Appl Microbiol Biotechnol (2009) 84:37–53
DOI 10.1007/s00253-009-2101-x
MINI-REVIEW
Ethanol production from xylose in engineered
Saccharomyces cerevisiae strains: current
state and perspectives
Akinori Matsushika & Hiroyuki Inoue &
Tsutomu Kodaki & Shigeki Sawayama
Received: 28 April 2009 / Revised: 18 June 2009 / Accepted: 18 June 2009 / Published online: 2 July 2009
# Springer-Verlag 2009
Abstract Bioethanol production from xylose is important
for utilization of lignocellulosic biomass as raw materials.
The research on yeast conversion of xylose to ethanol has
been intensively studied especially for genetically engi-
neered Saccharomyces cerevisiae during the last 20 years.
S. cerevisiae, which is a very safe microorganism that plays
a traditional and major role in industrial bioethanol
production, has several advantages due to its high ethanol
productivity, as well as its high ethanol and inhibitor
tolerance. However, this yeast cannot ferment xylose,
which is the dominant pentose sugar in hydrolysates of
lignocellulosic biomass. A number of different strategies
have been applied to engineer yeasts capable of efficiently
producing ethanol from xylose, including the introduction
of initial xylose metabolism and xylose transport, changing
the intracellular redox balance, and overexpression of
xylulokinase and pentose phosphate pathways. In this
review, recent progress with regard to these studies is
discussed, focusing particularly on xylose-fermenting
strains of S. cerevisiae. Recent studies using several
promising approaches such as host strain selection and
adaptation to obtain further improved xylose-utilizing S.
cerevisiae are also addressed.
A. Matsushika (*) : H. Inoue : S. Sawayama
Biomass Technology Research Center (BTRC), National Institute
of Advanced Industrial Science and Technology (AIST),
2-2-2 Hirosuehiro,
Kure, Hiroshima 737-0197, Japan
e-mail: a-matsushika@aist.go.jp
T. Kodaki
Institute of Advanced Energy, Kyoto University,
Gokasho,
Uji, Kyoto 611-0011, Japan
Keywords Ethanol production . Xylose fermentation .
Saccharomyces cerevisiae . Lignocellulosic biomass .
Metabolic engineering
Introduction
Bioethanol production from plant biomass has received
considerable attention recently in order to mitigate global
warming and demands for petroleum. Currently, bioethanol
is produced mainly from sugar-containing or starchy
biomass such as sugarcane and corn as the raw material.
As sugar-containing and starchy biomass is used for food
and animal feed, there is competition for its use as both
food and fuel. Due to this competition, lignocellulosic
bioethanol production has been eagerly researched world-
wide. Lignocellulosic biomass, such as woods and agricul-
tural residues, is an attractive feedstock for bioethanol
production because of its large amount of potentially
fermentable sugars. The main structural components of
lignocellulosic biomass are cellulose, hemicellulose, and
lignin. Of these, only cellulose and hemicellulose can be
used as raw materials to produce ethanol by fermentation of
carbohydrates obtained by chemical or enzymatic hydroly-
sis (saccharification). The main component of lignocellu-
losic hydrolysates is glucose, a hexose sugar derived from
cellulose and hemicellulose. Although the proportion of
monosaccharides in hemicellulose hydrolysates varies
depending on the raw material and the hydrolysis procedure
(Lee 1997; Hendriks and Zeeman 2009), they all contain
both pentose sugars, such as D-xylose and L-arabinose and
hexose sugars. D-Xylose is the second most abundant
carbohydrate and its content is particularly high in grass
and hardwood. Thus, a substantial number of the hydro-
lysates obtained from lignocellulosic biomass contain
38
xylose, requiring an economic conversion of biomass into
ethanol through xylose utilization.
One of the most effective ethanol-producing micro-
organisms for hexose sugars including glucose, mannose,
and galactose is Saccharomyces cerevisiae, a yeast with
high ethanol productivity, high tolerance to ethanol, and
tolerance to inhibitory compounds present in the hydroly-
sate of lignocellulosic biomass (Olsson and Hahn-Hägerdal
1993; Olsson and Nielsen 2000). Although S. cerevisiae
can grow well even at relatively low pH, which prevents
contamination by other bacteria, native strains are unable to
utilize xylose for growth or fermentation. Instead, it
metabolizes D-xylulose, an isomerization product of D-
xylose (Wang and Schneider 1980; Wang et al. 1980;
Chiang et al. 1981). Some yeast strains have been reported
to ferment xylose into ethanol (Schneider et al. 1981;
Jeffries 1981; Slininger et al. 1982), but the rate and yield
of ethanol production from xylose in these xylose-utilizing
yeast strains are considerably low compared to their glucose
fermentation. Therefore, genetic engineering and/or adap-
tation may be promising methods to develop sufficient
xylose fermentation in S. cerevisiae.
To date, numerous studies regarding the metabolic
engineering of S. cerevisiae for xylose utilization have
been reported, and many reviews have already addressed
the current advancement in metabolic engineering of
xylose-fermenting strains and factors which affect xylose
metabolism in yeasts (Jeffries and Shi 1999; Jeffries and Jin
2004; Jeffries 2006; Gong et al. 1999; Aristidou and
Penttilä 2000; Hahn-Hägerdal et al. 2001, 2007a, b; Dien
et al. 2003; van Maris et al. 2006, 2007; Lin and Tanaka
2006; Chu and Lee 2007; Almeida and Hahn-Hägerdal
2009). However, there are still some fundamental issues
linked to its industrial use. This review focuses on the
progress in recent studies to address these issues in the
industry.
Xylose utilization pathways
Figure 1 schematically illustrates the initial metabolic
pathways for xylose utilization in fungi and bacteria. In
most fungi and xylose-fermenting yeasts (e.g., Pichia
stipitis, Pachysolen tannophilus, and Candida shehatae),
D-xylose is converted to D-xylulose by two oxidoreductases
involving cofactors NAD(P)H and NAD(P)+ through the
type I pathway. That is, D-xylose is initially reduced to
xylitol by NAD(P)H-dependent xylose reductase (XR; EC
1.1.1.21; Bruinenberg et al. 1984; Verduyn et al. 1985;
Bolen et al. 1986; Rizzi et al. 1988), and then xylitol is
oxidized to D-xylulose by NAD+-dependent xylitol dehy-
drogenase (XDH; EC 1.1.1.9; Bolen et al. 1986; Rizzi et al.
1988, 1989; Wang and Jeffries 1990). Finally, xylulokinase
Appl Microbiol Biotechnol (2009) 84:37–53
(XK; EC 2.7.1.17) phosphorylates D-xylulose into D-
xylulose 5-phosphate (X5P; Deng and Ho 1990;
Rodriguez-Pena et al. 1998), which is further metabolized
through the pentose phosphate pathway (PPP). As this
reaction uses adenosine triphosphate (ATP), the reaction
rate is dependent on the energy charge and the phosphor-
ylation potential of the cell. Although S. cerevisiae cannot
utilize xylose, the genes encoding XR (YHR104w, GRE3;
Träff et al. 2002), XDH (YLR070c, ScXYL2; Richard et al.
1999), and XK (XKS1; Yang and Jeffries 1997; Richard et
al. 2000) are present in its genome; however, their
expression levels are too low to allow for xylose utilization.
In fact, only very slow growth on xylose has been observed
even when the endogenous genes are overexpressed in S.
cerevisiae (Toivari et al. 2004).
On the other hand, in most bacteria (e.g., Escherichia
coli and Streptomyces sp.), D-xylose is directly isomerized
to D-xylulose by xylose isomerase (XI; EC 5.3.1.5) through
the type II pathway. As in fungi, D-xylulose is phosphor-
ylated to D-xylulose 5-phosphate by XK. Both yeast
(Vongsuvanlert and Tani 1988) and some fungi (Banejee
et al. 1994; Harhangi et al. 2003) have been reported to
harbor XI activity.
XR and XDH expression
Anaerobic xylose fermentation by S. cerevisiae was first
demonstrated by heterologous expression of XYL1 (Rizzi
et al. 1988) and XYL2 (Rizzi et al. 1989) genes encoding
XR and XDH from P. stipitis (referred to as PsXR
and PsXDH, respectively; Kötter and Ciriacy 1993;
Tantirungkij et al. 1993). While P. stipitis can ferment
xylose to ethanol at theoretical yields under anaerobic
conditions, it requires oxygen for efficient xylose utiliza-
tion and has a slower sugar consumption rate compared to
S. cerevisiae (Jeffries et al. 2007; Agbogbo and Coward-
Kelly 2008). On the other hand, most other natural
xylose-fermenting yeasts excrete substantial amounts of
the by-product xylitol. The excretion of xylitol has been
mainly ascribed to the difference in coenzyme specific-
ities between the mainly NADPH-dependent XR and
strictly NAD+-dependent XDH. Thus, during the XR and
XDH reactions, excess NADH accumulates leaving a
shortage of NAD+, resulting in an intracellular redox
imbalance (Bruinenberg et al. 1983; Kötter and Ciriacy
1993). Despite the PsXR preference for NADPH (Verduyn
et al. 1985), P. stipitis is one of the few types of yeast that
possess both NADPH- and NADH-specific XR cofactors,
which provide the yeast with the ability to excrete less
xylitol during xylose fermentation. P. tannophilus also
possesses this dual specific XR, but produces more xylitol
than P. stipitis (Debus et al. 1983).
Appl Microbiol Biotechnol (2009) 84:37–53 39

Fig. 1 Outline of D-xylose Type-I (XR-XDH) Pathway Type-II (XI) Pathway

metabolic pathway in fungi and
[FUNGI] [BACTERIA]
bacteria
D-Xylose D-Xylose
NADH NADPH
Xylose reductase
(XR; EC 1.1.1.21) NAD+ NADP+

Xylose isomerase
Xylitol (XR; EC 5.3.1.5)
NAD+
Xylitol dehydrogenase
(XDH; EC 1.1.1.19) NADH

D-Xylulose D-Xylulose
ATP ATP
Xylulokinase Xylulokinase
(XK; EC 2.7.1.17) ADP (XK; EC 2.7.1.17) ADP

D-Xylulose-5-P D-Xylulose-5-P

Pentose phosphate Pentose phosphate

pathway pathway

Ethanol Ethanol

A considerable amount of xylitol is produced in
recombinant S. cerevisiae strains expressing PsXR and
PsXDH and its production reduces the ethanol yield (Kötter
and Ciriacy 1993; Tantirungkij et al. 1993). Moreover, the
rate of xylose utilization by recombinant S. cerevisiae is
generally one to two orders of magnitude lower than that of
glucose (Hahn-Hägerdal et al. 2005). Therefore, several
metabolic engineering strategies have attempted to improve
xylose fermentation using PsXR- and PsXDH-expressing
S. cerevisiae strains, as discussed in the following sections.
The higher level of XDH relative to XR has been
experimentally shown to decrease the xylitol yield and
increase the ethanol yield (Walfridsson et al. 1997; Eliasson
et al. 2001; Jin and Jeffries 2003; Karhumaa et al. 2007a).
Recent studies have also found that high activity of both
PsXR and PsXDH is important for generating an efficient
xylose-fermenting recombinant S. cerevisiae strain (Jeppsson
et al. 2003b; Karhumaa et al. 2007a; Matsushika and
Sawayama 2008).
XI expression
Expression of functional bacterial XI, encoded by the xylA
gene, in S. cerevisiae has proven difficult. Heterologous
expression of XI genes from Actinoplanes missouriensis
(Amore et al. 1989) and Clostridium thermosulfurogenes
(Moes et al. 1996) in S. cerevisiae generated inactive
proteins, even though their messenger RNA could be
detected. Meanwhile, other studies reported heterologous
expression of xylA from E. coli (Sarthy et al. 1987),
Bacillus subtilis (Amore et al. 1989), and Streptomyces
rubiginosus (Gárdonyi and Hahn-Hägerdal 2003) in S.
cerevisiae that resulted in mainly insoluble proteins which
were catalytically inactive. Unsuccessful heterologous
expression in S. cerevisiae is most likely due to protein
misfolding, posttranslational modifications, and disulfide-
bridge formation.
The first functional XI expressed in S. cerevisiae was
from Thermus thermophilus (Walfridsson et al. 1996).
Although the XI genetic background has been modified to
reduce xylitol excretion (Träff et al. 2001), growth
conditions in S. cerevisiae could not achieve maximum
specific activity of this enzyme (which occurs at 85°C),
resulting in poor xylose fermentation. Fungal XI genes
from Piromyces (Kuyper et al. 2003) and Orpinomyces
(Madhavan et al. 2009a) have also been functionally
expressed in S. cerevisiae at high levels, but the growth
on xylose was very slow. To address these difficulties,
adaptation (Kuyper et al. 2004, 2005b; Madhavan et al.
2009b) or extensive genetic engineering (Kuyper et al.
2005a; Hughes et al. 2009; Madhavan et al. 2009a) has
been applied to improve the xylose fermentation to ethanol.
These studies have also established a few bacterial and
40
fungal XI pathways in S. cerevisiae. In fact, a recent study
has reported successful expression of a new highly active
kind of bacterial XI from Clostridium phytofermentans
showing far less inhibition by xylitol in S. cerevisiae (Brat
et al. 2009).
Introducing the XI pathway instead of the XR–XDH
pathway is one solution to reduce xylitol excretion
(Walfridsson et al. 1996; Kuyper et al. 2003; Lönn et al.
2003). XI requires no redox cofactors and does not produce
an intracellular redox imbalance during xylose fermenta-
tion. Direct comparison of the xylose-fermenting abilities
has shown a lower xylitol yield and higher ethanol yield in
the XI-expressing recombinant S. cerevisiae strain than in
the XR–XDH-expressing strain (Karhumaa et al. 2007b);
however, the rate of xylose fermentation (i.e., xylose
consumption rate and ethanol production rate) was much
lower in the XI-expressing strains. Moreover, XI has only
been expressed under strong promoters in multicopy
plasmids, indicating that the specific activity of these XIs
is insufficient to generate stable chromosomally integrated
recombinant strains. Therefore, some other driving force is
necessary to address these issues.
Improving ethanol fermentation by xylose-utilizing
S. cerevisiae
In addition to the introduction of xylose-to-xylulose
conversion pathways in S. cerevisiae, a number of strategies
for metabolic engineering and genetic modification to
enhance the rapid and efficient fermentation of xylose to
ethanol have been investigated. Here, the most important
engineering strategies are addressed; they include over-
expression of native XK, changing the intracellular redox
balance, engineering the xylose transport, and enhancing
the PPP. Table 1 summarizes the xylose fermentation
performance including the ethanol and xylitol yields of
different recombinant Saccharomyces strains used in
various studies. Figure 2 outlines the metabolic reactions
that have been engineered to improve ethanol formation
from xylose by S. cerevisiae.
Xylulokinase
S. cerevisiae is able to metabolize an isomerization product
of D-xylose, D-xylulose, albeit at low rates (Wang and
Schneider 1980). Slow xylulose consumption is ascribed to
low levels of endogenous XK activity which in part limits
xylose fermentation (Chang and Ho 1988; Deng and Ho
1990). Ho et al. (1998) were the first to generate a
recombinant Saccharomyces strain, Saccharomyces sp.
1400 (pLNH32), in which the XKS1 gene encoding XK
from S. cerevisiae (ScXK) is overexpressed using a yeast-E.
Appl Microbiol Biotechnol (2009) 84:37–53
coli shuttle plasmid, along with PsXR and PsXDH genes
(Table 1). Stable recombinant S. cerevisiae strains
(TMB3001, Table 1), constructed by chromosomal integra-
tion of the genes for PsXR, PsXDH, and ScXK, have also
been developed (Eliasson et al. 2000b). These recombinant
strains were able to ferment xylose as well as coferment
glucose and xylose, albeit with slightly lower ethanol yields
(Ho et al. 1998; Eliasson et al. 2000b). Toivari et al. (2001)
also reported ethanol production from xylose through
overexpression of the ScXK gene in S. cerevisiae integrat-
ing the PsXR and PsXDH genes (strain H1691, Table 1).
Although these efforts have improved the yield of ethanol
from D-xylose in recombinant Saccharomyces strains,
xylitol is still a major by-product.
With regards to the level of XK activity, the optimal XK
expression level necessary for effective utilization of xylose
is still under debate (Ho et al. 1998; Eliasson et al. 2000b,
2001; Johansson et al. 2001; Toivari et al. 2001; Jin et al.
2003; Matsushika and Sawayama 2008). Ho et al. (1998)
previously reported that overexpression of the ScXK gene
along with the PsXR and PsXDH genes increases ethanol
production and decreases xylitol excretion from xylose.
Conversely, Rodriguez-Pena et al. (1998) showed that
overexpression of the ScXK gene inhibits growth on
xylulose, and Johansson et al. (2001) found that over-
expression of the ScXK gene considerably reduces xylose
consumption in recombinant S. cerevisiae, even though it
increases the yield of ethanol from xylose. Jin et al. (2003)
also showed that overexpression of the XYL3 gene encod-
ing XK from P. stipitis (PsXK; Jin et al. 2002), along with
high expression levels of the PsXR and PsXDH genes in S.
cerevisiae, inhibits cell growth on xylose, whereas recom-
binant S. cerevisiae expressing the PsXK at a moderate
level enhances growth. PsXK has higher specific activity
than ScXK (Jin et al. 2003). In contrasting studies, no
inhibitory effect was found for XK overexpression (Richard
et al. 2000; Toivari et al. 2001), and a recent study by
Matsushika and Sawayama (2008) showed increased
ethanol yield and decreased xylitol yield by moderate
ScXK activity with high activity of PsXR and PsXDH,
with no inhibitory effect on either growth or xylose
consumption from XK overexpression. Thus, only fine-
tuned overexpression of XK in S. cerevisiae appears to lead
to improved fermentation of xylose to ethanol. Ethanol
production may be inhibited when cells express high levels
of XK due to abnormal accumulation of X5P and depletion
of ATP (Johansson et al. 2001; Jin et al. 2003).
Interestingly, Jeffries and coworkers identified spontane-
ous or chemically induced mutants of engineered S.
cerevisiae that can overcome the growth inhibition caused
by overexpression of the ScXK or PsXK gene (Ni et al.
2007). Two prominent mutational events occurred: in-
creased expression of transaldolase gene TAL1 and deletion
Table 1 Batch fermentation performance of recombinant yeast strains in media containing xylose as a sole carbon source

Strain Description Strain backgrounda Condition Medium Xylose (g l−1) YE (g g−1) YX (g g−1) VE (g l−1 h−1) Reference

1400 (pLNH32) XYL1/XYL2/XKS1 Saccharomyces Fermentative C 50 0.30 0.08 NR Ho et al. (1998)

diastaticus/
Saccharomyces uvarum
H1691 XYL1/XYL2/XKS1 CEN.PK2 (H1346) Aerobic C 50 0.06 0.06 NR Toivari et al. (2001)
H1691 XYL1/XYL2/XKS1 CEN.PK2 (H1346) Microaerobic C 50 0.12 0.42 NR Toivari et al. (2001)
H1691 XYL1/XYL2/XKS1 CEN.PK2 (H1346) Anaerobic C 50 0.09 0.41 NR Toivari et al. (2001)
TMB3001 XYL1/XYL2/XKS1 CEN.PK 113-7A Anaerobic D 50 0.31 0.29 0.22 Jeppsson et al. (2002)
TMB3008 XYL1/XYL2/XKS1/Δgnd1 TMB3001 Anaerobic D 50 0.38 0.13 0.27 Jeppsson et al. (2002)
TMB3255 XYL1/XYL2/XKS1/Δzwf1 TMB3001 Anaerobic D 50 0.41 0.05 0.29 Jeppsson et al. (2002)
Appl Microbiol Biotechnol (2009) 84:37–53

TMB3253 XYL1/XYL2/XKS1 TMB3001 Anaerobic D 50 0.28 0.34 NR Jeppsson et al. (2003a)

TMB3254 XYL1/XYL2/XKS1/CTH TMB3001 Anaerobic D 50 0.28 0.30 NR Jeppsson et al. (2003a)
FPL-YSX3 XYL1/XYL2/XYL3 L2612 Oxygen-limited M 40 0.09 0.33 0.05 Jin et al. (2002)
FPL-YSX3 XYL1/XYL2/XYL3 L2612 Oxygen-limited C 20 0.12 0.27 NR Jin et al. (2003)
H2674 XYL1/XYL2/XKS1 CEN.PK2 Anaerobic C 50 0.18 0.53 NR Verho et al. (2003)
H2673 XYL1/XYL2/XKS1/GDP1 CEN.PK2 Anaerobic C 50 0.23 0.48 NR Verho et al. (2003)
H2723 XYL1/XYL2/XKS1/Δzwf1 CEN.PK2 Anaerobic C 50 0.24 0.28 NR Verho et al. (2003)
H2684 XYL1/XYL2/XKS1/ CEN.PK2 Anaerobic C 50 0.41 0.34 NR Verho et al. (2003)
GDP1/Δzwf1
TMB3001C1 XYL1/XYL2/XKS1, TMB3001 Anaerobic D 10 0.24 0.32 NR Sonderegger and
xylose adaptation Sauer (2003)
TMB3399 XYL1/XYL2/XKS1 Saccharomyces USM21 Anaerobic D 20 0.05 0.59 0.03 Wahlbom et al. (2003a)
TMB3400 XYL1/XYL2/XKS1, TMB3399 Anaerobic D 20 0.18 0.25 0.04 Wahlbom et al. (2003a)
random mutagenesis
RW 202-AFX XYLAP, xylose adaptation CEN.PK113-7D Anaerobic D 20 0.42 0.021 NR Kuyper et al. (2004)
TMB3120 XYL1/XYL2/XKS1/ΔGRE3 TMB3001 Oxygen-limited D 10 0.46 0.09 0.06 Träff-Bjerre et al. (2004)
TMB3266 XYL1/XYL2/XKS1/GRE3 TMB3001 Oxygen-limited D 10 0.46 0.18 0.06 Träff-Bjerre et al. (2004)
TMB3050 XYLAT/XKS1/TAL1/TKL1/ CEN.PK2-1C Oxygen-limited D 50 0.29 0.23 NR Karhumaa et al. (2005)
RKI1/RPE1/ΔGRE3,
xylose adaptation
RWB 217 XYLAP/XKS1/TAL1/TKL1/ CEN.PK102-3A Anaerobic D 20 0.43 0.003 0.092 Kuyper et al. (2005a)
RKI1/RPE1/ΔGRE3
RWB 218 XYLAP/XKS1/TAL1/TKL1/ RWB217 Anaerobic D 20 0.41 0.001 NR Kuyper et al. (2005b)
RKI1/RPE1/ΔGRE3,
glucose and xylose
adaptation
TMB3270 XYL1(K270M one copy)/ CEN.PK113-11C Anaerobic D 50 0.36 0.17 0.32 Jeppsson et al. (2006)
XYL2/XKS1
TMB3271 XYL1(K270M two copies)/ TMB3270 Anaerobic D 50 0.31 0.09 0.28 Jeppsson et al. (2006)
XYL2/XKS1
41
 42

Table 1 (continued)

Strain Description Strain backgrounda Condition Medium Xylose (g l−1) YE (g g−1) YX (g g−1) VE (g l−1 h−1) Reference

TMB3057 XYL1/XYL2/XKS1/TAL1/ TMB3001 Anaerobic D 50 0.33 0.22 0.13 Karhumaa et al. (2007b)
TKL1/RKI1/RPE1/ΔGRE3
TMB3066 XYLAP/XKS1/TAL1/TKL1/ CEN.PK2-1C Anaerobic D 50 0.43 0.04 0.073 Karhumaa et al. (2007b)
RKI1/RPE1/ΔGRE3
TMB3400 XYL1/XYL2/XKS1, TMB3399 Anaerobic D 50 0.34 0.29 0.12 Karhumaa et al. (2007b)
random mutagenesis
L2612 (pRS314-X123) XYL1/XYL2/XYL3 L2612 Aerobic M 40 0.13 0.00 0.032 Ni et al. (2007)
DR PHO13 XYL1/XYL2/XYL3/Δpho13 L2612 Aerobic M 40 0.25 0.04 0.093 Ni et al. (2007)
(pRS314-X123)
CMB.JHV.XYL123 XYL1/XYL2/XYL3 CEN.PK 113-5D Low oxygen D 20 0.066 0.007 NR Van Vleet et al. (2008)
CMB.JHV.XYL123. XYL1/XYL2/XYL3/Δpho13 CMB.JHV.XYL123 Low oxygen D 20 0.315 0.006 NR Van Vleet et al. (2008)
pho13a
BP10001 CtXR(K274-N276D)/ CEN.PK 113-7D Oxygen-limited D 20 0.34 0.17 NR Petschacher and
GmXDH/XKS1 Nidetzky (2008)
BP10001 CtXR(K274/N276D)/ CEN.PK 113-7D Anaerobic D 20 0.34 0.19 NR Petschacher and
GmXDH/XKS1 Nidetzky (2008)
MA-N5 XYL1/XYL2(D207A/I208R/ INVSc1 Anaerobic C 45 0.36 0.06 0.244 Matsushika et al. (2008b)
F209S/N211R)/XKS1
INVSc1/pRS406XKS/ XYLAO/XKS1/SUT1 INVSc1 Anaerobic M 50 0.39 0.08 0.043 Madhavan et al. (2009a)
pILSUT1/pWOXYLA
ADAP8 XYLAO/XKS1/SUT1, INVSc1/pRS406XKS/ Anaerobic M 20 0.43 0.08 0.070 Madhavan et al. (2009b)
xylose adaptation pILSUT1/pWOXYLA
ADAP8 XYLAO/XKS1/SUT1, INVSc1/pRS406XKS/ Anaerobic C 20 0.35 0.16 0.060 Madhavan et al. (2009b)
xylose adaptation pILSUT1/pWOXYLA
MA-R4 XYL1/XYL2/XKS1 IR-2 Anaerobic C 45 0.35 0.048 0.36 Matsushika et al. (2009a, b)
MA-R5 XYL1/XYL2(D207A/I208R/ IR-2 Anaerobic C 45 0.37 0.038 0.50 Matsushika et al. (2009b)
F209S/N211R)/XKS1

NR not reported, YE ethanol yield, YX xylitol yield, VE volumetric ethanol production rate, C complex media, D defined minimal media (Verduyn et al. 1992), M minimal media, XYL1 xylose
reductase from Pichia stipitis, CtXR xylose reductase from Candida tenuis, XYL2 xylitol dehydrogenase from P. stipitis, GmXDH xylose reductase from Galactocandida mastotermitis, XYLAT
xylose isomerase from Thermus thermophilus, XYLAP xylose isomerase from Piromyces sp., XYLAO xylose isomerase from Orpinomyces sp., XYL3 xylulokinase from P. stipitis, XKS1
xylulokinase from Saccharomyces cerevisiae, GRE3 aldose reductase from S. cerevisiae, TAL1 transaldolase from S. cerevisiae, TKL1 transketolase from S. cerevisiae, RKI1 ribose-5-phosphate
ketol-isomerase from S. cerevisiae, RPE1 ribulose-5-phosphate 3-epimerase from S. cerevisiae, gnd1 6-phosphogluconate dehydrogenase from S. cerevisiae, zwf1 glucose-6-phosphate
dehydrogenase from S. cerevisiae, CTH cytoplasmic transhydrogenase from Azotobacter vinelandii, GDP1 glyceraldehyde-3-phosphate dehydrogenase from Kluyveromyces lactis, pho13 alkaline
phosphatase from S. cerevisiae, SUT1 sugar transporter from P. stipitis
a
S. cerevisiae strains, unless otherwise noted
Appl Microbiol Biotechnol (2009) 84:37–53
Appl Microbiol Biotechnol (2009) 84:37–53 43

Xylose Glucose
(intracellular) (intracellular)
NAD(P)H NADPH

XYL1* GRE3 HXK1

NAD(P)+ NADP+ HXK2
Xylitol XYLA* GLK1
CO2
NAD+
6-phospho-
XYL2* RPE1* Ribulose-5-P GND1 ZWF1 Glucose-6-P
gluconate
NADH NADPH NADH NAD+

RKI1* PGI1 PFK1

Xylulose ATP ADP
Xylulose-5-P PFK2
Ribose-5-P Fructose-6-P
XKS1*
FBP1 Fructose-1,6-BP
ATP ADP
TKL1*
FBA1
Sedoheptulose-7-P TAL1*

Erythrose-4-P Dihydroxy-
Glyceraldehyde-3-P TPI1
Fructose-6-P NAD+
acetone-P
TDH1
NADH NADH
TDH2
TKL1* GPD1 GUT2
NADH TDH3
NAD+ NAD+
3-phospho-
glyceroyl-P Glycerol-3-P
ADP ADP

PGK1 GPP1 GUT1

Glutamate ATP ATP
NADH NADPH
3-&2-phosphoglycerate Glycerol
GDH2* GDH1
NAD+ NADP+ ENO1
Acetate GPM1
2-oxoglutarate ENO2
ALD2
NAD(P)H ALD3 Phosphoenolypyruvate
ALD4 ADP
NAD(P)+ PDC1
NAD+ NADH
ALD5 PYK1
CO2 PDC5
ALD6 ATP
ADH1 PDC6
Ethanol Acetaldehyde Pyruvate
ADH2

NAD+ NADH

Fig. 2 Overview of the metabolic pathway in S. cerevisiae engineered
for improved xylose fermentation. The genes marked with an asterisk
have been overexpressed; crossed genes have been deleted. ADH1 and
ADH2, alcohol dehydrogenase; ALD2, ALD3, ALD4, ALD5, and ALD6,
aldehyde dehydrogenase; ENO1 and ENO2, enolase; FBA1, fructose
1,6-bisphosphate aldolase; FBP1, fructose-1,6-bisphosphatase; GDH1
and GDH2, glutamate dehydrogenase; GDP1, glycerol-3-phosphate
dehydrogenase; GLK1, glucokinase; GND1, 6-phosphogluconate dehy-
drogenase; GPM1, phosphoglycerate mutase; GPP1, glycerol-3-
phosphatase; GRE3, aldose reductase; GUT1, glycerol kinase; GUT2,
glycerol-3-phosphate dehydrogenase; HXK1 and HXK2, hexokinase;
PDC1, PDC5, and PDC6, pyruvate decarboxylase; PGI1, phosphoglu-
cose isomerase; PGK1, 3-phosphoglycerate kinase; PFK1 and PFK2,
phosphofructokinase; PYK1, pyruvate kinase; RKI1, ribose-5-phosphate
ketol-isomerase; RPE1, ribulose-5-phosphate 3-epimerase; TAL1, trans-
aldolase; TDH1, TDH2, and TDH3, glyceraldehyde-3-phosphate
dehydrogenase; TKL1, transketolase; TPI1, triose phosphate isom-
erase; XKS1, xylulokinase; XYL1, xylose reductase; XYL2, xylitol
dehydrogenase; XYLA, xylose isomerase; ZWF1, glucose-6-
phosphate dehydrogenase

of the PHO13 gene encoding alkaline phosphatase specific
for p-nitrophenyl phosphate (Ni et al. 2007). Deletion
of PHO13 in xylose-utilizing S. cerevisiae strains, DR
PHO13 (pRS314-X123) and CMB.JHV.XYL123.pho13a
(see Table 1), also improves growth on xylose and increases
ethanol production from xylose when PsXK gene is over-
expressed (Ni et al. 2007; Van Vleet et al. 2008); however,
little is known about the physiological function of Pho13.
Redox balance
The intracellular redox imbalance caused by the coenzyme
specificity differences between PsXR (with NADPH) and
PsXDH (with NAD+) may be a major factor in promoting
xylitol excretion (Bruinenberg et al. 1983). To reduce
xylitol formation during xylose fermentation, Jeppsson et
al. (2002) constructed recombinant S. cerevisiae strains
expressing PsXR and PsXDH and overproducing ScXK
that lower the oxidative PPP activity through the GND1 and
ZWF1 genes (Fig. 2). These genes encode the NADPH
producing 6-phosphogluconate dehydrogenase and glucose-
6-phosphate dehydrogenase, respectively. Their mutants
Δgnd1 and Δzwf1 (strains TMB3008 and TMB3255,
Table 1) increase ethanol yield, compared to the parent
strain (TMB3001, Table 1), and show a reduced rate of
xylose consumption. Thus, NADPH formation appears to
44
be necessary for xylose fermentation in S. cerevisiae, and
strains with enhanced XR activity may overcome this
limitation.
S. cerevisiae does not possess a transhydrogenase to
convert NADH to NADPH (Bruinenberg et al. 1985),
resulting in cytosolic NADH accumulation. Heterologous
expression of a transhydrogenase gene from Azotobacter
vinlandii (Nissen et al. 2001) in S. cerevisiae (TMB3254,
Table 1) reduces xylitol production compared to the control
strain (TMB3253, Table 1), but increases the by-product
glycerol rather than ethanol, perhaps due to the reoxida-
tion of NADH formed in the transhydrogenase reaction
(Jeppsson et al. 2003a). Thus, this approach using the
transhydrogenase reaction may not be useful for ethanolic
xylose fermentation. On the other hand, Verho et al. (2003)
showed that the overexpression of the Kluyveromyces lactis
GDP1 gene (Verho et al. 2002) encoding a NADP+-
dependent glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) in a xylose-fermenting S. cerevisiae strain
enhances ethanol production (strain H2673, Table 1). The
deletion of the ZWF1 gene in combination with over-
expression of GDP1 also showed improvements in ethanol
production from xylose (Verho et al. 2003; strain H2684,
Table 1). Similarly, Bro et al. (2006) found that a non-
phosphorylating NADP+-dependent GAPDH, encoded by
the gapN gene, from Streptococcus mutants in xylose-
metabolizing S. cerevisiae increased ethanol production by
using a genome-scale metabolic flux model. Such redi-
rection of the flux toward ethanol has been achieved by
modifying the ammonia assimilation pathway (Nissen et
al. 2000). Higher ethanol production accompanying
reduced xylitol production can also be obtained by
deleting the NADPH-dependent glutamate dehydrogenase
gene GDH1 and overexpressing the NADH-dependent
isoenzyme gene GDH2 (Fig. 2). These strategies to
redirect flux towards ethanol formation appear to improve
intracellular cofactor concentrations in S. cerevisiae.
However, overexpressing NADH kinase encoded by the
POS5 gene in the mitochondria is ineffective for the
anaerobic growth physiology of xylose-utilizing S. cer-
evisiae, and in the cytosol, it increases xylitol accumula-
tion (Hou et al. 2009).
Another promising approach for reducing xylitol
excretion and enhancing ethanol yield using recombi-
nant S. cerevisiae involves alterations in the coenzyme
specificity of XR and/or XDH by protein engineering. In
the case of XR, Jeppsson et al. (2006) reported enhanced
ethanol production accompanied by decreased xylitol
formation in recombinant S. cerevisiae expressing both
mutated PsXR having reduced affinity for NADPH
(K270M; Kostrzynska et al. 1998) and PsXDH and ScXK
(strains TMB3270 and TMB3271, Table 1). Several
NADH-preferring XR mutants have also been generated
Appl Microbiol Biotechnol (2009) 84:37–53
from Candida tenuis (CtXR; Kavanagh et al. 2002, 2003;
Leitgeb et al. 2005; Petschacher and Nidetzky 2005;
Petschacher et al. 2005), and one S. cerevisiae strain
harboring the K274R-N276D CtXR double mutant has
shown enhanced ethanol production with decreased xylitol
formation (Petschacher and Nidetzky 2008; strain
BP10001, Table 1). Several NADH-preferring PsXR
mutants, such as K270R and R276H, have also been
reported to have positive effects on ethanol production and
xylitol excretion (Watanabe et al. 2007a, b). Furthermore,
several PsXDH mutants have been successfully generated
with complete reversal of coenzyme specificity toward
NADP+ by multiple site-directed mutagenesis within the
coenzyme-binding domain (Watanabe et al. 2005) and
with increased thermostability by introducing a zinc-
binding site (Annaluru et al. 2007). One of these, a
quadruple ARSdR mutant (D207A/I208R/F209S/N211R),
showed more than 4,500-fold higher values of kcat/Km for
NADP+ than the wild-type enzyme and similar values of
kcat/Km for NAD+ as the wild-type. In addition, recombi-
nant S. cerevisiae strains, in which the ARSdR mutant was
under the control of a strong constitutive promoter,
increased ethanol production from xylose accompanied
by decreased xylitol excretion (Watanabe et al. 2007c;
Matsushika et al. 2008a, b, 2009b), most likely by
maintaining the intracellular redox balance (strains MA-
N5 and MA-R5, Table 1). Of these, the recombinant
industrial S. cerevisiae strain MA-R5 expressing NADP+-
dependent PsXDH rapidly showed particularly high
ethanol production from xylose and low xylitol yield by
fermentation of not only xylose as the sole carbon source,
but also a mixture of glucose and xylose (Matsushika et al.
2009b).
As S. cerevisiae harbors a non-specific aldose reductase
encoded by the GRE3 gene that is strictly NADPH-
dependent and reduces D-xylose to xylitol (Kuhn et al.
1995; Fig. 2), deletion of this gene decreases xylitol
excretion not only in XI-carrying strains (Träff et al.
2001; Lönn et al. 2003; Kuyper et al. 2005a), but also in
strains carrying XR and XDH (Träff-Bjerre et al. 2004;
strain TMB3120, Table 1). Deletion of the GRE3 gene is of
particular importance for XI-carrying strains because xylitol
inhibits the activity of XI (Yamanaka 1969). However, the
GRE3 gene is stress-induced (Kuhn et al. 1995), and its
deletion reduces biomass formation (Träff-Bjerre et al.
2004). Therefore, fine-tuning of GRE3 expression may be
more useful than its deletion.
Xylose transport
Lacking xylose-specific transporters, S. cerevisiae take up
xylose by facilitated diffusion mainly through non-specific
hexose transporters encoded by the HXT gene family
Appl Microbiol Biotechnol (2009) 84:37–53
(Kruckeberg 1996). These transporters have lower affinity
for xylose than for glucose (Kötter and Ciriacy 1993), and
their xylose transport properties have mainly been charac-
terized with regard to sugar affinity (Hamacher et al. 2002;
Lee et al. 2002; Saloheimo et al. 2007). That is, the
transport of xylose is competitively inhibited by glucose,
and thus, xylose is usually consumed only after depletion of
glucose (van Zyl et al. 1993; Sedlak and Ho 2004b). As S.
cerevisiae does not recognize xylose as a fermentable
carbon source (Jin et al. 2004; Salusjärvi et al. 2008),
heterologous expression of xylose-specific transporters in
recombinant xylose-utilizing S. cerevisiae strains may
prove very useful.
Reintroduction and constitutive expression of individual
HXT genes in a deletion mutant of all of the hexose-
transporter genes have emphasized the importance of high
and intermediate affinity transporters Hxt4, Hxt5, Hxt7, and
Gal2 as xylose-transporting proteins (Sedlak and Ho
2004b). As the HXT5 and HXT7 genes are only expressed
when grown on xylose as the sole carbon source, they are
thought to be particularly important for xylose transport
among the HXT gene family. Despite attempts to enhance
the xylose fermentation by overexpression of xylose trans-
porters such as Hxt7 and Gal2, no significant effects have
been observed for growth on xylose and the xylose
fermentation rate (Hamacher et al. 2002). Further investi-
gation is needed to identify the proteins that are essential
for improving xylose transport and fermentation in S.
cerevisiae; such studies may focus on overexpressing other
genes related to the sugar transport including Agt1 (an
inducible high-affinity maltose transporter) and Snf3 (a
plasma membrane glucose sensor that regulates glucose
transport; Sedlak and Ho 2004b). The AGT1 transporter has
already been identified as a factor in efficient maltotriose
fermentation in S. cerevisiae (Alves et al. 2008).
The SUT1 gene (Weierstall et al. 1999), coding for a
sugar transporter from P. stipitis, was successfully
expressed in S. cerevisiae (Katahira et al. 2008). Heterol-
ogous expression of transporters involved in xylose uptake
in Trichoderma reesei (Xlt1; Saloheimo et al. 2007) and
Arabidopsis thaliana (At5g59250 and At5g17010; Hector
et al. 2008) has also been reported. Moreover, the glucose/
xylose-facilitated diffusion transporter and glucose/xylose
symporter from Candida intermedia, respectively,
encoded by GXF1 and GXF1 genes (Leandro et al.
2006), have been expressed in S. cerevisiae (Leandro et
al. 2008), and the recombinant xylose-fermenting S.
cerevisiae strain harboring Gxf1 has shown faster xylose
uptake and ethanol production (Runquist et al. 2009).
These expressions of heterologous xylose transporters
provide great promise for future development of xylose-
fermenting S. cerevisiae strains possessing increased rates
of xylose utilization.
45
Pentose phosphate pathway
The non-oxidative PPP is the only way to introduce
xylulose into glycolysis. However, the flux through the
PPP is insufficient in S. cerevisiae compared to other yeast
species (Gancedo and Lagunas 1973; Fiaux et al. 2003) and
causes the accumulation of PPP metabolites (Schaaff et al.
1990; Senac and Hahn-Hägerdal 1991; Kötter and Ciriacy
1993; Zaldivar et al. 2002; Kuyper et al. 2003), most likely
resulting in lower rates of xylulose fermentation. Moreover,
S. cerevisiae produces ethanol exclusively from hexose
sugars. Therefore, overproduction of non-oxidative PPP
enzymes, such as transaldolase (EC 2.2.1.2), transketolase
(EC 2.2.1.1), ribulose-5-phosphate 3-epimerase (EC
5.1.3.1), and ribose-5-phosphate ketol-isomerase (EC
5.3.1.6), has been attempted to enhance the PPP activity
in xylose-utilizing S. cerevisiae.
Overexpression of endogenous transaldolase gene
(TAL1) or TAL1 from P. stipitis has been shown to improve
growth on xylose in S. cerevisiae (Walfridsson et al. 1995;
Jin et al. 2005), supporting previous findings that TAL in S.
cerevisiae limits growth on xylose (Senac and Hahn-
Hägerdal 1991; Kötter and Ciriacy 1993). In contrast,
overexpression of the gene for transketolase (TKL1) from P.
stipitis in a xylose-metabolizing S. cerevisiae strain showed
considerable reduction in growth on xylose (Metzger and
Hollenberg 1994). Interestingly, overexpression of the all
four non-oxidative PPP genes, including ribulose-5-
phosphate 3-epimerase (RPE1) and ribose-5-phosphate
ketol-isomerase (RKI1) as well as TAL1 and TKL1, in
recombinant S. cerevisiae expressing PsXR, PsXDH, and
ScXK improved xylulose rather than xylose consumption,
supposedly through their synergistic effect (Johansson and
Hahn-Hägerdal 2002a, b; Fig. 2).
Overproduction of all non-oxidative PPP enzymes,
together with the overexpression of endogenous XKS1 and
deletion of GRE3, improved growth on xylose not only in a
strain carrying a bacterial XI (Karhumaa et al. 2005; strain
TMB3045, Table 1) but also in a strain carrying the
Piromyces XI (Kuyper et al. 2005a; Karhumaa et al.
2007b; strains RWB 217 and TMB3066, Table 1). The
combination of these modifications in a strain with high
activity of both XR and XDH also increased the rate of
xylose consumption (Karhumaa et al. 2005, 2007a, b; strain
TMB3057, Table 1). Thus, a simultaneous increase in the
activity converting xylose to xylulose and the PPP activity
required for xylulose metabolism appears to be essential for
efficient xylose utilization in S. cerevisiae. Such a strain
possessing increased non-oxidative PPP activity may also
be useful as one of the host strains used to improve the flux
through the initial xylose pathway including a xylose
transport system (Karhumaa et al. 2005). The importance
of high PPP activity in xylose-utilizing S. cerevisiae was
46
further supported by a northern analysis (Toivari et al.
2004), metabolic flux analysis (Pitkänen et al. 2003;
Sonderegger et al. 2004a), and microarray analysis
(Wahlbom et al. 2003b). Optimization of the ratio of
overexpressed enzymes in the non-oxidative PPP should
also be considered in future studies (Lu and Jeffries 2007).
Other metabolic engineering
The phosphoketolase pathway that converts D-xylulose 5-
phosphate to glyceraldehyde 3-phosphate and acetyl phos-
phate has been detected in bacteria and Rhodotorula sp.
strain (Evans and Ratledge 1984). Heterologous expression
of phosphoketolase (xfp) from Bifidobacterium lactis in a
xylose-utilizing S. cerevisiae strain decreases the xylitol
formation, increases acetate accumulation, and reduces the
xylose uptake rate (Sonderegger et al. 2004c). Deletion of
the gene encoding NADPH-dependent aldehyde dehydro-
genase (ALD6) leads to improved ethanol yield and xylose
fermentation rate (Sonderegger et al. 2004c).
Carbon catabolite repression, in which glucose is
consumed preferentially to any other sugar including
xylose, is a well-investigated regulatory mechanism in S.
cerevisiae (Trumbly 1992; Gancedo 1998). The MIG1 and
MIG2 genes, coding for transcription factors involved in
carbon catabolite repression, were deleted in XR–XDH–
XK-carrying S. cerevisiae strain to produce the engineered
strains which co-consume glucose and xylose (Roca et al.
2004); however, performance in xylose utilization showed
limited improvement.
Selection of host strain for metabolically engineered
xylose fermentation
Selecting the host strain(s) for xylose-utilizing S.
cerevisiae that is suitable for ethanol fermentation from
xylose is of utmost importance for overall process
optimization as the ability to ferment xylulose and the
tolerance to inhibitors in lignocellulosic hydrolysates
differ between potential production strains (Martín and
Jönsson 2003; Brandberg et al. 2004). To date, many
different laboratory and industrial Saccharomyces strains
have been metabolically engineered for improved xylose
utilization, and their performance of fermentation from
xylose and hydrolysates have been compared (Zaldivar et
al. 2002; Sonderegger et al. 2004b; Olsson et al. 2006;
Matsushika et al. 2009a). Laboratory strains are useful for
studying molecular genetics, while industrial strains are
generally selected to have optimal performance under
industrial conditions. Industrial S. cerevisiae strains have
shown more tolerance to hydrolysates than laboratory
strains (Garay-Arroyo et al. 2004; Sonderegger et al.
Appl Microbiol Biotechnol (2009) 84:37–53
2004b; Nilsson et al. 2005), suggesting strain-dependent
tolerance to specific inhibitors for S. cerevisiae; however,
robustness varies between strains.
On the other hand, S. cerevisiae strains have different
xylulose fermentation abilities (Yu et al. 1995; Eliasson et al.
2000a; Matsushika et al. 2009a, b), indicating that inherent
differences exist in their capacities to ferment pentose sugars.
The difference in the ability to ferment xylulose is probably
due to the difference in the PPP flux linking the xylose-to-
xylulose pathway to glycolysis (Johansson and Hahn-
Hägerdal 2002a). A recent study has generated a flocculent
industrial strain of S. cerevisiae IR-2, which has the highest
xylulose-fermenting ability among many different industrial
diploid strains for genetically engineering xylose fermenta-
tion (Matsushika et al. 2009b). The MA-R4 strain (Table 1),
engineered from IR-2 by chromosomal integration to express
the PsXR and PsXDH genes along with the ScXK gene,
showed superior xylose fermentation ability compared to
other recombinant industrial strains (Matsushika et al.
2009a). Therefore, evaluating the ability to ferment xylulose
may be another useful engineering approach directed at
anaerobic xylose fermentation.
Industrial xylose-fermenting strains and fermentation
of lignocellulosic hydrolysates
Industrial S. cerevisiae strains are generally superior ethanol
producers in view of their inhibitor tolerance and high
ethanol productivity under industrial conditions. To date,
only a limited number of industrial S. cerevisiae strains that
ferment xylose have been generated (Ho et al. 1999; Zaldivar
et al. 2002; Wahlbom et al. 2003a; Sedlak and Ho 2004a;
Sonderegger et al. 2004b; Karhumaa et al. 2006; see Hahn-
Hägerdal et al. 2007a for a review). The fermentation
performance of these industrial strains has also been
evaluated in various lignocellulosic residue or hemicellulose
hydrolysate pretreated by diluted acid or SO2 (Sedlak and Ho
2004a; Sonderegger et al. 2004b; Katahira et al. 2006;
Öhgren et al. 2006; Rudolf et al. 2008; Tomás-Pejó et al.
2008; Lu et al. 2009; Bertilsson et al. 2009). Using cell
surface display technology, Kondo and collaborators generat-
ed the xylose- and cellobiose-assimilating recombinant S.
cerevisiae strain (Katahira et al. 2006) and demonstrated an
effective cofermentation process (Nakamura et al. 2008).
Only recently has ethanol fermentation from lignocellulosic
hydrolysates prepared by ammonia fiber expansion (Lau et
al. 2008) been reported using industrial xylose-fermenting
strains (Lau and Dale 2009; Lu et al. 2009). Matsushika et al.
(2009a) showed that the MA-R4 strain had high ethanol
productivity using non-sulfuric acid hydrolysate of eucalyp-
tus wood chips (Inoue et al. 2008) as well as using mixed
carbon source of glucose and xylose.
Appl Microbiol Biotechnol (2009) 84:37–53
The pretreatment of lignocellulosic material results in the
formation of weak acids, furan derivatives, and phenolic
derivatives that act synergistically to inhibit ethanol fermen-
tation (Larsson et al. 1999; Palmqvist and Hahn-Hägerdal
2000). Furaldehydes are especially known for their biolog-
ical effects and their roles as inhibitors in fermentation
processes (Almeida et al. 2009). Thus, tolerance to these
inhibitors is a common indication for choosing industrial
strains suitable for lignocellulosic ethanol fermentation.
The kind and concentration of these inhibitors included in
each hydrolysate from cellulosic materials depend on the
pretreatment method. Thus, future research should consider
the inhibitor tolerance of the hydrolysate required for the
industrial strains followed by establishment of a pretreat-
ment method, including detoxification.
In addition to inhibitor tolerance, strain stability is also an
essential factor for designing recombinant industrial strains.
Strains carrying multicopy plasmids are generally not suitable
for industrial applications due to their instability (Futcher and
Cox 1984; Zhang et al. 1996). Multicopy plasmids require
auxotrophic or antibiotic resistance markers to be retained in
the cell, both of which are unsuitable for growth in industrial
media, which contain complex nutrients and are used in large
volumes. Thus, chromosomal integration of genes encoding
the xylose utilization pathway enzymes into industrially
applied yeast strains is necessary for large-scale fermentation
of xylose present in lignocellulosic biomass to ethanol.
Although industrial yeast strains usually carry no auxotro-
phic mutations, the integration of genes encoding the
enzymes of the xylose utilization pathway into the industrial
strains can be used as a positive selection for growth on
xylose (Ho et al. 1998; Wahlbom et al. 2003a), as well as
antibiotics such as zeocin (Zaldivar et al. 2002; Sonderegger
et al. 2004b) and aureobasidin A (Matsushika et al. 2009a,
2009b). Chromosomal integration using ribosomal DNA
locus can introduce multiple gene copies, which is effective
for optimal expression of heterologous enzyme genes
possessing low-specific activities (Lopes et al. 1996; Ho et
al. 1999; Karhumaa et al. 2006). Application of traditional
mating and breeding technology employed in the brewing
industry in combination with methods used to improve
xylose fermentation may also be used to introduce beneficial
genes in laboratory strains.
Adaptation of S. cerevisiae strains for efficient xylose
metabolism
In addition to targeted metabolic engineering, methods of
natural selection and random mutation are alternatives to
obtain improved xylose-utilizing yeast. These evolutionary
engineering approaches (Sauer 2001) have been success-
fully applied to a number of S. cerevisiae strains for
47
effective xylose fermentation. Although the first reported
recombinant S. cerevisiae TMB3001 was capable of
producing ethanol from xylose anaerobically (Eliasson et
al. 2000b), this strain cannot grow on xylose alone under
these conditions; the reason for which is not well
understood. Through a long-term, multistep chemostat
evolution experiment, Sonderegger and Sauer (2003)
obtained adapt strains that anaerobically grow on xylose
as the sole carbon source and that exhibit increased xylose
consumption rates (strain TMB3001C1, Table 1). Wahlbom
et al. (2003a) also maintained the industrial, diploid S.
cerevisiae strain TMB3399 (Table 1) in continuous culture
under aerobic, oxygen-limited, and anaerobic conditions
after treatment with chemical mutagen to obtain mutants of
improved recombinant S. cerevisiae (strain TMB3400,
Table 1). Pitkänen et al. (2005) used aerobic chemostat
cultivation on xylose alone and then changed to a xylose/
glucose mixture before switching back to xylose to generate
strain variants with improved xylose utilization.
Improved growth rates on xylose and xylose fermenta-
tion by evolutionary adaptation have also been accom-
plished for XI-carrying strains (Kuyper et al. 2004, 2005b;
Karhumaa et al. 2005; Madhavan et al. 2009b; strains RW
202-AFX, RWB 218, TMB3050, ADAP8, Table 1). In
addition, adaptation of a xylose-utilizing S. cerevisiae strain
TMB3001 to sugarcane bagasse hydrolysates with increas-
ing inhibitor concentration resulted in improved tolerance
to inhibitors and ethanol production (Martín et al. 2007).
Recently, Wisselink et al. (2009) have developed an
evolved strain (IMS0010) exhibiting improved specific
rates of consumption of xylose and arabinose using a novel
evolutionary engineering strategy. Microarray analysis,
metabolic flux analysis, and enzyme and metabolite
analyses using these evolved strains offer important
comparative information about bottlenecks in the metabo-
lism of xylose (Sonderegger et al. 2004a; Wahlbom et al.
2003b; Pitkänen et al. 2005; Bengtsson et al. 2008);
however, the results of these multiple changes are difficult
to interpret.
Concluding remarks and future prospects
Successful fermentation of lignocellulosic biomass to
ethanol is dependent on efficient utilization of D-xylose,
which is the second most common fermentable sugar in
hydrolysates. Many challenges in ethanol production
from xylose using metabolically engineered S. cerevisiae
have been extensively described in the literature. Several
approaches have been successfully employed to engineer
xylose metabolism. Nevertheless, these approaches are
insufficient for industrial bio-processes mainly due to the
low fermentation rate of xylose in comparison with that of
48
glucose. In spite of the fact that S. cerevisiae have all the
enzymes needed for a full xylose metabolic pathway (Batt
et al. 1986), its rate and yield of ethanol production from
xylose are considerably lower than those from glucose.
Studies have addressed this fundamental issue, yet no
clear solution has been achieved. One reason may be that
xylose is used as a non-fermentable carbon source in
metabolically engineered S. cerevisiae, as suggested in
some investigations using high-throughput experimental
techniques (Jin et al. 2004; Salusjärvi et al. 2008).
Functional genomics, including the transcriptome,
proteome, metabolome, and fluxome, are powerful tools
for targeting metabolic changes to enhance the rate and
yield of ethanol production from xylose (for a review see
Otero et al. 2007). This is an interesting area of future
research for improving xylose fermentation. Proteome
analysis of xylose fermentations has already revealed 22
proteins such as Adh2p, Ald4p, and Ald6p showing
significantly higher levels relative to glucose fermentation
(Salusjärvi et al. 2003). Furthermore, Salusjärvi et al.
(2008) found that xylose represses only partial metabolic
genes and decreases the expression of several genes
repressed by glucose via the Snf1p/Mig1p pathway. Global
expression studies using microarray technology with xylose
metabolizing recombinant yeast also provided important
information for further improving xylose fermentation
(Sedlak et al. 2003; Sonderegger et al. 2004a; Jin et al.
2004; Salusjärvi et al. 2006; Bengtsson et al. 2008);
however, these transcriptome results are often too complex
to interpret for practical use. Approaches that combine
functional genomics analysis with metabolic engineering
may provide several clues for developing robust S.
cerevisiae strains which can tolerate various inhibitors and
ferment xylose at similar rates to glucose. Moreover,
genome characteristics and regulatory patterns obtained
from the recently determined genome sequence of P. stipitis
(Jeffries et al. 2007) may serve as important guides for
further development of engineered S. cerevisiae or natural
xylose-fermenting yeasts. Further intensive studies are thus
required to develop engineered yeast that are capable of
efficiently fermenting all sugars including D-xylose found
in lignocellulosic hydrolysates to ethanol at a industrial
scale.
Acknowledgments The authors thank Dr. Katsuji Murakami,
Mr. Osamu Takimura, Mr. Shinichi Yano, Dr. Kenichiro Tsukahara,
Dr. Ohgiya Satoru (AIST), and Dr. Keisuke Makino and Dr. Seiya
Watanabe (Kyoto University) for their useful discussions. Many
thanks are due also to Dr. Takeshi Mizuno and Dr. Takafumi
Yamashino (Nagoya University) for their encouragement and
useful discussions. This study was supported in part by the New
Energy and Industrial Technology Development Organization
(NEDO), Japan.
Appl Microbiol Biotechnol (2009) 84:37–53
References
Agbogbo FK, Coward-Kelly G (2008) Cellulosic ethanol production
using the naturally occurring xylose-fermenting yeast, Pichia
stipitis. Biotechnol Lett 30:1515–1524
Almeida JR, Hahn-Hägerdal B (2009) Developing Saccharomyces
cerevisiae strains for second generation bioethanol: improving
xylose fermentation and inhibitor tolerance. Int Sugar J 111:172–
180
Almeida JR, Bertilsson M, Gorwa-Grauslund MF, Gorsich S, Lidén G
(2009) Metabolic effects of furaldehyde and impacts on biotech-
nological processes. Appl Microbiol Biotechnol 82:625–638
Alves SL Jr, Herberts RA, Hollatz C, Trichez D, Miletti LC, de Araujo
PS, Stambuk BU (2008) Molecular analysis of maltotriose active
transport and fermentation by Saccharomyces cerevisiae reveals a
determinant role for the AGT1 permease. Appl Environ Microbiol
74:1494–1501
Amore R, Wilhelm M, Hollenberg CP (1989) The fermentation of
xylose—an analysis of the expression of Bacillus and Actino-
planes xylose isomerase genes in yeast. Appl Microbiol
Biotechnol 30:351–357
Annaluru N, Watanabe S, Pack SP, Saleh AA, Kodaki T, Makino K
(2007) Thermostabilization of Pichia stipitis xylitol dehydroge-
nase by mutation of structural zinc-binding loop. J Biotechnol
129:717–722
Aristidou A, Penttilä M (2000) Metabolic engineering applications to
renewable resource utilization. Curr Opin Biotechnol 11:187–198
Banejee S, Archana A, Satyanarayana T (1994) Xylose metabolism in
a thermophilic mould Malbranchea pulchella var. sulfurea
TMD8. Curr Microbiol 29:349–352
Batt CA, Carvallo S, Easson DD Jr, Akedo M, Sinskey AJ (1986)
Direct evidence for a xylose metabolic pathway in Saccharomy-
ces cerevisiae. Biotechnol Bioeng 28:549–553
Bengtsson O, Jeppsson M, Sonderegger M, Parachin NS, Sauer U,
Hahn-Hägerdal B, Gorwa-Grauslund MF (2008) Identification of
common traits in improved xylose-growing Saccharomyces
cerevisiae for inverse metabolic engineering. Yeast 25:835–847
Bertilsson M, Olofsson K, Liden G (2009) Prefermentation improves
xylose utilization in simultaneous saccharification and co-
fermentation of pretreated spruce. Biotechnol Biofuels 2:8
Bolen PL, Roth KA, Freer SN (1986) Affinity purifications of aldose
reductase and xylitol dehydrogenase from the xylose-fermenting
yeast Pachysolen tannophilus. Appl Environ Microbiol 52:660–
664
Brandberg T, Franzén CJ, Gustafsson L (2004) The fermentation
performance of nine strains of Saccharomyces cerevisiae in batch
and fed-batch cultures in dilute-acid wood hydrolysate. J Biosci
Bioeng 98:122–125
Brat D, Boles E, Wiedemann B (2009) Functional expression of a
bacterial xylose isomerase in Saccharomyces cerevisiae. Appl
Environ Microbiol 75:2304–2311
Bro C, Regenberg B, Förster J, Nielsen J (2006) In silico aided
metabolic engineering of Saccharomyces cerevisiae for improved
bioethanol production. Metab Eng 8:102–111
Bruinenberg PM, de Bot PHM, van Dijken JP, Scheffers WA
(1983) The role of redox balances in the anaerobic fermenta-
tion of xylose by yeasts. Eur J Appl Microbiol Biotechnol
18:287–292
Bruinenberg PM, de Bot PHM, van Dijken JP, Scheffers WA (1984)
NADH-linked aldose reductase: the key to anaerobic fermenta-
tion of xylose by yeasts. Appl Microbiol Biotechnol 19:256–260
Bruinenberg PM, Jonker R, van Dijken JP, Scheffers WA (1985)
Utilization of formate as an additional energy source by glucose-
limited chemostat cultures of Candida utilis CBS 621 and
Saccharomyces cerevisiae CBS 8066—evidence for the absence
Appl Microbiol Biotechnol (2009) 84:37–53
of transhydrogenase activity in yeasts. Arch Microbiol 142:302–
306
Chang SF, Ho NW (1988) Cloning the yeast xylulokinase gene for the
improvement of xylose fermentation. Appl Biochem Biotechnol
17:313–318
Chiang LC, Gong CS, Chen LF, Tsao GT (1981) D-xylulose
fermentation to ethanol by Saccharomyces cerevisiae. Appl
Environ Microbiol 42:284–289
Chu BC, Lee H (2007) Genetic improvement of Saccharomyces
cerevisiae for xylose fermentation. Biotechnol Adv 25:425–441
Debus D, Methner H, Shulze D, Dellweg H (1983) Fermentation of
xylose with the yeast Pachysolen tannophilus. Eur J Appl
Microbiol Biotechnol 17:287–291
Deng XX, Ho NW (1990) Xylulokinase activity in various yeasts
including Saccharomyces cerevisiae containing the cloned
xylulokinase gene. Appl Biochem Biotechnol 24–25:193–199
Dien BS, Cotta MA, Jeffries TW (2003) Bacteria engineered for fuel
ethanol production: current status. Appl Microbiol Biotechnol
63:258–266
Eliasson A, Boles E, Johansson B, Österberg M, Thevelein JM,
Spencer-Martins I, Juhnke H, Hahn-Hägerdal B (2000a) Xylu-
lose fermentation by mutant and wild-type strains of Zygosac-
charomyces and Saccharomyces cerevisiae. Appl Microbiol
Biotechnol 53:376–382
Eliasson A, Christensson C, Wahlbom CF, Hahn-Hägerdal B (2000b)
Anaerobic xylose fermentation by recombinant Saccharomyces
cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium
chemostat cultures. Appl Environ Microbiol 66:3381–3386
Eliasson A, Hofmeyr J-HS, Pedler S, Hahn-Hägerdal B (2001) The
xylose reductase/xylitol dehydrogenase/xylulokinase ratio affects
product formation in recombinant xylose-utilising Saccharomy-
ces cerevisiae. Enzyme Microb Technol 29:288–297
Evans CT, Ratledge C (1984) Induction of xylulose-5-phosphate
phosphoketolase in a variety of yeasts grown on D-xylose:
the key to efficient xylose metabolism. Arch Microbiol
139:48–52
Fiaux J, Cakar ZP, Sonderegger M, Wüthrich K, Szyperski T, Sauer U
(2003) Metabolic-flux profiling of the yeasts Saccharomyces
cerevisiae and Pichia stipitis. Eukaryot Cell 2:170–180
Futcher AB, Cox BS (1984) Copy number and the stability of 2-μm
circle-based artificial plasmids of Saccharomyces cerevisiae. J
Bacteriol 157:283–290
Gancedo JM (1998) Yeast carbon catabolite repression. Microbiol Mol
Biol Rev 62:334–361
Gancedo JM, Lagunas R (1973) Contribution of the pentose
phosphate pathway to glucose metabolism in Saccharomyces
cerevisiae: a critical analysis on the use of labelled glucose. Plant
Sci Lett 1:193–200
Gárdonyi M, Hahn-Hägerdal B (2003) The Streptomyces rubiginosus
xylose isomerase is misfolded when expressed in Saccharomyces
cerevisiae. Enzyme Microb Technol 32:252–259
Garay-Arroyo A, Covarrubias AA, Clark I, Niño I, Gosset G,
Martinez A (2004) Response to different environmental stress
conditions of industrial and laboratory Saccharomyces cerevisiae
strains. Appl Microbiol Biotechnol 63:734–741
Gong CS, Cao NJ, Du J, Tsao GT (1999) Ethanol production
from renewable resources. Adv Biochem Eng Biotechnol
65:207–241
Hahn-Hägerdal B, Wahlbom CF, Gárdonyi M, van Zyl WH, Cordero
Otero RR, Jönsson LJ (2001) Metabolic engineering of Saccha-
romyces cerevisiae for xylose utilization. Adv Biochem Eng
Biotechnol 73:53–84
Hahn-Hägerdal B, Karhumaa K, Larsson CU, Gorwa-Grauslund MF,
Görgens J, van Zyl WH (2005) Role of cultivation media in the
development of yeast strains for large scale industrial use. Microb
Cell Fact 4:31
49
Hahn-Hägerdal B, Karhumaa K, Fonseca C, Spencer-Martins I,
Gorwa-Grauslund MF (2007a) Towards industrial pentose-
fermenting yeast strains. Appl Microbiol Biotechnol 74:937–953
Hahn-Hägerdal B, Karhumaa K, Jeppsson M, Gorwa-Grauslund MF
(2007b) Metabolic engineering for pentose utilization in Saccha-
romyces cerevisiae. Adv Biochem Eng Biotechnol 108:147–177
Hamacher T, Becker J, Gárdonyi M, Hahn-Hägerdal B, Boles E
(2002) Characterization of the xylose-transporting properties of
yeast hexose transporters and their influence on xylose utiliza-
tion. Microbiology 148:2783–2788
Harhangi HR, Akhmanova AS, Emmens R, van der Drift C, de Laat
WT, van Dijken JP, Jetten MS, Pronk JT, Op den Camp HJ
(2003) Xylose metabolism in the anaerobic fungus Piromyces sp.
strain E2 follows the bacterial pathway. Arch Microbiol
180:134–141
Hector RE, Qureshi N, Hughes SR, Cotta MA (2008) Expression of a
heterologous xylose transporter in a Saccharomyces cerevisiae
strain engineered to utilize xylose improves aerobic xylose
consumption. Appl Microbiol Biotechnol 80:675–684
Hendriks AT, Zeeman G (2009) Pretreatments to enhance the
digestibility of lignocellulosic biomass. Bioresour Technol
100:10–18
Ho NW, Chen Z, Brainard AP (1998) Genetically engineered
Saccharomyces yeast capable of effective cofermentation of
glucose and xylose. Appl Environ Microbiol 64:1852–1859
Ho NW, Chen Z, Brainard AP, Sedlak M (1999) Successful design
and development of genetically engineered Saccharomyces
yeasts for effective cofermentation of glucose and xylose from
cellulosic biomass to fuel ethanol. Adv Biochem Eng Biotechnol
65:163–192
Hou J, Vemuri GN, Bao X, Olsson L (2009) Impact of overexpressing
NADH kinase on glucose and xylose metabolism in recombinant
xylose-utilizing Saccharomyces cerevisiae. Appl Microbiol Bio-
technol 82:909–919
Hughes SR, Sterner DE, Bischoff KM, Hector RE, Dowd PF, Qureshi
N, Bang SS, Grynaviski N, Chakrabarty T, Johnson ET, Dien BS,
Mertens JA, Caughey RJ, Liu S, Butt TR, LaBaer J, Cotta MA,
Rich JO (2009) Engineered Saccharomyces cerevisiae strain for
improved xylose utilization with a three-plasmid SUMO yeast
expression system. Plasmid 61:22–38
Inoue H, Yano S, Endo T, Sakaki T, Sawayama S (2008) Combining
hot-compressed water and ball milling pretreatments to improve
the efficiency of the enzymatic hydrolysis of eucalyptus.
Biotechnol Biofuels 1:2
Jeffries TW (1981) Conversion of xylose to ethanol under aerobic
conditions by Candida tropicalis. Biotechnol Lett 3:213–218
Jeffries TW (2006) Engineering yeasts for xylose metabolism. Curr
Opin Biotechnol 17:320–326
Jeffries TW, Shi NQ (1999) Genetic engineering for improved xylose
fermentation by yeasts. Adv Biochem Eng Biotechnol 65:117–
161
Jeffries TW, Jin YS (2004) Metabolic engineering for improved
fermentation of pentoses by yeasts. Appl Microbiol Biotechnol
63:495–509
Jeffries TW, Grigoriev IV, Grimwood J, Laplaza JM, Aerts A,
Salamov A, Schmutz J, Lindquist E, Dehal P, Shapiro H, Jin
YS, Passoth V, Richardson PM (2007) Genome sequence of the
lignocellulose-bioconverting and xylose-fermenting yeast Pichia
stipitis. Nat Biotechnol 25:319–326
Jeppsson M, Johansson B, Hahn-Hägerdal B, Gorwa-Grauslund MF
(2002) Reduced oxidative pentose phosphate pathway flux in
recombinant xylose-utilizing Saccharomyces cerevisiae strains
improves the ethanol yield from xylose. Appl Environ Microbiol
68:1604–1609
Jeppsson M, Johansson B, Jensen PR, Hahn-Hägerdal B, Gorwa-
Grauslund MF (2003a) The level of glucose-6-phosphate
50
dehydrogenase activity strongly influences xylose fermentation
and inhibitor sensitivity in recombinant Saccharomyces cerevi-
siae strains. Yeast 20:1263–1272
Jeppsson M, Träff K, Johansson B, Hahn-Hägerdal B, Gorwa-
Grauslund MF (2003b) Effect of enhanced xylose reductase
activity on xylose consumption and product distribution in
xylose-fermenting recombinant Saccharomyces cerevisiae.
FEMS Yeast Res 3:167–175
Jeppsson M, Bengtsson O, Franke K, Lee H, Hahn-Hägerdal B,
Gorwa-Grauslund MF (2006) The expression of a Pichia stipitis
xylose reductase mutant with higher KM for NADPH increases
ethanol production from xylose recombinant Saccharomyces
cerevisiae. Biotechnol Bioeng 93:665–673
Jin YS, Jeffries TW (2003) Changing flux of xylose metabolites by
altering expression of xylose reductase and xylitol dehydroge-
nase in recombinant Saccharomyces cerevisiae. Appl Biochem
Biotechnol 105–108:277–286
Jin YS, Jones S, Shi NQ, Jeffries TW (2002) Molecular cloning of
XYL3 (D-xylulokinase) from Pichia stipitis and characterization
of its physiological function. Appl Environ Microbiol 68:1232–
1239
Jin YS, Ni H, Laplaza JM, Jeffries TW (2003) Optimal growth and
ethanol production from xylose by recombinant Saccharomyces
cerevisiae require moderate D-xylulokinase activity. Appl Envi-
ron Microbiol 69:495–503
Jin YS, Laplaza JM, Jeffries TW (2004) Saccharomyces cerevisiae
engineered for xylose metabolism exhibits a respiratory response.
Appl Environ Microbiol 70:6816–6825
Jin YS, Alper H, Yang YT, Stephanopoulos G (2005) Improvement of
xylose uptake and ethanol production in recombinant Saccharo-
myces cerevisiae through an inverse metabolic engineering
approach. Appl Environ Microbiol 71:8249–8256
Johansson B, Christensson C, Hobley T, Hahn-Hägerdal B (2001)
Xylulokinase overexpression in two strains of Saccharomyces
cerevisiae also expressing xylose reductase and xylitol dehydro-
genase and its effect on fermentation of xylose and lignocellu-
losic hydrolysate. Appl Environ Microbiol 67:4249–4255
Johansson B, Hahn-Hägerdal B (2002a) The non-oxidative pentose
phosphate pathway controls the fermentation rate of xylulose but
not of xylose in Saccharomyces cerevisiae TMB3001. FEMS
Yeast Res 2:277–282
Johansson B, Hahn-Hägerdal B (2002b) Overproduction of pentose
phosphate pathway enzymes using a new CRE-loxP expression
vector for repeated genomic integration in Saccharomyces
cerevisiae. Yeast 19:225–231
Karhumaa K, Hahn-Hägerdal B, Gorwa-Grauslund MF (2005)
Investigation of limiting metabolic steps in the utilization of
xylose by recombinant Saccharomyces cerevisiae using metabol-
ic engineering. Yeast 22:359–368
Karhumaa K, Wiedemann B, Hahn-Hägerdal B, Boles E, Gorwa-
Grauslund MF (2006) Co-utilization of L-arabinose and D-xylose
by laboratory and industrial Saccharomyces cerevisiae strains.
Microb Cell Fact 5:18
Karhumaa K, Fromanger R, Hahn-Hägerdal B, Gorwa-Grauslund MF
(2007a) High activity of xylose reductase and xylitol dehydro-
genase improves xylose fermentation by recombinant Saccharo-
myces cerevisiae. Appl Microbiol Biotechnol 73:1039–1046
Karhumaa K, Garcia Sanchez R, Hahn-Hägerdal B, Gorwa-Grauslund
MF (2007b) Comparison of the xylose reductase-xylitol dehy-
drogenase and the xylose isomerase pathways for xylose
fermentation by recombinant Saccharomyces cerevisiae. Microb
Cell Fact 6:5
Katahira S, Mizuike A, Fukuda H, Kondo A (2006) Ethanol
fermentation from lignocellulosic hydrolysate by a recombinant
xylose- and cellooligosaccharide-assimilating yeast strain. Appl
Microbiol Biotechnol 72:1136–1143
Appl Microbiol Biotechnol (2009) 84:37–53
Katahira S, Ito M, Takema H, Fujita Y, Tanino T, Tanaka T, Fukuda H,
Kondo A (2008) Improvement of ethanol productivity during
xylose and glucose co-fermentation by xylose-assimilating S.
cerevisiae via expression of glucose transporter Sut1. Enzyme
Microb Technol 43:115–119
Kavanagh KL, Klimacek M, Nidetzky B, Wilson DK (2002) The
structure of apo and holo forms of xylose reductase, a dimeric
aldo-keto reductase from Candida tenuis. Biochemistry 41:8785–
8795
Kavanagh KL, Klimacek M, Nidetzky B, Wilson DK (2003) Structure
of xylose reductase bound to NAD+ and the basis for single and
dual co-substrate specificity in family 2 aldo-keto reductases.
Biochem J 373:319–326
Kostrzynska M, Sopher CR, Lee H (1998) Mutational analysis of the
role of the conserved lysine-270 in the Pichia stipitis xylose
reductase. FEMS Microbiol Lett 159:107–112
Kötter P, Ciriacy M (1993) Xylose fermentation by Saccharomyces
cerevisiae. Appl Microbiol Biotechnol 38:776–783
Kruckeberg AL (1996) The hexose transporter family of Saccharo-
myces cerevisiae. Arch Microbiol 166:283–292
Kuhn A, van Zyl C, van Tonder A, Prior BA (1995) Purification and
partial characterization of an aldo-keto reductase from Saccha-
romyces cerevisiae. Appl Environ Microbiol 61:1580–1585
Kuyper M, Harhangi HR, Stave AK, Winkler AA, Jetten MS, de Laat
WT, den Ridder JJ, Op den Camp HJ, van Dijken JP, Pronk JT
(2003) High-level functional expression of a fungal xylose
isomerase: the key to efficient ethanolic fermentation of xylose
by Saccharomyces cerevisiae? FEMS Yeast Res 4:69–78
Kuyper M, Winkler AA, van Dijken JP, Pronk JT (2004) Minimal
metabolic engineering of Saccharomyces cerevisiae for efficient
anaerobic xylose fermentation: a proof of principle. FEMS Yeast
Res 4:655–664
Kuyper M, Hartog MM, Toirkens MJ, Almering MJ, Winkler AA, van
Dijken JP, Pronk JT (2005a) Metabolic engineering of a xylose-
isomerase-expressing Saccharomyces cerevisiae strain for rapid
anaerobic xylose fermentation. FEMS Yeast Res 5:399–409
Kuyper M, Toirkens MJ, Diderich JA, Winkler AA, van Dijken JP,
Pronk JT (2005b) Evolutionary engineering of mixed-sugar
utilization by a xylose-fermenting Saccharomyces cerevisiae
strain. FEMS Yeast Res 5:925–934
Larsson S, Palmqvist E, Hahn-Hägerdal B, Tengborg C, Stenberg K,
Zacchi G, Nilvebrant N-O (1999) The generation of fermentation
inhibitors during dilute acid hydrolysis of softwood. Enzyme
Microb Technol 24:151–159
Lau MW, Dale BE (2009) Cellulosic ethanol production from AFEX-
treated corn stover using Saccharomyces cerevisiae 424A(LNH-
ST). Proc Natl Acad Sci U S A 106:1368–1373
Lau MW, Dale BE, Balan V (2008) Ethanolic fermentation of
hydrolysates from ammonia fiber expansion (AFEX) treated corn
stover and distillers grain without detoxification and external
nutrient supplementation. Biotechnol Bioeng 99:529–539
Leandro MJ, Gonçalves P, Spencer-Martins I (2006) Two glucose/
xylose transporter genes from the yeast Candida intermedia: first
molecular characterization of a yeast xylose-H+ symporter.
Biochem J 395:543–549
Leandro MJ, Spencer-Martins I, Gonçalves P (2008) The expression
in Saccharomyces cerevisiae of a glucose/xylose symporter from
Candida intermedia is affected by the presence of a glucose/
xylose facilitator. Microbiology 154:1646–1655
Lee J (1997) Biological conversion of lignocellulosic biomass to
ethanol. J Biotechnol 56:1–24
Lee WJ, Kim MD, Ryu YW, Bisson LF, Seo JH (2002) Kinetic studies
on glucose and xylose transport in Saccharomyces cerevisiae.
Appl Microbiol Biotechnol 60:186–191
Leitgeb S, Petschacher B, Wilson DK, Nidetzky B (2005) Fine tuning
of coenzyme specificity in family 2 aldo-keto reductases revealed
Appl Microbiol Biotechnol (2009) 84:37–53
by crystal structures of the Lys-274→Arg mutant of Candida
tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+.
FEBS Lett 579:763–767
Lin S, Tanaka S (2006) Ethanol fermentation from biomass resources:
current state and prospects. Appl Microbiol Biotechnol 69:627–
642
Lönn A, Träff-Bjerre KL, Cordero Otero RR, van Zyl WH, Hahn-
Hägerdal B (2003) Xylose isomerase activity influences xylose
fermentation with recombinant Saccharomyces cerevisiae strains
expressing mutated xylA from Thermus thermophilus. Enzyme
Microb Technol 32:567–573
Lopes TS, de Wijs IJ, Steenhauer SI, Verbakel J, Planta RJ (1996)
Factors affecting the mitotic stability of high-copy-number
integration into the ribosomal DNA of Saccharomyces cerevisiae.
Yeast 12:467–477
Lu C, Jeffries T (2007) Shuffling of promoters for multiple genes to
optimize xylose fermentation in an engineered Saccharomyces
cerevisiae strain. Appl Environ Microbiol 73:6072–6077
Lu Y, Warner R, Sedlak M, Ho N, Mosier NS (2009) Comparison of
glucose/xylose cofermentation of poplar hydrolysates processed
by different pretreatment technologies. Biotechnol Prog 25:349–
356
Madhavan A, Tamalampudi S, Ushida K, Kanai D, Katahira S,
Srivastava A, Fukuda H, Bisaria VS, Kondo A (2009a) Xylose
isomerase from polycentric fungus Orpinomyces: gene sequenc-
ing, cloning, and expression in Saccharomyces cerevisiae for
bioconversion of xylose to ethanol. Appl Microbiol Biotechnol
82:1067–1078
Madhavan A, Tamalampudi S, Srivastava A, Fukuda H, Bisaria
VS, Kondo A (2009b) Alcoholic fermentation of xylose and
mixed sugars using recombinant Saccharomyces cerevisiae
engineered for xylose utilization. Appl Microbiol Biotechnol
82:1037–1047
Martín C, Jönsson LJ (2003) Comparison of the resistance of
industrial and laboratory strains of Saccharomyces cerevisiae
and Zygosaccharomyces to lignocellulose-derived fermentation
inhibitors. Enzyme Microb Technol 32:386–395
Martín C, Marcet M, Almazán O, Jönsson LJ (2007) Adaptation of a
recombinant xylose-utilizing Saccharomyces cerevisiae strain to
a sugarcane bagasse hydrolysate with high content of fermenta-
tion inhibitors. Bioresour Technol 98:1767–1773
Matsushika A, Sawayama S (2008) Efficient bioethanol production
from xylose by recombinant Saccharomyces cerevisiae requires
high activity of xylose reductase and moderate xylulokinase
activity. J Biosci Bioeng 106:306–309
Matsushika A, Watanabe S, Kodaki T, Makino K, Sawayama S
(2008a) Bioethanol production from xylose by recombinant
Saccharomyces cerevisiae expressing xylose reductase, NADP+-
dependent xylitol dehydrogenase, and xylulokinase. J Biosci
Bioeng 105:296–299
Matsushika A, Watanabe S, Kodaki T, Makino K, Inoue H, Murakami
K, Takimura O, Sawayama S (2008b) Expression of protein
engineered NADP+-dependent xylitol dehydrogenase increase
ethanol production from xylose in recombinant Saccharomyces
cerevisiae. Appl Microbiol Biotechnol 81:243–255
Matsushika A, Inoue H, Murakami K, Takimura O, Sawayama S
(2009a) Bioethanol production performance of five recombinant
strains of laboratory and industrial xylose-fermenting Saccharo-
myces cerevisiae. Bioresour Technol 100:2392–2398
Matsushika A, Inoue H, Watanabe S, Kodaki T, Makino K, Sawayama
S (2009b) Efficient bioethanol production by recombinant
flocculent Saccharomyces cerevisiae with genome-integrated
NADP+-dependent xylitol dehydrogenase gene. Appl Environ
Microbiol 75:3818–3822
Metzger MH, Hollenberg CP (1994) Isolation and characterization of
the Pichia stipitis transketolase gene and expression in a xylose-
51
utilising Saccharomyces cerevisiae transformant. Appl Microbiol
Biotechnol 42:319–325
Moes CJ, Pretorius IS, van Zyl WH (1996) Cloning and expression of
the Clostridium thermosulfurogenes D-xylose isomerase gene
(xylA) in Saccharomyces cerevisiae. Biotechnol Lett 18:269–274
Nakamura N, Yamada R, Katahira S, Tanaka T, Fukuda H, Kondo A
(2008) Effective xylose/cellobiose co-fermentation and ethanol
production by xylose-assimilating S. cerevisiae via expression of
β-glucosidase on its cell surface. Enzyme Microb Technol
43:233–236
Ni H, Laplaza M, Jeffries TW (2007) Transposon mutagenesis to
improve the growth of recombinant Saccharomyces cerevisiae on
D-xylose. Appl Environ Microbiol 73:2061–2066
Nilsson A, Gorwa-Grauslund MF, Hahn-Hägerdal B, Lidén G (2005)
Cofactor dependence in furan reduction by Saccharomyces
cerevisiae in fermentation of acid-hydrolyzed lignocellulose.
Appl Environ Microbiol 71:7866–7871
Nissen TL, Kielland-Brandt MC, Nielsen J, Villadsen J (2000)
Optimization of ethanol production in Saccharomyces cerevisiae
by metabolic engineering of the ammonium assimilation. Metab
Eng 2:69–77
Nissen TL, Anderlund M, Nielsen J, Villadsen J, Kielland-Brandt MC
(2001) Expression of a cytoplasmic transhydrogenase in Saccha-
romyces cerevisiae results in formation of 2-oxoglutarate due to
depletion of the NADPH pool. Yeast 18:19–32
Öhgren K, Bengtsson O, Gorwa-Grauslund MF, Galbe M, Hahn-
Hägerdal B, Zacchi G (2006) Simultaneous saccharification and
co-fermentation of glucose and xylose in steam-pretreated corn
stover at high fiber content with Saccharomyces cerevisiae
TMB3400. J Biotechnol 126:488–498
Olsson L, Hahn-Hägerdal B (1993) Fermentative performance of
bacteria and yeast in lignocellulose hydrolysates. Process
Biochem 28:249–257
Olsson L, Nielsen J (2000) The role of metabolic engineering in the
improvement of Saccharomyces cerevisiae: utilization of indus-
trial media. Enzyme Microb Tech 26:785–792
Olsson L, Soerensen HR, Dam BP, Christensen H, Krogh KM, Meyer
AS (2006) Separate and simultaneous enzymatic hydrolysis and
fermentation of wheat hemicellulose with recombinant xylose
utilizing Saccharomyces cerevisiae. Appl Biochem Biotechnol
129–132:117–129
Otero JM, Panagiotou G, Olsson L (2007) Fueling industrial
biotechnology growth with bioethanol. Adv Biochem Eng
Biotechnol 108:1–40
Palmqvist E, Hahn-Hägerdal B (2000) Fermentation of lignocellulosic
hydrolysates. II: Inhibitors and mechanisms of inhibition. Bio-
resour Technol 74:25–33
Petschacher B, Nidetzky B (2005) Engineering Candida tenuis xylose
reductase for improved utilization of NADH: antagonistic effects
of multiple side chain replacements and performance of site-
directed mutants under simulated in vivo conditions. Appl
Environ Microbiol 71:6390–6393
Petschacher B, Nidetzky B (2008) Altering the coenzyme preference
of xylose reductase to favor utilization of NADH enhances
ethanol yield from xylose in a metabolically engineered strain of
Saccharomyces cerevisiae. Microb Cell Fact 7:9
Petschacher B, Leitgeb S, Kavanagh KL, Wilson DK, Nidetzky B
(2005) The coenzyme specificity of Candida tenuis xylose
reductase (AKR2B5) explored by site-directed mutagenesis and
X-ray crystallography. Biochem J 385:75–83
Pitkänen JP, Aristidou A, Salusjärvi L, Ruohonen L, Penttilä M (2003)
Metabolic flux analysis of xylose metabolism in recombinant
Saccharomyces cerevisiae using continuous culture. Metab Eng
5:16–31
Pitkänen JP, Rintala E, Aristidou A, Ruohonen L, Penttilä M (2005)
Xylose chemostat isolates of Saccharomyces cerevisiae show
52
altered metabolite and enzyme levels compared with xylose,
glucose, and ethanol metabolism of the original strain. Appl
Microbiol Biotechnol 67:827–837
Richard P, Toivari MH, Penttilä M (1999) Evidence that the gene
YLR070c of Saccharomyces cerevisiae encodes a xylitol dehy-
drogenase. FEBS Lett 457:135–138
Richard P, Toivari MH, Penttilä M (2000) The role of xylulokinase in
Saccharomyces cerevisiae xylulose catabolism. FEMS Microbiol
Lett 190:39–43
Rizzi M, Erlemann P, Bui-Thanh NA, Dellweg H (1988) Xylose
fermentation by yeasts. 4. Purification and kinetic studies of
xylose reductase from Pichia stipitis. Appl Microbiol Biotechnol
29:148–154
Rizzi M, Harwart K, Erlemann P, Bui-Thanh NA, Dellweg H (1989)
Purification and properties of the NAD+-xylitol-dehydrogenase
from the yeast Pichia stipitis. J Ferment Bioeng 67:20–24
Roca C, Haack MB, Olsson L (2004) Engineering of carbon catabolite
repression in recombinant xylose fermenting Saccharomyces
cerevisiae. Appl Microbiol Biotechnol 63:578–583
Rodriguez-Pena JM, Cid VJ, Arroyo J, Nombela C (1998) The
YGR194c (XKS1) gene encodes the xylulokinase from the
budding yeast Saccharomyces cerevisiae. FEMS Microbiol Lett
162:155–160
Rudolf A, Baudel H, Zacchi G, Hahn-Hägerdal B, Lidén G (2008)
Simultaneous saccharification and fermentation of steam-
pretreated bagasse using Saccharomyces cerevisiae TMB3400
and Pichia stipitis CBS6054. Biotechnol Bioeng 99:783–790
Runquist D, Fonseca C, Radstrom P, Spencer-Martins I, Hahn-
Hägerdal B (2009) Expression of the Gxf1 transporter from
Candida intermedia improves fermentation performance in
recombinant xylose-utilizing Saccharomyces cerevisiae. Appl
Microbiol Biotechnol 82:123–130
Saloheimo A, Rauta J, Stasyk OV, Sibirny AA, Penttilä M,
Ruohonen L (2007) Xylose transport studies with xylose-
utilizing Saccharomyces cerevisiae strains expressing heterol-
ogous and homologous permeases. Appl Microbiol Biotechnol
74:1041–1052
Salusjärvi L, Poutanen M, Pitkänen JP, Koivistoinen H, Aristidou A,
Kalkkinen N, Ruohonen L, Penttilä M (2003) Proteome analysis
of recombinant xylose-fermenting Saccharomyces cerevisiae.
Yeast 20:295–314
Salusjärvi L, Pitkänen JP, Aristidou A, Ruohonen L, Penttilä M (2006)
Transcription analysis of recombinant Saccharomyces cerevisiae
reveals novel responses to xylose. Appl Biochem Biotechnol
128:237–261
Salusjärvi L, Kankainen M, Soliymani R, Pitkänen JP, Penttilä M,
Ruohonen L (2008) Regulation of xylose metabolism in
recombinant Saccharomyces cerevisiae. Microb Cell Fact 7:18
Sarthy AV, McConaughy BL, Lobo Z, Sundstrom JA, Furlong CE,
Hall BD (1987) Expression of the Escherichia coli xylose
isomerase gene in Saccharomyces cerevisiae. Appl Environ
Microbiol 53:1996–2000
Sauer U (2001) Evolutionary engineering of industrially important
microbial phenotypes. Adv Biochem Eng Biotechnol 73:129–169
Schaaff I, Hohmann S, Zimmermann FK (1990) Molecular analysis of
the structural gene for yeast transaldolase. Eur J Biochem
188:597–603
Schneider H, Wang PY, Chan YK, Maleszka R (1981) Conversion of
D-xylose into ethanol by the yeast Pachysolen tannophilus.
Biotechnol Lett 3:89–92
Sedlak M, Ho NW (2004a) Production of ethanol from cellulosic
biomass hydrolysates using genetically engineered Saccharomy-
ces yeast capable of cofermenting glucose and xylose. Appl
Biochem Biotechnol 113–116:403–416
Sedlak M, Ho NW (2004b) Characterization of the effectiveness of
hexose transporters for transporting xylose during glucose and
Appl Microbiol Biotechnol (2009) 84:37–53
xylose co-fermentation by a recombinant Saccharomyces yeast.
Yeast 21:671–684
Sedlak M, Edenberg HJ, Ho NW (2003) DNA microarray analysis of
the expression of the genes encoding the major enzymes in
ethanol production during glucose and xylose co-fermentation by
metabolically engineered Saccharomyces yeast. Enzyme Microb
Technol 33:19–28
Senac T, Hahn-Hägerdal B (1991) Effects of increased transaldolase
activity on D-xylulose and D-glucose metabolism in Saccharo-
myces cerevisiae cell extracts. Appl Environ Microbiol 57:1701–
1706
Slininger PJ, Bothast RJ, Van Cauwenberge JE, Kurtzman CP (1982)
Conversion of D-xylose to ethanol by the yeast Pachysolen
tannophilus. Biotechnol Bioeng 24:371–384
Sonderegger M, Sauer U (2003) Evolutionary engineering of
Saccharomyces cerevisiae for anaerobic growth on xylose. Appl
Environ Microbiol 69:1990–1998
Sonderegger M, Jeppsson M, Hahn-Hägerdal B, Sauer U (2004a)
Molecular basis for anaerobic growth of Saccharomyces cerevi-
siae on xylose, investigated by global gene expression and
metabolic flux analysis. Appl Environ Microbiol 70:2307–2317
Sonderegger M, Jeppsson M, Larsson C, Gorwa-Grauslund MF, Boles
E, Olsson L, Spencer-Martins I, Hahn-Hägerdal B, Sauer U
(2004b) Fermentation performance of engineered and evolved
xylose-fermenting Saccharomyces cerevisiae strains. Biotechnol
Bioeng 87:90–98
Sonderegger M, Schümperli M, Sauer U (2004c) Metabolic engineer-
ing of a phosphoketolase pathway for pentose catabolism in
Saccharomyces cerevisiae. Appl Environ Microbiol 70:2892–
2897
Tantirungkij M, Nakashima N, Seki T, Yoshida T (1993) Construction
of xylose-assimilating Saccharomyces cerevisiae. J Ferment
Bioeng 75:83–88
Toivari MH, Aristidou A, Ruohonen L, Penttilä M (2001) Conversion
of xylose to ethanol by recombinant Saccharomyces cerevisiae:
importance of xylulokinase (XKS1) and oxygen availability.
Metab Eng 3:236–249
Toivari MH, Salusjarvi L, Ruohonen L, Penttilä M (2004) Endoge-
nous xylose pathway in Saccharomyces cerevisiae. Appl Environ
Microbiol 70:3681–3686
Tomás-Pejó E, Oliva JM, Ballesteros M, Olsson L (2008) Comparison
of SHF and SSF processes from steam-exploded wheat straw for
ethanol production by xylose-fermenting and robust glucose-
fermenting Saccharomyces cerevisiae strains. Biotechnol Bioeng
100:1122–1131
Träff KL, Otero Cordero RR, van Zyl WH, Hahn-Hägerdal B (2001)
Deletion of the GRE3 aldose reductase gene and its influence on
xylose metabolism in recombinant strains of Saccharomyces
cerevisiae expressing the xylA and XKS1 genes. Appl Environ
Microbiol 67:5668–5674
Träff KL, Jönsson LJ, Hahn-Hägerdal B (2002) Putative xylose and
arabinose reductases in Saccharomyces cerevisiae. Yeast
19:1233–1241
Träff-Bjerre KL, Jeppsson M, Hahn-Hägerdal B, Gorwa-Grauslund
MF (2004) Endogenous NADPH-dependent aldose reductase
activity influences product formation during xylose consumption
in recombinant Saccharomyces cerevisiae. Yeast 21:141–150
Trumbly RJ (1992) Glucose repression in the yeast Saccharomyces
cerevisiae. Mol Microbiol 6:15–21
van Maris AJ, Abbott DA, Bellissimi E, van den Brink J, Kuyper M,
Luttik MA, Wisselink HW, Scheffers WA, van Dijken JP, Pronk
JT (2006) Alcoholic fermentation of carbon sources in biomass
hydrolysates by Saccharomyces cerevisiae: current status.
Antonie van Leeuwenhoek 90:391–418
van Maris AJ, Winkler AA, Kuyper M, de Laat WT, van Dijken JP,
Pronk JT (2007) Development of efficient xylose fermentation in
Appl Microbiol Biotechnol (2009) 84:37–53
Saccharomyces cerevisiae: xylose isomerase as a key component.
Adv Biochem Eng Biotechnol 108:179–204
Van Vleet JH, Jeffries TW, Olsson L (2008) Deleting the para-
nitrophenyl phosphatase (pNPPase), PHO13, in recombinant
Saccharomyces cerevisiae improves growth and ethanol produc-
tion on D-xylose. Metab Eng 10:360–369
van Zyl C, Prior BA, Kilian SG, Brandt EV (1993) Role of D-ribose
as a cometabolite in D-xylose metabolism by Saccharomyces
cerevisiae. Appl Environ Microbiol 59:1487–1494
Verduyn C, Van Kleef R, Frank J, Schreuder H, Van Dijken JP,
Scheffers WA (1985) Properties of the NAD(P)H-dependent
xylose reductase from the xylose-fermenting yeast Pichia stipitis.
Biochem J 226:669–677
Verduyn C, Postma E, Scheffers WA, Van Dijken JP (1992) Effect of
benzoic acid on metabolic fluxes in yeasts: a continuous-culture
study on the regulation of respiration and alcoholic fermentation.
Yeast 8:501–517
Verho R, Richard P, Jonson PH, Sundqvist L, Londesborough J,
Penttilä M (2002) Identification of the first fungal NADP–
GAPDH from Kluyveromyces lactis. Biochemistry 41:13833–
13838
Verho R, Londesborough J, Penttilä M, Richard P (2003) Engineering
redox cofactor regeneration for improved pentose fermentation in
Saccharomyces cerevisiae. Appl Environ Microbiol 69:5892–
5897
Vongsuvanlert V, Tani Y (1988) Purification and characterization of
xylose isomerase of a methanol yeast, C. boidinii, which is
involved in sorbitol production from glucose. Agric Biol Chem
52:1817–1824
Wahlbom CF, van Zyl WH, Jönsson LJ, Hahn-Hägerdal B, Cordero
Otero RR (2003a) Generation of the improved recombinant
xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random
mutagenesis and physiological comparison with Pichia stipitis
CBS 6054. FEMS Yeast Res 3:319–326
Wahlbom CF, Cordero Otero RR, van Zyl WH, Hahn-Hägerdal B,
Jönsson LJ (2003b) Molecular analysis of a Saccharomyces
cerevisiae mutant with improved ability to utilize xylose shows
enhanced expression of proteins involved in transport, initial
xylose metabolism, and the pentose phosphate pathway. Appl
Environ Microbiol 69:740–746
Walfridsson M, Hallborn J, Penttilä M, Keränen S, Hahn-Hägerdal B
(1995) Xylose-metabolizing Saccharomyces cerevisiae strains
overexpressing the TKL1 and TAL1 genes encoding the pentose
phosphate pathway enzymes transketolase and transaldolase.
Appl Environ Microbiol 61:4184–4190
Walfridsson M, Bao X, Anderlund M, Lilius G, Bülow L, Hahn-
Hägerdal B (1996) Ethanolic fermentation of xylose with
Saccharomyces cerevisiae harboring the Thermus thermophilus
xylA gene, which expresses an active xylose (glucose) isomerase.
Appl Environ Microbiol 62:4648–4651
Walfridsson M, Anderlund M, Bao X, Hahn-Hägerdal B (1997)
Expression of different levels of enzymes from the Pichia stipitis
53
XYL1 and XYL2 genes in Saccharomyces cerevisiae and its
effects on product formation during xylose utilisation. Appl
Microbiol Biotechnol 48:218–224
Wang PY, Schneider H (1980) Growth of yeasts on D-xylulose. Can J
Microbiol 26:1165–1168
Wang PY, Shopsis C, Schneider H (1980) Fermentation of a pentose
by yeasts. Biochem Biophys Res Commun 94:248–254
Wang VW, Jeffries T (1990) Purification and properties of xylitol
dehydrogenase from the xylose-fermenting Candida shehatae.
Appl Biochem Biotechnol 26:197–206
Watanabe S, Kodaki T, Makino K (2005) Complete reversal of
coenzyme specificity of xylitol dehydrogenase and increase of
thermostability by the introduction of structural zinc. J Biol
Chem 280:10340–10349
Watanabe S, Pack SP, Saleh AA, Annaluru N, Kodaki T, Makino K
(2007a) Positive effect of the decreased NADPH-preferring
activity of xylose reductase from Pichia stipitis on ethanol
production using xylose-fermenting recombinant Saccharomyces
cerevisiae. Biosci Biotechnol Biochem 71:1365–1369
Watanabe S, Saleh AA, Pack SP, Annaluru N, Kodaki T, Makino K
(2007b) Ethanol production from xylose by recombinant Sac-
charomyces cerevisiae expressing protein engineered NADH-
preferring xylose reductase from Pichia stipitis. Microbiology
153:3044–3054
Watanabe S, Saleh AA, Pack SP, Annaluru N, Kodaki T, Makino K
(2007c) Ethanol production from xylose by recombinant Sac-
charomyces cerevisiae expressing protein engineered NADP+-
dependent xylitol dehydrogenase. J Biotechnol 130:316–319
Weierstall T, Hollenberg CP, Boles E (1999) Cloning and character-
ization of three genes (SUT1–3) encoding glucose transporters of
the yeast Pichia stipitis. Mol Microbiol 31:871–883
Wisselink HW, Toirkens MJ, Wu Q, Pronk JT, van Maris AJ
(2009) A novel evolutionary engineering approach for accel-
erated utilization of glucose, xylose and arabinose mixtures by
engineered Saccharomyces cerevisiae. Appl Environ Microbiol
75:907–914
Yamanaka K (1969) Inhibition of D-xylose isomerase by pentitols and
D-lyxose. Arch Biochem Biophys 131:502–506
Yang VW, Jeffries TW (1997) Regulation of phosphotransferases in
glucose- and xylose-fermenting yeasts. Appl Biochem Biotech-
nol 63–65:97–108
Yu S, Jeppsson H, Hahn-Hägerdal B (1995) Xylulose fermentation by
Saccharomyces cerevisiae and xylose-fermenting yeast strains.
Appl Microbiol Biotechnol 44:314–320
Zaldivar J, Borges A, Johansson B, Smits HP, Villas-Bôas SG, Nielsen
J, Olsson L (2002) Fermentation performance and intracellular
metabolite patterns in laboratory and industrial xylose-fermenting
Saccharomyces cerevisiae. Appl Microbiol Biotechnol 59:436–
442
Zhang Z, Moo-Young M, Chisti Y (1996) Plasmid stability in
recombinant Saccharomyces cerevisiae. Biotechnol Adv
14:401–435