Vol.1, No.3, 210-215 (2009)
Copyright © 2009 Openly accessible at http://www.scirp.org/journal/NS/
Natural Science
Development on ethanol production from xylose by
recombinant Saccharomyces cerevisiae
Jin-Ying Yang, Jian-Ren Lu*, Hong-Yue Dang, Yan Li, Bao-Sheng Ge
Center for Bioengineering and Biotechnology of China University of Petroleum, Qingdao, China
Received 26 August 2009; revised 28 September 2009; accepted 30 September 2009.
Xylose is the second major fermentable sugar
present in lignocellulosic hydrolysates, so its
fermentation is essential for the economic con-
version of lignocellulose to ethanol. However,
the traditional ethanol production strain Sacch-
aromyces cerevisiae does not naturally use xy-
lose as a substrate. A number of different ap-
proaches have been used to engineer yeasts to
reconstruct the gene background of S. cerevi-
siae in recent years. The recombinant strains
showed better xylose fermentation quality by
comparison with the natural strains. This review
examines the research on S. cerevisiae strains
that have been genetically modified or adapted
to ferment xylose to ethanol from three aspects
including construction of xylose transportation,
xylose-metabolic pathway and inhibitor toler-
ance improvement of S. cerevisiae.
Keywords: Sacchromyces cerevisiae; Xylose;
Ethanol; Metabolic Engineering
Rising concerns over the cost of petroleum and the
prospect of global warming are driving the development
of technologies for the production of alternative fuels
such as ethanol [1]. Cellulosic biomass is an attractive
feedstock for fuel ethanol production since it is readily
available, e.g., as a waste from the pulp and paper or
agricultural industries, and also due to the fact that it is
renewable with cycles many orders of magnitude shorter
compared with those of fossil fuels. Hydrolysates of
cellulosic biomass will contain mixtures of sugars, inclu-
ding glucose, galactose, mannose, xylose and arabinose,
and other constituents in variable proportions depending
on the source [2]. Successful industrial production of
ethanol from lignocellulosic hydrolysate depends on the
quantitative conversion of carbon present in the biomass.
It is well known that one of the most effective etha-
nol-producing organisms for hexose sugars is Sac-
charomyces cerevisiae, which shows high ethanol pro-
ductivity, high tolerance to ethanol, and tolerance to in-
hibitory compounds present in the hydrolysate of ligno-
cellulosic biomass [3-5]. However, S. cerevisiae does not
naturally use xylose as a substrate. Only a few yeasts
such as Pichia stipitis [6] and Pachysolen tannophilus [7]
are able to ferment xylose. Genetic engineering can be
used to enable S. cerevisiae to transport and ferment
xylose including modeling, mutation, deletion and so on.
The pentose phosphate pathway (PPP) [8] is a process
that serves to generate NADPH for reductive biosynthe-
sis reactions within cells and the synthesis of pentose
(5-carbon) sugars for the synthesis of the nucleotides and
nucleic acids. There are two distinct phases in the path-
way. The oxidative phase converts the hexose, D-glucose
6P, into the pentose, D-ribulose 5P, plus CO2 and
NADPH. The non-oxidative phase converts D-ribulose
5P into D-ribose 5P, D-xylulose 5P, D- sedoheptulose 7P,
D-erythrose 4P, D-fructose 6P and D- glyceraldehyde 3P.
D-Xylose and L-arabinose enter the PPP through
D-xylulose (Figure 1)
In bacteria, xylose is directly isomerized to xylulose
by xylose isomerase (XI) before entering pentose phos-
phate pathway. In xylose-fermenting yeasts, xylose is
first reduced to xylitol by xylose reductase (XR) and
then oxidized to xylulose by xylitol dehydrogenase
(XDH) [9]. Xylulokinase (XK) phosphorylates xylulose
to xylulose 5-phosphate, which is then metabolized
through the PPP and glycolysis (Figure 2). S. cerevisiae
is not able to metabolize xylose due to the lack of XR
and XDH activity, but it can utilize the isomeric form
2.1. Xylose Uptake
Xylose is not readily fermentable in wild-type strains of
S. cerevisiae. To circumvent this problem, different
J. Y. Yang et al. / Natural Science 1 (2009) 210-215
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Engineering pentose metabolism in yeasts. The pentose phosphate pathway (PPP) in yeasts contains the oxidative phase, which
consists of glucose 6-phosphate dehydrogenase (ZWF1) and 6-phosphogluconate dehydrogenase (GND1), and the
non-oxidative phase, which is carried out by D-ribulose-5-phosphate 3-epimerase (RPE1), ribose-5-phosphate ketol-isomerase
(RKI1), transketolase (TKL1) and transaldolase (TAL1).
Figure 1. The pentose phosphate pathway.
metabolic engineering strategies have been applied to
enable xylose metabolism, and pentose-fermenting str-
ains of S. cerevisiae have been created principally by
engineering the pathways for converting xylose to xylu-
lose-5-phosphate [10,11]. However, fermentation of xy-
lose still remains significantly less efficient than that of
glucose by these strains. The uptake of xylose into the
cell is one of the reasons.
The S. ceresiave genome contains 20 genes that en-
code for hexose transporters but does not contain genes
for xylose-specific transport system like natural xy-
lose-utilizing yeasts [12]. Uptake of xylose by S. cere-
visiae has been proposed to be mediated more or less
unspecifically by its hexose-transport system. This is
composed of a large family of 18 related transporter
proteins called Hxts and additional sugar transporters
with broader substrate specificity [13,14].
Hamacher et al. [12] found that after deletion of all of
the 18 hexose-transporter genes, the ability of S. cere-
visiae cells to take up and to grow on xylose was lost.
Re-introduction and constitutive expression of individual
HXT genes in strain TMB3201 revealed that at 2% xy-
lose concentrations, high- (Hxt7 and Gal2) and interme-
diate-affinity (Hxt4 and Hxt5) glucose transporters are
required for xylose uptake.
Several studies have indicated that in S. cerevisiae
glucose and xylose appear to share the same transport
facilities and competitively inhibit their mutual transport
[15,16]. Competition with glucose restricts xylose as-
similation, so heterologous expression of a specific xy-
lose transporter could be very useful.
Researchers have tried to identify genes target for im-
proved xylose assimilation. Two genes (GXF1and GSX1)
encoding xylose/glucose transporters from Candida in-
XR: Xylose reductase; XDH : xylitol dehydrogenase; XK : Xylulo-
Figure 2. The metabolism of xylose in bacteria.
termedia were isolated by Leandro et al. [17], and ex-
pressed in S. cerevisiae. Gsx1 is the first yeast xy-
lose/glucose–H + symporter to be characterized in
Arabidopsis thaliana at the molecular level. Except
GSX1, xylose transporters from Arabidopsis thaliana
J. Y. Yang et al. / Natural Science 1 (2009) 210-215
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(At5g59250) and Escherichia coli (xylE) were also ex-
pressed in S. ceresiave TMB3120 and failed to support
vigorous growth of the recipient S. cerevisiae strain on
xylose. Even though, the results warrant further investi-
gations for the development of efficient bioethanol pro-
duction processes from lignocellulosic materials.
The presence of three sugar transporters in P. stipitis,
Sut1, Sut2 and Sut3 has been reported. Although all
three transporters have a higher affinity for glucose than
for xylose, the Sut1 transporter has a higher Vmax for
xylose uptake compared to other two Sut transporters
and for hexose transporters [18,19]. Satoshi Katahira [20]
et al. introduced SUT1 into a xylose-assimilating S. cer-
evisiae strain that expresses xylose reductase, xylosede-
hydrogenase and xylulokinase. The results showed that
expression of Sut1 in xylose-assimilating S. cerevisiae
increased both xylose uptake ability and ethanol produc-
tivity during xylose fermentation. Also, the enhancement
of xylose uptake enables to accelerate the ethanol pro-
ductivity during xylose/glucose co-fermentation. How-
ever, there are researchers with different opinions. Gár-
donyi et al. [21,22] concluded that xylose transport in S.
cerevisiae strains has low control over the rate of xylose
utilization, unless the xylose pathway is significantly
improved. In-depth study of the transporters’ mechanism
along with new modeling should continue to drive this
field forward.
2.2. Construction of Recomnant S.
Cerevisiae Strains with
Xylose-Fermenting Ability
2.2.1. Recombinant S. cerevisiae Expressing XR,
XDH, and XK
Researchers have engaged in the development of engi-
neered yeast strains capable of xylose fermentation by
introducing XR and XDH into S
cerevisiae. Both of
these enzymes have been isolated and characterized due
to the central role they play in xylose metabolism.
The purified monomeric XR was NADPH-dependent
with an apparent MW of 37 kDa, which was firstly puri-
fied and characterized by Kuhn et al. [23] XDHs also
have been purified and characterized from various xy-
lose-fermenting yeasts.
Kötter et al. [24] first reported the construction of a S.
cerevisiae strain expressing the XR- and XDH-encoding
genes XYL1 and XYL2 derived from the xylose-utilizing
yeast P. stipitis. Walfridsson et al. [25] also genetically
engineered S. cerevisiae to utilize xylose by introducing
the XYL1 and XYL2 genes on either multicopy plas-
mids or by integrating them into the chromosome.
Although these strains can ferment xylose to ethanol,
the excretion of xylitol occurs unless a co-metabolizable
carbon source such as glucose is added. One of the most
important reason is intercellular redox imbalance due to
a different coenzyme specificity of xylose reductase
(with NADPH+) and XDH (with NAD+) [26]. Protein-
engineering of NADPH+-preferring XR and/or NAD+-
dependent XDH is an alternative approach to solve the
Anderlund [27] constructed four chimeric genes en-
coding fusion proteins of XYL1 and XYL2 with different
orders of the enzymes and different linker lengths. These
genes were expressed in S. cerevisiae. The fusion pro-
teins exhibited both XR and XDH activity when XYL1
was fused downstream of XYL2. The results showed
that the xylitol yield was lower in these strains than in
strains expressing only native XR and XDH monomers,
0.55 and 0.62, respectively, and the ethanol yield was
By analyzing the amino acid of coenzyme-binding
domain of XDH, Watanabe [28] modified XDH from P.
stipitis by three- and four-site direct mutagenesis. The
triple mutant (D207A/I208R/F209S) and quadruple mu-
tant (D207A/I208R/F209S/N211R) showed more than
4500-fold higher values in kcat/Km with NADP+ than
the wild-type enzyme, reaching values comparable with
kcat/Km with NAD+ of the wild-type enzyme.
In recent years, the research group introduced these
mutated PsXDHs with the PsXR WT to S. cerevisiae and
estimated effect(s) of the functional modication(s) of
PsXDH on fermentation of xylose to ethanol in recom-
binant S. cerevisiae [29]. The results showed that re-
combinant yeast strains gave the highest ethanol produc-
tion and the lowest xylitol excretion.
Zeng et al. [30] altered the coenzyme specificity of P.
stipitis XR via rational design based on the 3D structure.
Lys21, the only one amino acid that has hydrogen bind-
ing interaction with NADP+ but not with NAD+ in the
binding pocket, were changed to Ala and Arg respec-
tively. The results showed that the coenzyme depend-
ence of K21A was completely reversed to NADH+.
2.2.2. Recombinant S. cerevisiae Expressing
Xylose Isomerase
Xylose isomerase (XI), encoded by the xylA gene, cata-
lyzes the isomerization of xylose to xylulose in bacteria
and some fungi [31]. xylA has been cloned into S. cere-
visiae from several bacteria. However the XI produced
by the recombinant S. cerevisiae strains was inactive.
Improper protein folding, postranslational modifications,
inter- and intramolecular disulfide bridge formation, and
the internal pH of yeast have been suggested as possible
reasons [32].
In 1996, Walfridsson et al. [33] cloned the Thermus
thermophilus xylA gene encoding xylose (glucose)
isomerase and successfully expressed in S. cerevisiae
under the control of the yeast PGK1 promoter. The re-
combinant xylose isomerase showed the highest activity
at 85 with a specific activity of 1.0 U/mg. It was the
first successful attempt to express the procaryotic gene
xylA for the enzyme XI in the eucaryote S. cerevisiae,
J. Y. Yang et al. / Natural Science 1 (2009) 210-215
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which could be due to the relatedness between the two
organisms. The recombinant strains could not covert
xylose to ethanol efficiently because the temperature and
pH optimum for the recombinant enzyme are high.
The XylA gene from the anaerobic fungus Piromyces
sp. E2 (ATCC 76762) was functionally expressed in S.
cerevisiae by Marko Kuyper et al. [34]. After prolonged
cultivation on xylose, a mutant strain was obtained that
grew aerobically and anaerobically on xylose. The an-
aerobic ethanol yield was 0.42 g ethanol /g. xylose and
also by-product formation was comparable to that of
glucose-grown anaerobic cultures.
In 2009, Brat et al. [35] cloned and successfully ex-
pressed a highly active new kind of xylose isomerase
from the anaerobic bacterium Clostridium phytofermen-
tans in S. cerevisiae. The recombinant yeast cells with
heterologous expression got the ability to metabolize
D-xylose and to use it as the sole carbon and energy
source. The new enzyme has low sequence similarities to
the XI Thermus thermophilus and Piromyces sp. E2,
which were the only two xylose isomerases previously
functionally expressed in S. cerevisiae. Importantly, the
new enzyme is far less inhibited by xylitol, which ac-
crues as a side-product during xylose fermentation. The
findings provided an excellent starting point for further
improvement of xylose fermentation in industrial yeast
3.1. Xylulokinase
Although recombinant strains containing genes cod-
ing for XR and XDH from the xylose-utilizing yeast
P. stipitis have been reported, such strains ferment
xylose to ethanol poorly. One reason for this may be
the low capacity of xylulokinase, the third enzyme in
the xylose pathway [36,37].
Xylulokinase is an enzyme that catalyzes the
chemical reaction: ATP + D-xylulose ADP +
D-xylulose 5-phosphate. In 1989, Ho et al. [38]
cloned the xylulokinase (xks1) gene from S. cere-
visiae and firstly overexpressed in S. cerevisiae.
Toivari et al. [39] also overexpressed the endogenous
gene for xylulokinase (xks1) in S. cerevisiae along
with the P. stipitis genes for XR and XDH. The me-
tabolism of this recombinant yeast was further inves-
tigated in pure xylose bioreactor cultivation at various
oxygen levels. The results clearly indicated that
overexpression of xks1 significantly enhanced the
specific rate of xylose utilization. In addition, the
XK-overexpressing strain can more efficiently con-
vert xylose to ethanol under all aeration conditions
studied. These two studies represented an important
step in efforts to improve xylose metabolism in S.
cerevisiae, as their results strongly indicated that na-
tive XK activity was insufficient for xylose or xylu-
lose fermentation, and overexpression was required to
obtain high ethanol yields.
3.2. Transketolase and Ransaldolase
Transketolase and transaldolase catalyze transfer of
2-C and 3-C molecular fragments respectively, in
each case from a ketose donor to an aldose acceptor.
The two enzymes have been implicated as being
rate-limiting for xylose and xylulose fermentation.
The TKL1 and TAL1 genes encoding transketolase
and transaldolase were overexpressed individually and
together in the S. cerevisiae strain containing XYL and
XYL2. The strain overexpressing TAL1 showed consid-
erably enhanced growth on xylose compared with a
strain containing only XYL1 and XYL2. Overexpression
of only TKL1 did not inuence growth. The results in-
dicated that the transaldolase level in S. cerevisiae is
insufcient for the efcient utilization of pentose phos-
phate pathway metabolites [40]. Bao et al. [41] also
found that the S. cerevisiae strain overexpressing the
TAL1 and TKL1 showed considerably good growth on
the xylose plate.
The growth of S. cerevisiae and ethanol production were
limited by multiple inhibitors, including furan deriva-
tives, 5-hydroxymethylfurfural (HMF), weak acids, and
phenolic compounds produced during biomass-to-ethanol
processing. The PPP is an important pathway incarbo-
hydrate metabolism, and a lot of previous studies have
shown a correlation between several PPP genes and spe-
cific stresses such as oxidative [42], sorbic acid [43], and
osmotic [44].
To improve production of fuel ethanol from renew-
able raw materials, laccase from the white rot fungus
Trametes versicolor was expressed under control of the
PGK1 promoter in S. cerevisiae to increase its resistance
to phenolic inhibitors in lignocellulose hydrolysates [45].
To identify target genes involved in furfural tolerance,
Gorsich [46] screened a S. cerevisiae gene disruption
library for mutants with growth deficiencies in the pres-
ence of furfural. As a result, more than 62 genes were
found to be associated with sensitivity to furfural. They
also further showed that overexpression of ZWF1 in S.
cerevisiae allowed growth at furfural concentrations that
are normally toxic, which demonstrated a strong rela-
tionship between PPP genes and furfural tolerance.
Adapting strains is also an alternative to improve the
performance of microorganisms. Liu [47] improved bio-
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transformation by newly developed strains adapted to
tolerate the challenges of furfural and HMF in batch
cultures compared with the parental strains. The results
suggest a possible in situ detoxification of the inhibitors
for bioethanol fermentation using improved yeast strains.
Although they have not been tested against inhibitor
complexes such as those in a biomass hydrolysate, the
development and study of such strains provided neces-
sary materials for further studies of the mechanisms of
the stress tolerance at molecular and genomic levels.
The bioconversion of cellulose and hemicellulose to
biofuels and chemicals is being actively researched with
the aim of developing technically and economically vi-
able processes. D-Xylose is the major product of the
hydrolysis of hemicellulose and considerable research
efforts has been focused on the development of xylose-
fermenting recombinant S. cerevisiae. Significant im-
provements in ethanol productivity from xylose have
been achieved through metabolic engineering. However,
there are still unidentified limiting steps in the xylose
metabolism of metabolically engineered S. cerevisiae,
such as lower ethanol yield, more byproducts, the suit-
ability of these recombinant strains and so on. There are
still many tasks that left in the xylose-metabolic engi-
neering. So far the recombinant S. cerevisiae were con-
structed base on the laboratory strains, which are less
complex in genetic background, growth characters, and
physiological characters comparing with the industrial
yeast strains. To get strains easy to be industrialized,
more emphasis should be focused on the reconstruction
of the wild type yeasts. Further improve the expression
and stability of the heterogenous genes in yeasts can be
expected for higher ethanol yield.
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