in revised form 6 August 2002; accepted 7 August 2002
First published online 7 November 2002
Abstract
The fungal pathway for L-arabinose catabolism converts L-arabinose to D-xylulose 5-phosphate in five steps. The intermediates are, in
this order: L-arabinitol, L-xylulose, xylitol and D-xylulose. Only some of the genes for the corresponding enzymes were known. We have
recently identified the two missing genes for L-arabinitol 4-dehydrogenase and L-xylulose reductase and shown that overexpression of all
the genes of the pathway in Saccharomyces cerevisiae enables growth on L-arabinose. Under anaerobic conditions ethanol is produced
from L-arabinose, but at a very low rate. The reasons for the low rate of L-arabinose fermentation are discussed.
6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords :
L-Arabinose ;
Pentose fermentation; L-Arabinitol 4-dehydrogenase; L-Xylulose reductase ; Saccharomyces cerevisiae
1. Introduction
The two most widespread pentose sugars found in our
biosphere are D-xylose and L-arabinose [1]. Pathways for
pentose catabolism are relevant for micro-organisms living
on decaying plant material, but also in biotechnology
when cheap raw materials such as plant hydrolysates are
to be fermented to ethanol. Common in the catabolism of
these two pentose sugars in all micro-organisms is that the
sugar is converted to D-xylulose 5-phosphate. However,
the pathways to convert L-arabinose and D-xylose to Dxylulose 5-phosphate are distinctly di¡erent in bacteria
and fungi. In bacteria, D-xylose is converted to D-xylulose
by an isomerase and then phosphorylated by xylulokinase.
In bacteria, L-arabinose is ¢rst converted to L-ribulose by
an isomerase, then phosphorylated by ribulokinase and
the L-ribulose 5-phosphate is then converted to D-xylulose
5-phosphate by an epimerase. In fungi, these pentose sugars go through oxidation and reduction reactions before
they are phosphorylated by xylulokinase. D-Xylose is ¢rst
* Corresponding author. Tel. : +358 (9) 456 7190;
Fax : +358 (9) 455 2103.
E-mail address : peter.richard@vtt.¢ (P. Richard).
reduced to xylitol by an reduced nicotinamide adenine
dinucleotide phosphate (NADPH)-consuming reaction.
Xylitol is then oxidised by an NADþ -consuming reaction
to form D-xylulose. In fungi, L-arabinose goes through
four redox reactions. Two oxidations are coupled to
NADþ consumption and two reductions to NADPH consumption (Figs. 1 and 3).
Several attempts have been made, by means of genetic
engineering, to generate strains of Saccharomyces cerevisiae able to ferment these pentose sugars. The expression of
the bacterial pentose pathways in yeast has had only limited success in the past. The xylose isomerases expressed in
S. cerevisiae (or in Schizosaccharomyces pombe) have
shown no or only very low activity [2^8]. In an attempt
to overexpress the bacterial L-arabinose pathway, the
individual enzymes showed activity when produced in
S. cerevisiae ; however, the yeast strain carrying all three
bacterial genes neither grew on L-arabinose nor fermented
it to ethanol [9].
The expression of the fungal D-xylose pathway has been
more successful : in S. cerevisiae it led to growth on Dxylose as well as to ethanol production from D-xylose
[10^13]. The expression of the fungal L-arabinose pathway
has not been possible until recently because not all genes
of this pathway were known.
All enzymes of the fungal D-xylose pathway can also be
used in the L-arabinose pathway. The ¢rst enzyme is an
1567-1356 / 02 / $22.00 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S1567-1356(02)00184-8
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P. Richard et al. / FEMS Yeast Research 3 (2003) 185^189
aldose reductase (EC 1.1.1.21). This enzyme has been
characterised after puri¢cation from S. cerevisiae [14]
and from Pichia stipitis [15]. Both enzymes have similar
activities with D-xylose and L-arabinose. Also, the corresponding genes for the S. cerevisiae enzyme (GRE3 [14])
and the P. stipitis enzyme (XYL1, [16]) are known. The Dxylulose reductase (EC 1.1.1.19) and the xylulokinase (EC
2.7.1.17) are also involved in both pathways. Genes coding
for D-xylulose reductases are known in various fungi
[17,18]. Genes coding for xylulokinase are known for
S. cerevisiae [19] and P. stipitis [20].
We have identi¢ed the missing two genes for L-arabinitol 4-dehydrogenase and L-xylulose reductase. The ¢rst
has been puri¢ed from the mould Trichoderma reesei (Hypocrea jecorina) and the corresponding gene (lad1) identi¢ed [21]. To identify the gene for the L-xylulose reductase,
all the other genes of the L-arabinose pathway, i.e. genes
coding for aldose reductase, L-arabinitol 4-dehydrogenase,
D-xylulose reductase and xylulokinase had been expressed
in S. cerevisiae. This strain was then transformed with a
cDNA library from T. reesei and screened for growth on
L-arabinose. In this way a gene coding for an L-xylulose
reductase (lxr1) has been identi¢ed [22]. The strain
harbouring all the genes of the L-arabinose pathway
could grow on L-arabinose [22]. In this paper we were
interested if such a strain could also ferment L-arabinose
to ethanol.
2. Materials and methods
2.1. Strains
The cultivations were done with S. cerevisiae strain
H2561 carrying all the genes of the L-arabinose pathway
(XYL1, lad1, lxr1, XYL2 and XKS1) and a control strain
H2562, which was identical except that it did not contain
the lxr1, but an empty vector instead. XYL1 and XYL2
are from P. stipitis coding for aldose reductase and Dxylulose reductase, respectively. XKS1 is from S. cerevisiae
and codes for xylulokinase; lad1 and lxr1 are from T.
reesei and code for L-arabinitol 4-dehydrogenase and Lxylulose reductase, respectively. XYL1 (under PGK promoter), XYL2 (under ADH promoter) and XKS1 (under
ADH promoter) were integrated into the chromosomes by
targeted integration: lad1 and lxr1 (both under TPI promoter) were on two di¡erent plasmids. The lad1 was on a
plasmid with URA3, the lxr1 on a plasmid with LEU2 as
selection marker. The control strain was a similar construct with two plasmids, except that it did not contain
the lxr1 gene. The construction of the strains has been
described earlier [21,22].
2.2. Fermentation conditions
Batch cultivations were performed in two 1.8-l Chemap
CMF fermenters (Chemap, Switzerland). The culture volume was 0.9 l during the ¢rst 2 days and the medium was
synthetic complete medium [23] lacking uracil and leucine,
with 2% glucose as the carbon source. After 2 days 0.3 l of
synthetic medium with 20% L-arabinose was added to give
a ¢nal L-arabinose concentration of 5%. The temperature
was 30 ‡C and agitation speed was 500 rpm. During cultivation on D-glucose the medium was sparged with air at
a £ow rate of 1.0 l min31 and pH was adjusted to 5.00
with 2-M potassium hydroxide. When the L-arabinose
was added the air £ow was changed to nitrogen at 0.1 l
min31 to have anaerobic conditions. Samples were taken
at di¡erent time intervals. They were analysed for dry
weight and ethanol content. The dry weight was measured
for 10 ml of cell suspension. Cells were harvested by centrifugation, washed twice with water and dried overnight
at 95 ‡C. The ethanol concentration was measured enzymatically using a commercial kit (Roche Diagnostics,
Mannheim, Germany). The analysis was done in a Cobas
Mira automated analyser (Roche).
3. Results
3.1. L-Arabinose fermentation under anaerobic conditions
Yeast cells were ¢rst grown on the medium with D-glucose under aerobic conditions for 2 days to generate biomass. After this time most of the ethanol had been used.
Synthetic medium containing L-arabinose was then added
to give a concentration of 5% L-arabinose. The fermenter
was then switched from air to nitrogen and the fermentation monitored for 70 h. In earlier trials we harvested the
cells, washed them in phosphate bu¡er and resuspended
them in the L-arabinose medium before introducing them
to the fermenter. We observed that under these conditions
the cells lysed so that after 70 h only 50% of the initial
biomass remained. Without this harvesting and washing,
we found that the biomass remained approximately constant during the 70 h. The L-arabinose was added when
most of the ethanol had been used because it was then
more accurate to measure small changes in the ethanol
concentration. In Fig. 2 the ethanol production is shown
during the anaerobic L-arabinose fermentation for the
strain where the complete pathway is expressed and for
a control strain. As a control we used a construct where
only the lxr1 was omitted. The initial ethanol concentration in both cases was below 0.05 g l31 .
The strain with the complete pathway produced about
0.1 g l31 ethanol during the 70-h period, whereas the
ethanol production in the control strain was below our
detection limit. The biomass of the strain with the complete pathway was 4 g l31 , i.e. the productivity was 0.35
mg ethanol per g dry weight and hour. The productivity of
the control strain was below 0.05 mg ethanol per g dry
weight and hour.
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Fungal Pathways:
D-xylose
Bacterial pathways:
L-arabinose
aldose reductase
EC 1.1.1.21
xylitol
D-xylose
aldose reductase
EC 1.1.1.21
L-arabinitol
D-xylulose reductase
EC 1.1.1.9
D-xylulose
D-xylulose 5-phosphate
xylose isomerase
EC 5.3.1.5
D-xylulose
L-arabinitol
4-dehydrogenase
EC 1.1.1.12
L-xylulose
xylulokinase
EC 2.7.1.17
187
xylulokinase
EC 2.7.1.17
D-xylulose 5-phosphate
L-arabinose
L-arabinose isomerase
EC 5.3.1.4.
L-ribulose
ribulokinase
EC 2.7.1.16
L-ribulose 5-phosphate
L-ribulosephosphate
4-epimerase
EC 5.1.3.4
D-xylulose 5-phosphate
L-xylulose reductase
EC 1.1.1.10
xylitol
D-xylulose reductase
EC 1.1.1.9
D-xylulose
xylulokinase
EC 2.7.1.17
D-xylulose 5-phosphate
Fig. 1. The fungal and bacterial pathways for the utilisation of the pentose sugars D-xylose and L-arabinose.
4. Discussion
4.1. The fungal L-arabinose pathway
A fungal pathway for the metabolism of L-arabinose
was ¢rst described for the mould Penicillium chrysogenum
in 1960 by Chiang and Knight [24]. The same pathway has
been found in the mould Aspergillus niger [25]. There is
also evidence that this pathway is active in the yeast
P. stipitis. Shi et al. [26] have found that a deletion in the
D-xylulose reductase gene of P. stipitis prevents growth on
L-arabinose. This indicates that yeasts and moulds, i.e.
fungi, in general use the same pathway for L-arabinose
consumption, which is distinctly di¡erent from the bacterial pathway. We have combined the genes of this pathway
from di¡erent fungi, from the yeast P. stipitis XYL1 and
XYL2, from the yeast S. cerevisiae XKS1 and from the
¢lamentous fungus T. reesei lad1 and lxr1. We have overexpressed them in S. cerevisiae, which is a yeast species
that cannot use L-arabinose. The resulting strain showed
activities of all the enzymes of the pathway and could
grow on L-arabinose, although at a very low rate. When
applying anaerobic conditions we could demonstrate for
the ¢rst time that in a recombinant strain ethanol was
produced from L-arabinose. However, the ethanol production under these conditions was very slow. There are various factors that might limit the L-arabinose fermentation,
such as the imbalance of redox cofactors or the L-arabinose transport into the cell.
4.2. The imbalance of redox cofactors
produced ethanol / g/l
0,14
complete pathway
control
0,12
0,10
0,08
0,06
0,04
0,02
0,00
0
20
40
60
80
time / hours
Fig. 2. Ethanol production from L-arabinose. At time zero the fermenter
is switched to anaerobiosis and L-arabinose at a ¢nal concentration of
5% is added. The open triangles show the strain where the complete
L-arabinose pathway is expressed. The open squares show the control
strain, which is a similar construct lacking the lxr1 gene.
The fungal L-arabinose pathway consists of two oxidations and two reductions, i.e. the conversion of L-arabinose to D-xylulose is redox neutral. However, the reductions are NADPH-linked and the oxidations NADþ linked so that there is an imbalance of redox cofactors.
This imbalance of redox cofactors could be solved by a
transhydrogenase activity facilitating the equilibration between NADH/NADPþ and NADþ /NADPH. Yeasts are
believed not to have such activity [27]. It remains an
open question how fungal micro-organisms cope with
this cofactor imbalance. It has been suggested [28] that
NADPH is mainly regenerated through the oxidative
part of the pentose phosphate pathway. The ¢lamentous
fungus A. niger exhibited higher activities of glucose 6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase when growing on pentoses [28]. In the oxidative
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NADH
NADP +
G6P
D-xylulose
NAD +
NADP +
xylitol
NADH
L-xylulose
NADPH
NADP +
L-arabinitol
NAD +
L-arabinose
NADPH
NADPH
CO2
pentose
phosphate
pathway
F6P
X5P
GAP
NAD +
NADH
NADH
NAD +
ethanol + CO2
Fig. 3. Redox cofactor requirement in L-arabinose catabolism. L-Arabinose conversion to equimolar amounts of CO2 and ethanol is redox neutral, i.e.
anaerobic fermentation to ethanol should be possible. However, the conversion of L-arabinose to D-xylulose requires NADPH and NADþ and produces
NADH and NADPþ . NADPH is mainly regenerated in the oxidative part of the pentose phosphate pathway, where the reduction of NADPþ is
coupled to CO2 production. The abbreviations are: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; X5P, D-xylulose 5-phosphate ; GAP, D-glyceraldehyde 3-phosphate.
part of the pentose phosphate pathway, however, the reduction of NADPþ is coupled to CO2 production (Fig. 3).
In this case the anaerobic conversion of L-arabinose to
CO2 and ethanol is no longer redox neutral, i.e. the fermentation of L-arabinose is inhibited by an accumulation
of reduced redox cofactors. A possible strategy to solve
this imbalance of cofactors and thereby facilitate L-arabinose fermentation would be to introduce transhydrogenase
activity.
4.3. L-Arabinose transport into the cell
The ethanol production was about 0.1 g per 70 h at a
biomass dry weight of 4 g l31 , which corresponds to 0.13
nmol per mg dry weight and minute. For such an ethanol
production rate an L-arabinose uptake rate of 0.08 nmol
per mg of dry weight and minute would be required, i.e.
only if the maximal uptake rate is less than this order of
magnitude, it can be a limiting factor. We are not aware of
any information in the literature dealing with the uptake
rate of L-arabinose in S. cerevisiae. D-Xylose however, another pentose sugar that is also not a natural substrate for
S. cerevisiae, is taken up. Both D-xylose and glucose are
transported by facilitated di¡usion. The maximal uptake
rate for D-xylose has been estimated to be 100^240 nmol
(mg min)31 [10]. This is in the same order of magnitude as
the maximal glucose uptake rate, which is 200^400 nmol
(mg min)31 [29]. The only report about L-arabinose transport in yeast is from Lucas and van Uden [30], who observed that L-arabinose was transported in Candida shehatae by a speci¢c proton symport mechanism.
It remains to be seen whether the L-arabinose fermenta-
tion can be stimulated by addressing the problems of redox cofactor imbalance and L-arabinose transport. Addressing the problem of cofactor imbalance can also be
bene¢cial for D-xylose fermentation, since a strain with
the L-arabinose pathway would also be able to ferment
D-xylose.
Acknowledgements
This work was supported by the ‘Sustainable Use of
Natural Resources’ (SUNARE) programme of the Academy of Finland and the research programme ‘VTT Industrial Biotechnology’ (Academy of Finland; Finnish Centre
of Excellence programme, 2000^2005, Project no. 64330).
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