Bioethanol

Kevin A Gray1, Lishan Zhao1 and Mark Emptage2

Alternatives to petroleum-derived fuels are being sought in
order to reduce the worlds dependence on non-renewable
resources. The most common renewable fuel today is ethanol
derived from corn grain (starch) and sugar cane (sucrose). It is
expected that there will be limits to the supply of these raw
materials in the near future, therefore lignocellulosic biomass is
seen as an attractive feedstock for future supplies of ethanol.
However, there are technical and economical impediments to
the development of a commercial processes utilizing biomass.
Technologies are being developed that will allow cost-effective
conversion of biomass into fuels and chemicals. These
technologies include low-cost thermochemical pretreatment,
highly effective cellulases and hemicellulases and efficient and
robust fermentative microorganisms. Many advances have
been made over the past few years that make
commercialization more promising.
Addresses
1
Diversa Corporation, 4955 Directors Place, San Diego CA, 92121,
USA
2
DuPont Central Research & Development, Experimental Station
E328, Wilmington, DE 19880, USA
Corresponding author: Gray, Kevin A (kgray@diversa.com)

Current Opinion in Chemical Biology 2006, 10:141146

This review comes from a themed issue on
Biocatalysis and biotransformation
Edited by Ben Davis and Grace DeSantis
Available online 7th March 2006
1367-5931/$ see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2006.02.035

Introduction
Currently the United States consumes approximately 20
million barrels of crude oil daily of which about 60% is
imported. Liquid transportation fuels including gasoline,
diesel and jet fuel account for almost 70% of the total.
The US Energy Policy Act of 2005 (http://www.ferc.gov)
states that the oil industry is required to blend 7.5 billion
gal of renewable fuels into gasoline by 2012. In addition,
many states have passed renewable fuels standards that
require the sale of 10% and 20% blends (E10 and E20) by
certain dates [1]. By far the most common renewable fuel
is ethanol, and annual ethanol production in the United
States recently surpassed 4 billion gal, with global production twice that. In the United States, the major raw
material for ethanol is corn grain (starch). The US has the
capacity to produce 13 billion gallons per year from corn
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alone and will probably reach the 7.5 billion gal per year
goal much sooner than expected. However, any further
increases in ethanol production will have to come from
feedstocks other than corn grain because of limitations in
supply. These feedstocks are typically grouped under the
heading of biomass and include agricultural residues,
wood, municipal solid waste and dedicated energy crops.
The US Department of Agriculture and Department of
Energy have estimated that the US has the resource
potential to produce over 1 billion tons of biomass
annually [2], thus accounting for close to 30% displacement of current fossil fuel usage (about 80 billion gal).
Unlike corn grain where the major carbohydrate is starch,
biomass is composed of cellulose (4050%), hemicellulose (2535%) and lignin (1520%). Starch processing is a
fairly mature technology utilizing enzymatic liquefaction
and saccharification, which produces a relatively clean
glucose stream that is then fermented to ethanol by
Saccharomyces yeasts. Recent advances in starch processing have improved the economics and efficiency of the
process. One example has been the development of low
pH a-amylases that simplify the process and reduce
chemical costs as well as improving ethanol yield [3].
The other major advance is the development of enzymes
that function on raw, uncooked starch, thereby improving
overall process economics [4,5].
Starch is a storage compound consisting of glucose linked
via a-1,4 and a-1,6 glycosidic linkages (amylose and amylopectin), whereas cellulose is a structural compound
composed exclusively of glucose linked via b-1,4 glycosidic bonds. Because of the b-1,4 linkage, cellulose is highly
crystalline and compact making it very resistant to biological attack. In general, hemicellulose consists of a main
chain xylan backbone (b-1,4 linkages) with various
branches of mannose, arabinose, galactose, glucuronic
acid, etc (Figure 1). The degree of branching and identity
of the minor sugars in hemicellulose tends to vary depending upon the type of plant. Furthermore, lignin can be
covalently linked to hemicellulose via ferulic acid ester
linkages. The compactness and complexity of lignocellulose makes it much more difficult than starch to enzymatically degrade to fermentable sugars. Hence, the cost of
producing a gallon of ethanol from biomass is higher than
production from starch [6]. In order to be cost competitive
with grain-derived ethanol, the enzymes used for biomass
hydrolysis must become more efficient and far less expensive. In addition, the presence of non-glucose sugars in the
feedstock complicates the fermentation process because
conversion of pentose sugars into ethanol is less efficient
than conversion of the hexose sugars.
Current Opinion in Chemical Biology 2006, 10:141146

142 Biocatalysis and biotransformation

Figure 1

Schematic of the basic structure of hemicellulose. A, arabinose; FeA, ferulic acid; G, galactose; Glc, glucuronic acid; X, xylose.

In this review, we focus on advances over the past several

years in the development of processes to more effectively
and efficiently convert lignocellulosic materials into ethanol. There are three major steps in the conversion process
(Figure 2): first, thermochemical pretreatment a preprocessing step that improves enzyme access to the
cellulose; second, enzymatic saccharification use of
cellulases and on some occasions hemicellulases; and
thirdly, fermentation of the released sugars by specialized
organisms.

Thermochemical pretreatment
Raw, untreated biomass is extremely recalcitrant to
enzymatic digestion. Therefore, a number of thermochemical pretreatment methods have been developed to
improve digestibility [7]. Pretreatment disrupts the
plant cell wall and improves enzymatic access to the
Figure 2

polysaccharides. Studies have shown a direct correlation

between the removal of lignin and hemicellulose and the
digestibility of cellulose [8]. Pretreatment chemistries
vary from very acidic to quite alkaline, thereby having
different effects upon the major constituents in biomass.
For example, the acidic pretreatments will hydrolyze the
hemicellulose fraction while leaving the cellulose and
lignin intact in the residual solids [911]. The most
common approaches utilize sulfuric acid, though other
strong acids have also been tried. The alkaline approaches
tend to have more of an effect on the lignin component
and leave both hemicellulose and cellulose intact [1214].
These methods require enzymes to digest both hemicellulose and cellulose, thereby increasing the enzyme
usage in the process. Pretreatment chemistry also affects
the non-sugar composition of the hydrolysate; for example, acidic pretreatments may result in high concentrations of furfurals in the liquid phase, whereas the alkaline
methods may result in high concentrations of ferulate and
acetate in the hydrolysate. These compounds will be
present in the sugar stream and may have deleterious
effects on that fermentative microorganism. Technoeconomic analyses have been performed on the majority of
pretreatments to assess cost and performance [15]. The
focus lately has been the development of low-cost reactors and processes such that pretreatment represents a
relatively small portion of the total ethanol production
costs. Several comprehensive reviews have been written
on pretreatment strategies and should be referred to
[16,17].

Enzymatic depolymerization of cellulose and

hemicellulose
Schematic of biomass and starch processing that could occur in a
biorefinery.
Current Opinion in Chemical Biology 2006, 10:141146

Cellulases comprise three types of enzymes: endoglucanases (EC 3.2.1.4), which cleave internal b-1,4-glucosidic
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Bioethanol Gray, Zhao and Emptage 143

bonds; exoglucanases (EC 3.2.1.91), which processively

act on the reducing and non-reducing ends of cellulose
chains to release short-chain cello-oligosaccharides; and
b-glucosidases (EC 3.2.1.21), which hydrolyze soluble
cellooligosaccharides (e.g. cellobiose) to glucose. Hemicellulases include enzymes that break down both b-1,4xylan (xylanases EC 3.2.1.8 and b-xylosidases EC
3.2.1.37) and various side chains (a-l-arabinofuranosidases EC 3.2.1.55, a-glucuronidases EC 3.2.1.139, acetyl
xylan esterases EC 3.1.1.72, ferulic acid esterases EC
3.1.1.73, and a-galactosidases EC 3.2.1.22). The cellulases and hemicellulases have been grouped into a
number of sequence and structurally related families
(http://afmb.cnrs-mrs.fr/cazy/). Plant cell-wall degrading
enzymes exist either in complexed or non-complexed
systems. The complexed systems are known as cellulosomes and tend to be present in anaerobic bacteria and
fungi, whereas the aerobic microorganisms produce discrete enzymes secreted into the growth media. Cellulosomes are outside the scope of this review so readers
should refer to some recent, more comprehensive articles
[18,19,20].
In the past few years, new cellulases and hemicellulases
from both bacterial and fungal sources have continued to
be isolated [21,22], and the regulation of cellulase production in microorganisms has also been extensively
investigated [23,24]. Furthermore, significant progress
has been made in the cost reduction of cellulases, particularly the non-complexed cellulases. For example, cellulases from the aerobic fungus Trichoderma reesei have
been the focus of research for 50 years and are the most
commonly used cellulases in lab and pilot-scale bioethanol production. A greater than 10-fold cost reduction for
T. reesei cellulases was recently reported [25], which
leaves the enzyme costs in the range of 10 to 20 cents
per gallon of ethanol produced. Cost reduction was
achieved by a combination of enzyme engineering and
fermentation process development.

Several mutants that were expressed in T. reesei showed

improved thermostability and reversibility.
Another approach is to introduce heterologous enzymes
into an existing system such as T. reesei so that the overall
performance of the system can be enhanced. Bower [29]
introduced several bacterial endoglucanases into T. reesei.
One of them, GH5A from Acidothermus cellulolyticus, was
fused to T. reesei cellobiohydrolase CBH1. The fusion
protein was expressed in T. reesei and found to be more
effective in the saccharification of pretreated corn stover,
needing 6 h to obtain 20% cellulose conversion, compared
with 10 h for the same conversion with the parent cellulase. In addition, Wu and co-workers [30,31] discovered a
large number of CBH I and II homologs from a variety of
fungi that may be useful in biomass hydrolysis.

Enzymesubstrate interaction
Cellulases often contain carbohydrate-binding modules
(CBMs) to facilitate the interaction between the enzyme
and the substrate surface. Similar to catalytic domains of
glycosyl hydrolases, CBMs are divided into families on
the basis of amino acid similarities and crystal structures
(http://afmb.cnrs-mrs.fr/CAZY). Subtle differences in the
structure of CBMs can lead to very different ligand
specificity [32]. Through binding to the surface of
crystalline cellulose, CBMs target their cognate catalytic
domains to specific substrates and enhance catalytic efficiency by increasing the effective concentration at the
surface. In addition to targeting, it has been proposed that
CBMs may actually be capable of disrupting the structure
of polysaccharides, thereby enhancing the rate of hydrolysis. Recently, Vaaje-Kolstad et al. [33,34] showed that
efficient chitin degradation depends upon the presence of
a small non-catalytic protein, CBP21, that binds to the
crystalline substrate. They suggested that CBP21 binding
leads to structural changes in the substrate and promotes
hydrolysis. It is possible that cellulase performance may
be improved by the assistance of similar CBMs that also
possess disruptive function.

Cellulase engineering
Several approaches have been utilized to improve cellulase performance and decrease the amount of enzyme
needed to saccharify biomass substrates. The primary
target for cellulase engineering has been the cellobiohydrolases, as they tend to constitute 6080% of natural
cellulase systems [26]. Teter et al. [27] used a combination of site-directed mutagenesis, site-saturation mutagenesis, error-prone PCR and DNA shuffling to generate
variants of T. reesei Cel7A. The mutants were expressed in
S. cerevisiae and screened for improved thermal stability
and thermal activity. One of the variants identified was
expressed in T. reesei in place of the wild-type Cel7A. The
recombinant strain outperformed the parent T. reesei in
the hydrolysis of pretreated corn stover. Day et al. [28] also
used site-directed mutagenesis to generate novel Hypocrea jecorena cellobiohydrolase Cel7A (CBH1) variants.
www.sciencedirect.com

The lignin component of the biomass material poses

challenges for enzymatic degradation because of its nonproductive binding and inactivation of cellulases. Berlin
et al. [35] proposed a novel approach to improve activity of
cellulases for lignocellulosic hydrolysis by using weak
lignin-binding enzymes. They showed that naturally
occurring cellulases with similar catalytic activity on a
model cellulosic substrate differ significantly in their
affinities for lignin, thereby affecting performance on
native substrates. Palonen [36] found that the location
and structure of lignin affects the enzymatic hydrolysis
more than the absolute amount of lignin. The study
showed that modification of lignin surfaces by oxidative
treatments with laccase alone and delignification treatment with a laccase-mediator system leads to increased
hydrolysis of lignocellulose.
Current Opinion in Chemical Biology 2006, 10:141146

144 Biocatalysis and biotransformation

Hemicellulases
The efficient degradation of hemicellulose requires the
synergistic action of many enzymes. More importantly,
hemicellulases facilitate cellulose hydrolysis by exposing
the cellulose fibers, thus making them more accessible
[21]. Commercial development for hemicellulases in
lignocellulose hydrolysis is not as advanced as cellulases
because current commercial preparations have been primarily developed on dilute-acid pretreated biomass
where hemicellulose is removed before saccharification.
However, with the development of non-acid pretreatment methods [16] where the hemicellulose fraction
remains intact, hemicellulases are required. Current cellulases such as those from T. reesei tend to have weak
hemicellulase activity and are not adequate for complete
conversion to monomer sugar. In the past several years,
continued progress has been made in understanding the
structure/function of xylanases [37,38], enzyme specificity [39,40], and hemicellulase CBMs [32,41]. Development of low-cost, commercial hemicellulases that work
synergistically with cellulases for bioethanol production is
expected to be the focus in the near future.

Hexose and pentose fermentation

Production of ethanol from sugar derived from starch and
sucrose has been commercially dominated by the yeast S.
cerevisiae [42]. However, sugar derived from biomass is a
mixture of hexoses (primarily glucose) and pentoses
(primarily xylose) and most wild-type strains of S. cerevisiae do not metabolize xylose. Researchers have basically
taken two approaches to increase fermentation yields of
ethanol derived from biomass sugars. The first approach
has been to add to yeast and other natural ethanologens
additional pentose metabolic pathways by genetic engineering. The second approach is to improve ethanol
yields by genetic engineering in microorganisms that
have the ability to ferment both hexoses and pentoses
[43,44]. Although both approaches have been nominally successful, rates and yields on mixed sugars derived
from biomass have not yet reached commercially viable
targets. In addition, in contrast to the clean sugar streams
derived from starch and sucrose, hydrolysates derived
from biomass tend to have fermentation inhibitors (acetic
acid, furfural, etc.) that either must be removed when
concentrations are too high or require the development of
robust strains that are resistant to the inhibitors.
Because the major fermentable sugars in biomass hydrolysate are glucose and xylose (with significantly less
amounts of arabinose, galactose and mannose), the initial
efforts to produce a commercially viable ethanologen
have focused on co-fermentation of glucose and xylose.
In the first approach, xylose-metabolizing genes have
been engineered into wild-type ethanologens such as
yeast and the bacterium Zymomonas mobilis [43,44].
Recombinant strains of S. cerevisiae with the ability to
co-ferment glucose and xylose have been constructed by
Current Opinion in Chemical Biology 2006, 10:141146

adding Pichia stipitis genes (XYL1, XYL2) for an

NADPH-dependent xylose reductase and a NAD+dependent xylitol dehydrogenase, and by enhancing
expression of the endogenous xylulokinase [44]. Thus,
in three steps, xylose is converted to xylulose-5-phosphate, which is a central metabolite of the endogenous
pentose phosphate pathway. While improved recombinant yeast strains have been developed to ferment biomass hydrolysates with this added xylose pathway
[44,45,46], the anaerobic cofermentation of glucose
and xylose is still below commercial requirements. Xylose
fermentation in these recombinant yeast strains is
thought to be limited by redox cofactor imbalances
caused by xylose reductase utilizing NADPH, whereas
xylitol dehydrogenase requires NAD+. Metabolic flux
analysis suggests that the redox cofactor imbalance limits
ATP production and thus growth [47,48]. The redox
imbalance has recently been addressed by either adding
an NADP+-dependent D-glyceraldehyde-3-phosphate
dehydrogenase to help regenerate NADPH [49] or by
replacing the xylose reductase with a mutant enzyme that
has a higher affinity for NADH and thus uses less
NADPH [50]. A third approach that bypasses the redox
imbalance issue is to replace the reductase and dehydrogenase with one enzyme xylose isomerase that can be
expressed in yeast [51,52,53,54]. The isomerase is
inhibited by xylitol and deletion of the gene for an aldose
reductase (gre3) is necessary to minimize xylitol production from xylulose and achieve higher yields and rates
[54]. These latest results show great progress in improving xylose fermentation with recombinant yeast and may
soon lead to strains that will produce ethanol from biomass hydrolysates at commercially viable rates, titers and
yields. Progress in the transformation of other fungal and
bacterial strains into mixed-sugar fermenting ethanologens has been adequately reviewed recently [43,44],
and is not covered here.

Conclusions
Significant progress has been made in the past several
years in all aspects of lignocellulosic conversion to ethanol. The key to the establishment of a commercial process
is a reduction in capital and operating costs of each of the
unit operations. There is a much better understanding of
the capital costs associated with pretreatment, suggesting
ways to further reduce costs without compromising performance. Similarly enzyme costs have been reduced by a
combination of protein engineering and process development. However, further cost reductions are required
and will more than likely come from novel, tailored
cocktails of enzymes with higher specific activities than
current commercial enzymes. Finally, improvements
have been made in microorganisms capable of converting
multiple sugars into ethanol. A commercial organism
must, however, be able to withstand the rigors of a
full-scale process, including potential toxic compounds
present in the sugar hydrolysate.
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Bioethanol Gray, Zhao and Emptage 145

References and recommended reading

Papers of particular interest, published within the annual period of
review, have been highlighted as:
 of special interest
 of outstanding interest
1.

Kotrba R: Keeping pace with policy. Ethanol Producer Magazine

2005, 1:58-62.

2.

Perlack RD, Wright LL, Turhollow AF, Graham RL, Stokes BJ,
Erbach DC: Biomass as feedstock for a bioenergy and
bioproducts industry: the technical feasibility of a billion-ton
annual supply. 2005, http://www.osti.gov/bridge.

3.

Richardson TH, Tan X, Frey G, Callen W, Cabell M, Lam D,

Macomber J, Short JM, Robertson DE, Miller C: A novel, high
performance enzyme for starch liquefaction. Discovery and
optimization of a low pH, thermostable alpha-amylase.
J Biol Chem 2002, 277:26501-26507.

18. Demain AL, Newcomb M, Wu JHD: Cellulase, clostridia, and


ethanol. Microbiol Mol Biol Rev 2005, 69:124-154.
A comprehensive review on a complexed cellulase system from Clostriduim thermocellum.
19. Doi RH, Kosugi A: Cellulosomes: plant-cell-wall-degrading
enzyme complexes. Nat Rev Microbiol 2004, 2:541-551.
20. Lynd LR, van Zyl WH, McBride JE, Laser M: Consolidated

bioprocessing of cellulosic biomass: an update. Curr Opin
Biotechnol 2005, 16:577-583.
Review on the recent progress on consolidated bioprocessing for
bioethanol production.
21. Shallom D, Shoham Y: Microbial hemicellulases. Curr Opin

Microbiol 2003, 6:219-228.
Comprehensive review on the recent developments of microhemicellulases, covering enzyme structures and biotechnology applications.
22. Hilden L, Johansson G: Recent developments on cellulases and
carbohydrate-binding modules with cellulose affinity.
Biotechnol Lett 2004, 26:1683-1693.

4.

Shetty JK, Lantero OJ, Dunn-Coleman N: Technological

advances in ethanol production. Int Sugar J 2005, 107: 605606,608.

23. Aro N, Pakula T, Penttilae M: Transcriptional regulation of plant

cell wall degradation by filamentous fungi. FEMS Microbiol Rev
2005, 29:719-739.

5.

Bhargava S, Frisner H, Bisgard-Frantzen H, Tams JW (Novozymes

North America IU, Novozymes A/S): Production of ethanol from
enzymatically hydrolyzed starch. 2005, patent 2005113785.

6.

Wyman CE: Potential synergies and challenges in refining

cellulosic biomass to fuels, chemicals, and power.
Biotechnol Prog 2003, 19:254-262.

24. Foreman PK, Brown D, Dankmeyer L, Dean R, Diener S,

Dunn-Coleman NS, Goedegebuur F, Houfek TD, England GJ,
Kelley AS et al.: Transcriptional regulation of biomassdegrading enzymes in the filamentous fungus Trichoderma
reesei. J Biol Chem 2003, 278:31988-31997.

7.


Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR,

Lee YY: Coordinated development of leading biomass
pretreatment technologies. Bioresour Technol 2005,
96:1959-1966.
An excellent assessment of the leading pretreatment technologies being
evaluated by a USDA-DOE funding consortium.
8.

Kim S, Holtzapple MT: Effect of structural features on

enzyme digestibility of corn stover. Bioresour Technol 2006,
97:583-591.

9.

Liu C, Wyman CE: Partial flow of compressed-hot water

through corn stover to enhance hemicellulose sugar recovery
and enzymatic digestibility of cellulose. Bioresour Technol
2005, 96:1978-1985.

10. Lloyd TA, Wyman CE: Combined sugar yields for dilute sulfuric
acid pretreatment of corn stover followed by enzymatic
hydrolysis of the remaining solids. Bioresour Technol 2005,
96:1967-1977.
11. Mosier N, Hendrickson R, Ho N, Sedlak M, Ladisch MR:
Optimization of pH controlled liquid hot water pretreatment of
corn stover. Bioresour Technol 2005, 96:1986-1993.
12. Kim TH, Lee YY: Pretreatment and fractionation of corn stover
by ammonia recycle percolation process. Bioresour Technol
2005, 96:2007-2013.
13. Teymouri F, Laureano-Perez L, Alizadeh H, Dale BE: Optimization
of the ammonia fiber explosion (AFEX) treatment parameters
for enzymatic hydrolysis of corn stover. Bioresour Technol
2005, 96:2014-2018.
14. Kim S, Holtzapple MT: Lime pretreatment and enzymatic
hydrolysis of corn stover. Bioresour Technol 2005,
96:1994-2006.
15. Eggeman T, Elander RT: Process and economic analysis

of pretreatment technologies. Bioresour Technol 2005,
96:2019-2025.
A review of the economics of each of the leading pretreatment methods.
16. Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M,
Ladisch M: Features of promising technologies for
pretreatment of lignocellulosic biomass. Bioresour Technol
2005, 96:673-686.
17. Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR,
Lee YY: Comparative sugar recovery data from laboratory
scale application of leading pretreatment technologies to corn
stover. Bioresour Technol 2005, 96:2026-2032.
www.sciencedirect.com

25. Greer D: Spinning straw into fuel. Biocycle 2005, 46:61-65.

26. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS: Microbial
cellulose utilization: fundamentals and biotechnology.
Microbiol Mol Biol Rev 2002, 66:506-577.
27. Teter S, Cherry J, Ward C, Jones A, Harris P, Yi J (Novozymes
 Biotech IU): Variants of cellobiohydrolase I from Trichoderma
reesei with improved properties. 2004, 2005048619.
An excellent demonstration of utilizing multiple strategies in engineering
T. reesei CBH1.
28. Day AG (Genencor International IU): Novel variant Hypocrea
jecorina CBH I cellulases, their production with recombinant
cells, and their uses. 2003, patent 2004016760.
29. Bower BS (Genencor International IU): Fusion proteins of an
exocellobiohydrolase and an endoglucanase for use in the
saccharification of cellulose and hemicellulose. 2005. patent
2005093073.
30. Lange L, Wu W, Aubert D, Landvik S, Schnorr KM, Clausen IG
(Novozymes A/S D): Cloning and sequences of microbial
cellobiohydrolase I for use in DNA shuffling, ethanol prodn.,
transgenic plants, and detergents. 2002, patent 2003000941.
31. Wu W, Lange L, Skovlund DA, Liu Ye (Novozymes A/S D): Cloning
and sequences of cellobiohydrolase II from thermophilic fungi
and their use for ethanol production and in detergents. 2003,
patent 2004056981.
32. Boraston AB, Bolam DN, Gilbert HJ, Davies GJ: Carbohydrate binding modules: fine-tuning polysaccharide recognition.
Biochem J 2004, 382:769-781.
An excellent review on CBMs from a structural point of view to help
understand the mechanisms by which CBMs bind to their target ligands.
33. Vaaje-Kolstad G, Houston DR, Riemen AHK, Eijsink VGH,
van Aalten DMF: Crystal structure and binding properties of
the Serratia marcescens chitin-binding protein CBP21.
J Biol Chem 2005, 280:11313-11319.
34. Vaaje-Kolstad G, Horn SJ, van Aalten DMF, Synstad B,
Eijsink VGH: The non-catalytic chitin-binding protein CBP21
from Serratia marcescens is essential for chitin degradation.
J Biol Chem 2005, 280:28492-28497.
35. Berlin A, Gilkes N, Kurabi A, Bura R, Tu M, Kilburn D,
Saddler J: Weak lignin-binding enzymes. A novel approach to
improve activity of cellulases for hydrolysis of lignocellulosics.
Appl Biochem Biotechnol 2005, 121-124:163-170.
36. Palonen H: Role of lignin in the enzymatic hydrolysis of
lignocellulose. VTT Publications 2004, 520:1-80.
Current Opinion in Chemical Biology 2006, 10:141146

146 Biocatalysis and biotransformation

37. Kaneko S, Ichinose H, Fujimoto Z, Kuno A, Yura K, Go M,

Mizuno H, Kusakabe I, Kobayashi H: Structure and function of a
family 10 b-xylanase chimera of Streptomyces olivaceoviridis
E-86 FXYN and Cellulomonas fimi cex. J Biol Chem 2004,
279:26619-26626.

47. Sonderegger M, Jeppsson M, Hahn-Haegerdal B, Sauer U:

Molecular basis for anaerobic growth of Saccharomyces
cerevisiae on xylose, investigated by global gene expression
and metabolic flux analysis. Appl Environ Microbiol 2004,
70:2307-2317.

38. Pell G, Szabo L, Charnock SJ, Xie H, Gloster TM, Davies GJ,
Gilbert HJ: Structural and biochemical analysis of Cellvibrio
japonicus xylanase 10C: how variation in substrate-binding
cleft influences the catalytic profile of family GH-10 xylanases.
J Biol Chem 2004, 279:11777-11788.

48. Sonderegger M, Jeppsson M, Larsson C, Gorwa-Grauslund MF,

Boles E, Olsson L, Spencer-Martins I, Hahn-Haegerdal B,
Sauer U: Fermentation performance of engineered and
evolved xylose-fermenting Saccharomyces cerevisiae
strains. Biotechnol Bioeng 2004, 87:90-98.

39. Pell G, Taylor EJ, Gloster TM, Turkenburg JP, Fontes CMGA,
Ferreira LMA, Nagy T, Clark SJ, Davies GJ, Gilbert HJ: The
mechanisms by which family 10 glycoside hydrolases bind
decorated substrates. J Biol Chem 2004, 279:9597-9605.

49. Verho R, Londesborough J, Penttilae M, Richard P:

Engineering redox cofactor regeneration for improved
pentose fermentation in Saccharomyces cerevisiae.
Appl Environ Microbiol 2003, 69:5892-5897.

40. Vardakou M, Flint J, Christakopoulos P, Lewis RJ, Gilbert HJ,

Murray JW: A family 10 thermoascus aurantiacus xylanase
utilizes arabinose decorations of xylan as significant
substrate specificity determinants. J Mol Biol 2005,
352:1060-1067.

50. Jeppsson M, Bengtsson O, Franke K, Lee H, Hahn-Haegerdal B,

Gorwa-Grauslund MF: The expression of a Pichia stipitis xylose
reductase mutant with higher Km for NADPH increases
ethanol production from xylose in recombinant
Saccharomyces cerevisiae. Biotechnol Bioeng 2005. DOI:
10.1002/bit.20737.

41. Levasseur A, Navarro D, Punt PJ, Belaich JP, Asther M, Record E:

Construction of engineered bifunctional enzymes and their
overproduction in Aspergillus niger for improved enzymatic
tools to degrade agricultural by-products. Appl Environ
Microbiol 2005, 71:8132-8140.
42. Lin Y, Tanaka S: Ethanol fermentation from biomass resources:
current state and prospects. Appl Microbiol Biotechnol 2005, 69:
DOI: 10.1007/s00253-005-0229-x.
43. Dien BS, Cotta MA, Jeffries TW: Bacteria engineered for fuel

ethanol production: current status. Appl Microbiol Biotechnol
2003, 63:258-266.
A good short review covering the literature through early 2003 on the three
primary recombinant bacterial strains being developed (E. coli, Klebsiella
oxytoca, and Zymomonas mobilis) for producing ethanol from mixed sugars.
44. Jeffries TW, Jin YS: Metabolic engineering for improved
 fermentation of pentoses by yeasts. Appl Microbiol Biotechnol
2004, 63:495-509.
An excellent short review covering the literature through early 2003 on the
relevant problems and approaches to making recombinant yeast strains
that ferment mixed sugars.
45. Hahn-Hagerdal B, Pamment N: Microbial pentose metabolism.
Appl Biochem Biotechnol 2004, 113-116:1207-1209.
46. Sedlak M, Ho NWY: Production of ethanol from cellulosic
biomass hydrolysates using genetically engineered
Saccharomyces yeast capable of cofermenting glucose and
xylose. Appl Biochem Biotechnol 2004, 113-116:403-416.

Current Opinion in Chemical Biology 2006, 10:141146

51. Kuyper M, Harhangi HR, Stave AK, Winkler AA, Jetten MSM,
De Laat WTAM, Den Ridder JJJ, Op den Camp HJM,
van Dijken JP, Pronk JT: High-level functional expression
of a fungal xylose isomerase: the key to efficient ethanolic
fermentation of xylose by Saccharomyces cerevisiae?
FEMS Yeast Res 2003, 4:69-78.
52. Kuyper M, Toirkens MJ, Diderich JA, Winkler AA, van Dijken JP,
Pronk JT: Evolutionary engineering of mixed-sugar utilization
by a xylose-fermenting Saccharomyces cerevisiae strain.
FEMS Yeast Res 2005, 5:925-934.
53. Kuyper M, Hartog MMP, Toirkens MJ, Almering MJH, Winkler AA,
 van Dijken JP, Pronk JT: Metabolic engineering of a xyloseisomerase-expressing Saccharomyces cerevisiae strain for
rapid anaerobic xylose fermentation. FEMS Yeast Res 2005,
5:399-409.
It appears that Delft University of Technology has now generated a
recombinant yeast strain that has sufficient rates on mixed sugars to be
worthwhile for industrial development. Their earlier approach to eliminate
much of the redox imbalance issues by finding a yeast-compatible xylose
isomerase [51] has worked well.
54. Lonn A, Traff-Bjerre KL, Cordero Otero RR, van Zyl WH,
Hahn-Hagerdal B: Xylose isomerase activity influences
xylose fermentation with recombinant Saccharomyces
cerevisiae strains expressing mutated xylA from
Thermus thermophilus. Enzyme Microb Technol 2003,
32:567-573.

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