Microbial enzyme systems for biomass conversion: Emerging paradigms

Abstract

The contemporary relevance of biofuels as an attractive replacement for liquid fossil fuels has rekindled global interest in the conversion of cellulosic biomass - the most abundant renewable source of carbon and energy on our planet. In order to achieve efficient systems for such a formidable substrate, we take guidance from the native enzyme systems of the microbes that have evolved to rid the natural environment of plant-derived wastes. These cellulolytic bacteria and fungi employ a diversity of contrasting but complementary mechanisms for the hydrolysis of cellulose and other related complex plant cell wall polysaccharides. This review covers various known microbial approaches for attacking the recalcitrance problem in the conversion of cellulosic biomass to soluble sugars en route to a biofuels-based society.

Full text

future science group 325
ISSN 1759-7269
xx.x xxx/XXX.xx.XXX © 2010 Future Science Ltd
†Author for correspo ndence
1Biosciences Center, National Renewable Energy Laborator y, Golden, CO 80401, USA
2Bioenergy Sc ience Center (BESC), Oak Ridge Nationa l Laboratory, TN 37830, USA
3Departm ent of Molecular Microbiology and Biotechn ology, Tel Aviv Universit y, Ramat Aviv 69978, Israel
4Departm ent of Biological Chemistr y, Weizmann Instit ute of Science, Rehovot 76100, Israel
Tel.: +972 8 934 2373; Fax: +972 8 946 8256; E-mail: ed.bayer@weizmann.a c.il
Biofuels derived from lignocellulose, the most abundant
source of organic material on our planet, are an attrac-
tive alternative to current petroleum-based fuels, due to
their potential for sustainability as well as reduction of
greenhouse gas emissions. Plants have evolved mecha-
nisms over millennia to protect the structural forms of
polysaccharides from which their cell walls are com-
prised. Therefore, only a small fraction of microorgan-
isms possess the ability to degrade cellulose efficiently.
Fungi and bacteria are the dominant micro organisms
responsible for lignocellulose degradation in the bio-
sphere. These microbes show significant diversity in their
surviving environments and can be found in mesophilic
as well as thermophilic ecosystems where plant matter
is abundant, such as forest and pasture soils, hot spring
pools and decaying plant debris (Table1) [1]. In these
decay communities, degradation of the plant cell wall is
accomplished by complex suites of hydrolytic enzymes
that all cellulolytic microbes secrete outside of their cell
wall. In this article, we will review the major paradigms
of microbial enzyme systems for biomass conversion.
Lignocellulosic biomass: an abundant resource
Lignocellulosic biomass can be defined as crop resi-
dues, short rotation transgenic trees (e.g., poplars),
woody grasses (e.g., switchgrass), forestry waste, con-
struction waste, waste from pulp and paper produc-
tion, agricultural residues and municipal solid waste.
A recent study by Oak Ridge National Laboratory
(ORNL) determined that 1.3 billion tons of ligno-
cellulosic biomass was theoretically available in the
USA alone [2]. Although some challenges remain for
biomass-based biofuels processes, the sheer availability
of biomass does not appear to be a limiting factor.
Rather, the high cost of conversion is recognized as the
major deterrent for commercialization. Crop residues
consist primarily of plant tissues that are composed
of various types of cell walls and these plant tissues
pose significant resistance to chemical, enzymatic and
microbial deconstruction.
Plant cell wall biomass represents the structural
forms of monosaccharides, as opposed to starch and
amylopectin, which represent the storage forms [3]. The
Biofuel s (2010) 1(2), 325–343
Microbial enzyme systems for biomass conversion:
emergingparadigms
Michael E Himmel1,2, Qi Xu1,2, Yonghua Luo1, Shi-You Ding1,2, Raphael Lamed3 & Edward A Bayer4†
The contemporary relevance of biofuels as an attractive replacement for liquid fossil fuels has rekindled
global interest in the conversion of cellulosic biomass – the most abundant renewable source of carbon
and energy on our planet. In order to achieve ecient systems for such a formidable substrate, we take
guidance from the native enzyme systems of the microbes that have evolved to rid the natural environment
of plant-derived wastes. These cellulolytic bacteria and fungi employ a diversity of contrasting but
complementary mechanisms for the hydrolysis of cellulose and other related complex plant cell wall
polysaccharides. This review covers various known microbial approaches for attacking the recalcitrance
problem in the conversion of cellulosic biomass to soluble sugars en route to a biofuels-basedsociety.
Review

Biofuels (2010) 1(2) future science group
326
Review Himmel, Xu, Luo, Ding, Lamed & Bayer
predominant polysaccharide in the cell walls of plants
is cellulose, followed by the hemicelluloses and then the
pectins. In general, the most abundant weight fraction
cell wall type in plant tissue is the secondary cell wall,
produced after the cell has stopped growing. Secondary
cell walls contain structural polysaccharides, strength-
ened further with polymeric lignin covalently cross-
linked to hemicellulose.
Table1. Representative cellulolytic microbes isolated from diverse natural ecosystems.
Bacteria Fungi
Species Source Species Source
Aerobes (free , noncomplexed cellulases)
Mesophilic bacteria Mesophilic fungi
Bacillus brevis§Termite gut Aspergillus nidulans‡,
A. niger
Soil, wood rot
Cellulomonas mi§Soil Agaricus bisporus Compost
Cellvibrio japonicus Soil Coprinus truncorum Soil, compost
Cytophaga hutchinsonii‡Soil, compost Geotrichum candidum Soil, compost
Paenibacillus polymyxa Compost Penicillium chrysogenum Soil, wood rot
Pseudomonas uorescens‡,
P. putida
Soil, sludge Phanerochaete chrysosporium Compost
Saccharophagus degradans‡Rotting marsh grass Trichocladium canadense Soil
Sorangium cellulosum Soil Hypocrea jecorinaঠSoil, rotting canvas
Thermophi lic bacteria Thermophi lic fungi
Acidothermus cellulolyticus‡Hot spring Chaetomium thermophilum Soil
Thermobida fusca‡Compost Corynascus thermophilus Mush compost
Paecilomyces thermophila Soil, compost
Thielavia terrestris Soil
Anaerobes (complexed or free , noncomplexed cellulases)
Mesophilic bacteria Mesophilic fungi
Acetivibrio cellulolyticus Sewage Neocallimastix patriciarum Rumen
Bacteroides cellulosolvens Sewage Orpinomyces joyonii Rumen
Clostridium cellulolyticum*Compost Orpinomyces PC-2 Rumen
Clostridium cellulovorans Wood fermenter Piromyces equi Rumen
Clostridium josui Compost Piromyces E2 Feces
Clostridium papyrosolvens‡Mud (freshwater)
Clostridium phytofermentans‡Soil
Fibrobacter succinogenes Rumen
Prevotella ruminicola Rumen
Ruminococcus albus‡Rumen
Ruminococcus avefaciens‡Rumen
Thermophi lic bacteria
Anaerocellum thermophilum‡Hot spring
Caldicellulosiruptor saccharolyticus Hot spring
Clostridium thermocellum‡Sewage, soil, manure
Clostridium stercorarium Compost
Thermotoga maritima‡Mud (marine)
Rhodothermus marinus Hot spring
‡Microbial str ains whose genome sequenc ing has been completed.
§Most Cellulomonas and Bacillus strains are f acultative anaerobes that can also grow anaerobically.
¶Hypocree jecorina w as originally named Trichoderma reesei.

Microbial enzyme systems for biomass conversion: emergingparadigms Review
future science group www.future -science.com 327
Plant cellulose is a linear unbranched polymeric
chain, consisting solely of b- (1,4)-linked d- glucose
residues. The single chains can reach lengths of over
10,000 glucose units. Cellulose is synthesized by cel-
lulose synthase ‘rosettes’, which contain 36 enzyme
units located in the cell membrane. Immediately after
synthesis, the cellodextrin chains are directly deposited
into the cell wall as elementary fibrils that coalesce to
form successively larger microfibrils and in some cases,
macrofibrils [4]. Plant cellulose is comprised of cellulose
Ia and cellulose Ib. Cellulose Ia is a triclinic form with
one chain per unit cell and is of higher energy compared
with the more stable monoclinic Ib form [5,6 ]. The Ia
form is more susceptible to hydrolysis, but plant cell
wall cellulose consists mainly of the stable Ib form. In
cellulose, all of the glucosyl hydroxyl groups are in the
equatorial position, whereas all of the axial positions
are occupied by nonpolar (and nonhydrogen-bonding)
aliphatic protons. Consequently, the ‘sides’ of the ele-
mentary microfibrils are polar and hydrogen bonding
and the ‘tops and bottoms’ are hydrophobic. The rela-
tively extended solution structure of cello dextrins per-
mits them to aggregate with a regular crystalline pack-
ing, matching up hydrophobic faces as well as allowing
hydrogen bond formation between chains [7]. Currently,
the detailed mechanisms of cellulase attack on these
cellulose allomorphs and their respective crystal faces
remain unknown.
In addition to cellulose, the plant cell wall matrix
contains two additional major types of cell wall poly-
saccharides; the hemicelluloses and the pectins. Unlike
cellulose, both are synthesized in the Golgi apparatus,
delivered to the cell membrane via small vesicles and
secreted into the cell wall. Hemicelluloses are gener-
ally complex, branched carbohydrate polymers that
are formed from different monomeric sugars attached
through different linkages. Carbohydrate substituents
and noncarbohydrate components occur in hemicel-
luloses on either the main chain or on the carbohydrate
branches. The complex structures of the hemicelluloses
are thought to confer a wide range of biophysical and
biomechanical properties on the plant tissues in which
they occur, as well as on products made from these
tissues. The principal pentose sugar in the major plant
cell wall hemicellulose is b-d-xylopyranose, which
has only the position 2- and 3-carbons available for
O-linked substitution by substituent sugars when
b-(1,4) linked as in xylan. Hemicelluloses also include
xyloglucan, arabinoxylan and glucomannans, which
contain other sugars including the pentose arabinose
and the aldohexoses glucose, mannose and galactose.
The hemicelluloses can also be esterified by acetyla-
tion and/or cross-linked to lignins via p-coumaroyl
and feruloyl groups.
As a complex phenolic polymer,
lignins exist widely in cell walls of
plants and some algae. There are
three types of functional groups in
lignins, including p-hydroxyphenyl,
guaiacyl and syringyl, from which
the monolignols, 4 -hydroxycin-
namyl, coniferyl and sinapyl alco-
hols, respectively, are comprised. The
complex and highly variable chemi-
cal heterogeneity of lignin is due to
the diversity of substitution patterns
and intermolecular linkages utilized
during polymerization. Although
lignins enable critical functions for
the plant, including mechanical sup-
port, water transport and defense,
lignin is also an undesirable com-
ponent in the biomass conversion
process, due to its ability to shield polysaccharides from
enzymatic hydrolysis and generally impede diffusion into
plant tissue by chemicals and enzymes. In native plant
cell walls, lignins are covalently linked to hemicellulose,
which forms a matrixing layer around the cellulose com-
prising the microfibril core that further hinders cellulo-
lytic and hemicellulolytic enzymes. Moreover, many of
the lignin degradation products are either inhibitory or
generally detrimental to the plant cell wall polysaccha-
ride-degrading enzymes. It is therefore necessary to take
this into account when designing an enzymatic process
for degradation of lignocellulosic biomass.
Dominant paradigms for plant cell walls
Bacteria and fungi that decompose plant cell wall poly-
saccharides efficiently employ a multitude of remark-
able enzymes to accomplish this particularly challenging
feat. These highly specialized, often intricate enzyme
systems include the cellulases, hemicellulases and other
related glycoside hydrolases, as well as the polysaccha-
ride lyases and the carbohydrate esterases. The selected
microbes that have evolved to occupy different lignocel-
lulolytic ecosystems utilize a surprisingly varied set of
strategies to either compete or collaborate with other
bacteria and fungi and to, thus, survive and thrive in
their environment.
 Free enzyme systems
In cellulolytic bacteria and fungi, the cellulases all
hydrolyze the same type of bond of the cellulose chain
(i.e., the b-[1,4]-glucosidic bond). They do so, however,
using different modes of action. The definitive enzymatic
degradation of cellulose to glucose is generally accom-
plished by the synergistic action of three distinct classes
of enzymes:
Key terms
Lignocellulosic biomass: Biomass
feedstock containing cellulose,
hemicelluloses, pec tins and lignin but
not starch; usually crop residues or
wood waste
Cellulases: Enzymes that hydrolyze the
b-(1,4) bond in cellulose
Hemicellulases: Enzymes that
hydrolyze any portion of the various
complex, branched polysaccharides
known as hemicelluloses, such as the
b-(1,4) bond of the xylan main chain or
any of the various linkages in the
sidechains
Glycoside hydrolases: Enzymes that
hydrolyze a glycosidic bond between
two adjacent saccharide groups or
between a carbohydrate and a
noncarbohydrate moiety

Catalytic
module
CBM
Cellulose
Biofuels © Future Science Group (2010)
Biofuels (2010) 1(2) future science group
328
Review Himmel, Xu, Luo, Ding, Lamed & Bayer
The endo-b-(1,4)-glucanases or
b-(1,4)-d-glucan-4-glucanohydro-
lases (EC 3.2.1.4), which act ran-
domly on soluble and insoluble
b-(1,4)-glucan substrates and are
commonly measured by detecting
the reducing groups released from
carboxymethylcellulose;
The exo-b-(1,4)-d- glucanases,
including both the b- (1,4)-d-glu-
can glucohydrolases (EC 3.2.1.74), which liberate
d-glucose from b-(1,4) -d -glucans and hydroly ze
d-cellobiose slowly, and b-(1,4)-d- glucan cellobiohy-
drolase (EC 3.2.1.91), which liberates d-cellobiose in
a ‘processive’ manner (successive cleavage of product)
from b-(1,4)-glucans;
The b-d-glucosidases or b-d-glucoside glucohydro-
lases (EC 3.2.1.21), which act to release d- glucose
units from cellobiose and soluble cellodextrins, as
well as an array of glycosides. The above classification
scheme is not entirely rigid and a few enzymes have
properties that do not fit one of the above definitions.
Free cellulases frequently bear a cellulose-binding
carbohydrate-binding module (CBM) that delivers the
catalytic module to the surface of its crystalline cellulosic
substrate as shown schematically in Figure1 [8]. In aero-
bic fungi, the CBM is invariably from family 1, which is
very small (~30–35 amino acid residues). The ancillary
CBMs of bacterial cellulases are often from family 2
or 3, which are much larger than their fungal analogs,
comprising approximately 100 and 150 amino acid resi-
dues, respectively. Despite the differences in size, these
types of cellulose-binding CBMs all exhibit a planar
array of aromatic side chains located on a relatively flat
surface of the CBM molecule. These planar-strip resi-
dues are generally highly conserved and are believed to
align with the hydrophobic faces of the glucose rings
along the length of a single cellodextrin lying on the
cellulose surface, thus providing the structural rationale
for substrate binding of the CBM, the parent enzyme,
the cellulosome (see below) or the entire microbial cell
(in cases where a CBM is attached to the cell surface).
The cellulases and hemicellulases belong to the glyco-
side hydrolases (GHs), a large group of enzymes, which
hydrolyze the glycosidic bond between two or more car-
bohydrates or between a carbohydrate and a noncarbohy-
drate moiety. Classification schemes have been based pre-
viously on substrate specificities of an enzyme, but this
is inappropriate for glycoside hydrolases, since the same
protein fold often harbors several types of specificities. A
more appropriate classification scheme was instituted [9],
based on the amino acid sequence and consequent fold
of the protein. The various glycoside hydrolases form
115 different families to date and membership of a given
enzyme into a known GH family provides insight into its
comparative structural features within a family, its evo-
lutionary relationships with other family members and
its mechanism of action. A compendium of the glyco-
side hydrolases and related carbohydrate-active enzymes
(CAZymes) can be found on the CAZy Website [201]. In
addition to the GHs, the polysaccharide lyases are clas-
sified into 21 families and carbohydrate esterases into
16. The CBMs are also divided into families, currently
numbering 59 on the CAZy database.
Structurally, the topology of the active sites differs
between the endoglucanases and exoglucanases. The
active sites of endoglucanases typically attain a cleft-like
topology. Thus, a cellulose chain can be accessed in
random fashion by an endoglucanase and bond cleavage
can occur anywhere along the chain of the substrate. By
contrast, the active site of the exoglucanases resembles
a tunnel, formed by long loops of the protein molecule
that fold over the active site residues [10]. Consequently,
a single glycan chain is fed into one end of the tunnel-
like active site, followed by subsequent bond cleavage
inside the tunnel and release of cellobiose product from
the other end [11,12]. Since the chain is still fixed within
the active site tunnel, successive cleavage events can
continue processively in a unidirectional manner along
Figure1. Interaction of free cellulase system with cellulosic substrates.
The CBM of each enzyme delivers the catalytic module to the cellulosic
substrate and the various individual enzymes act in a synergistic manner
to degrade plant cell wall polysaccharides. Some CBMs are specic
for noncrystalline or hemicellulose portions of the plant cell wall and
their parent enzymes (and catalytic module) are directed towards the
appropriate substrate (not shown in the gure).
CBM: Cellulose-binding module.
Key terms
Endoglucanases: Enzymes that
hydrolyze the bonds in cellulose via
random cleavage at internal sites of the
cellulose chain
Exoglucanases: ‘Processive’ enzymes
that hydrolyze the bonds in cellulose via
sequential cleavage from either the
reducing or nonreducing free
chainends

Cellulose
Enzymatic subunits
Scaffoldin subunit
Cohesin
Dockerin
X
CBM
X
Biofuels © Future Science Group (2010)
Microbial enzyme systems for biomass conversion: emergingparadigms Review
future science group www.future -science.com 329
the glucan chain [13,14]. It seems, however, that some
differences in this mechanism may occur among the
different types of exoglucanases [15], perhaps reflecting
the length of the tunnel, the directionality of action
(from nonreducing to reducing end or vice versa) and
flexibility of the loops that form the tunnel.
Selected endoglucanases can also exhibit a distinc-
tive type of processive action on the substrate [16]. For
example, certain types of GH9 enzymes contain a sub-
family of CBM3, termed CBM3c, which is fused to the
catalytic module; the two modules are connected tightly
via a characteristic linker segment. The GH9-CBM3c
couple work in concert, such that the CBM3c serves
to feed a single cellulose chain into the active site cleft
of the endoglucanase, modifying its character from a
simple endoglucanase, which acts randomly along the
substrate, to one that acts successively along its chain.
In contrast to microbial degradation of cellulose, bac-
teria and fungi produce many different types of enzymes
that act very efficiently on the various types of hemicel-
lulose. The very complex structure of hemicelluloses,
however, requires a very large number of enzymes for
complete degradation. Hemicellulases can be placed
into three general categories:
The endo-acting enzymes attack polysaccharide
chains internally;
The exo-acting enzymes tend to act processively from
either the reducing or nonreducing termini;
The so-called ‘accessory’ enzymes required to
hydrolyze hemicellulose in native plant tissue.
The endo-acting hemicellulases exhibit very little
activity on short oligomers (i.e., degree of polymerization
[DP] less than 3). In contrast, some exo-acting enzymes
have preferences for short chain substrates (DP 2–4)
although others prefer larger substrates (DP >4). The
accessory enzymes include a variety of acetyl esterases
and esterases that hydrolyze lignin-linked glycoside
bonds, such as coumaric acid esterase and feruloyl acid
esterase [17]. The complexity of hemicellulose struc-
tures requires a high degree of coordination between
the enzymes involved in hemicellulose degradation.
Synergism in enzyme action is defined as a mixture
of enzymes that produce more end product than each
could produce separately. In the cellulase system, for
example, synergism can be easily shown for native and
artificial combinations of endo- and exo-glucanases [18].
As may be expected for an analogous, yet more complex
series of biopolymers, synergism has been demonstrated
between b-xylanases and acetylxylan esterase [19], a-l-
arabinofuranosidase [20] and b-glucuronidase [21]. Most
enzymes have very specific requirements for tight sub-
strate binding and precise transition state formation,
which usually leads to high catalytic turnover rates.
Cellulosomes
In general, the multienzyme cellulosome complex is
composed of two major types of subunit: the noncata-
lytic scaffoldin(s) and the enzymes (Figure 2) [22–24] .
The assembly of the enzymatic subunits into the cel-
lulosome complex is facilitated by the high-affinity
recognition between cohesin modules of the scaf-
foldin subunit and enzyme-borne dockerin modules.
Scaffoldins usually contain multiple cohesin modules,
thereby enabling numerous different enzymes to be
assembled into the cellulosome complex (Table 2). In
addition, a multiplicity of scaffoldins has been found
in some species, which lends a higher level of complex-
ity to cellulosome assembly. Theoretically, over 70 dif-
ferent dockerin-containing components can be assem-
bled into the cellulosome of Clostridium thermocellum
Figure2. Interaction of the cellulosome with cellulosic substrates.
Cellulosomal enzymes are integrated into the complex via the potent
protein–protein interaction between the single dockerin of each enzymatic
subunit and multiple cohesin modules on the scaoldin subunit. The
cohesins are separated on the scaoldin polypeptide by distinctive linker
segments. The entire cellulosome complex is delivered to the cellulosic
substrate through the action of the lone CBM of the scaoldin subunit.
Scaoldins can also carry one or more ‘X’ modules of unknown function.
Some dockerin-bearing cellulosomal enzymes also contain CBMs that
are selective for noncrystalline or hemicellulosic portions of the plant cell
wall and would therefore be directed towards these substrates during the
degradation process while tethered to the exible scaoldin subunit (not
shown in the gure).
CBM: Cellulose binding module.

Biofuels (2010) 1(2) future science group
330
Review Himmel, Xu, Luo, Ding, Lamed & Bayer
[25,2 6]. Since the scaffoldin subunit in this bacterium
contains only nine cohesin modules, the varied collec-
tion of individual cellulosomes is immensely hetero-
geneous. Another important scaffoldin-borne compo-
nent is the cellulose-specific CBM, which functions
as the major binding factor for specific recognition
of cellulosic substrates. The CBM of the scaffoldin
serves to deliver the entire complement of cellulosome
enzymes collectively to the lignocellulosic substrate,
thus fulfilling another important requirement for
efficient degradation.
In many aspects, cellulosomal enzymes are very
similar to their free counterparts, except their catalytic
modules are attached to a dockerin rather than a CBM.
The scaffoldin-based CBM serves
as a single cellulose-targeting agent
for all cellulosomal components.
Members of the same families of
cellulases and hemicellulases that
are involved in the free enzyme
systems also serve as cellulosomal
enzymes – with some exceptions. In
this context, the GH7 and GH45
cellulases that occur exclusively in
fungi never appear in the cellulo-
somal context. Intriguingly, how-
ever, GH6 enzymes that occur both
in fungi as well as some bacteria,
have not been found in native cellu-
losome systems. With so much hori-
zontal transfer of GH genes among
the microbes in a given ecosystem,
it is fascinating why members of
selected cellulase families would
be excluded from cellulosomal sys-
tems. Compared with free enzyme
systems, the cellulosome brings the
catalytic modules into close physi-
cal association with each other and,
collectively, to the cellulose surface,
thereby promoting their essential
synergistic action by concentrating
the enzymes with complementary
functions at defined sites on the lig-
nocellulosic substrate. Nevertheless,
enzyme proximity within a complex
could theoretically reduce synergism
as a consequence of conformational
restrictions imposed by the intricate
quaternary structure of the cellulo-
some. This potential obstacle, how-
ever, is countered by the plasticity
(or flexibility) of the cellulosome
that provides an important clue to
its functionality. Plasticity of the quaternary structure of
the cellulosome is thus a major functional rationale for
the observed synergy among its enzymatic subunits. The
following properties of native cellulosomal components
contribute to the observed plasticity of the complex:
The scaffoldin(s) of the cellulosome contains inter-
modular linkers of various lengths, but usually very
long [27];
The linker between the catalytic module and dock-
erin of the parent cellulase provides fine-tuned posi-
tioning of the catalytic module on the substrate [28] ;
The type I dockerin of the C. thermocellum cellulos-
omal cellulases can bind to the corresponding scaffol-
din-borne cohesins in two alternative modes, thereby
generating two opposing orientations for the associated
catalytic module on the cohesin–dockerin interface [29].
The purported conformational ‘flip-flop’ thus gener-
ated by this dual mode of binding provides significant
plasticity by further modulating the orientation of the
catalytic subunits within the supramolecular cellulosome
Key term
Cellulosomes: Discrete high-molecular
weight enzyme complexes secreted
from anaerobic bacteria, which contain
considerable diversity in content of
glycoside hydrolases and other related
plant cell wall-degrading enzymes and
subcomponents
Table2. Diversity of scaoldins derived from various cellulosome-producing bacteria.
Species Type of
scaoldin
composition
Protein Modular architecture Mol.
Wt.
(kDa)
Clostridium
cellulovorans
Single CbpA CBM3-X-Coh1-Coh2-X-Coh3-Coh4-Coh5-Coh6-
Coh7-Coh8-X-X-Coh9 186
Clostridium
cellulolyticum
Single CipC CBM3-X-Coh1-Coh2-Coh3-Coh4-Coh5-Coh6-
Coh7-X-Coh8
155
Clostridium josui Single CipJ CBM3-X-Coh1-Coh2-Coh3-Coh4-Coh5-Coh6 117
Clostridium
acetobulyticum
Single CipA CBM3-X-X-Coh1-X-Coh2-X-Coh3-X-Coh4-X-
Coh5
152
Bacteroides
cellulosolvens
Multiple (1) ScaA
(2) ScaB
(1) Coh1- Coh2-Coh3-Coh4 -Coh5-CBM3-Coh6-
Coh7-Coh8-Coh9-Coh10-Coh11-Doc
(2) Coh1-Coh2-Coh3-Coh4-Coh5-Coh6-Coh7-
Coh8-Coh9-Coh10-X-SLH
(1) 242
(2) 240
Clostridium
thermocellum
Multiple (1) CipA
(2) OlpB
(3) Orf2p
(4) SdbA
(1) Coh1-Coh2-CBM3-Coh3-Coh4-Coh5-Coh6-
Coh7-Coh8-Coh9-XDoc
(2) Coh1-Coh2-Coh3-Coh4-Coh5-Coh6-Coh7-SLH
(3) Coh1-Coh2-SLH
(4) Coh-X-SLH
(1) 194
(2) 245
(3) 245
(4) 66
Acetivibrio
cellulolyticus
Multiple (1) ScaA
(2) ScaB
(3) ScaC
(4) ScaD
(1) GH9-Coh1-Coh2-Coh3-CBM3-Coh4-Coh5-
Coh6-Coh7-XDoc
(2) Coh1-Coh2-Coh3-Coh4-Doc
(3) Coh1-Coh2-Coh3-SLH
(4) Coh1-Coh2-Coh3-SLH
(1) 197
(2) 97
(3) 124
(4) 89
Ruminococcus
avefaciens
Multiple
(1) ScaA
(2) ScaB
(3) ScaC
(4) ScaE
(1) X-Coh1-Coh2-Coh3-Doc
(2) Coh1-Coh2-Coh3-Coh4-Coh5-Coh6-Coh7-
X-Doc
(3) Coh-Doc
(4) Coh-Ssm
(1) 90
(2) 178
(3) 26
(4) 28
CBM3: Cellulose- binding family-3; Coh: Cohesin; Doc: D ockerin; GH9: Family-9 cellulase; SLH: S-layer homology m odule; Ssm:
Sortase -signal motif (SPKTG); X: Module o f unknown function; XDo c: Dyad of X-module and doc kerin.

Microbial enzyme systems for biomass conversion: emergingparadigms Review
future science group www.future -science.com 331
assembly, relative to the cellulosic substrate, as the cellu-
lolytic process proceeds [30,31]. This dual mode of binding
that characterizes integration of the enzymes into the
complex is contrasted by the apparent single mode of
cohesin-dockerin binding displayed by the attachment
of the scaffoldin subunit to the cell surface [32].
Conventional sequencing of genes identified in vari-
ous bacteria, together with associated bioinformatics and
biochemical analyses, has provided novel information
regarding the components and architecture of cellulo-
some systems from different anaerobic cellulolytic bacte-
ria [33]. Following the initial sequencing and description
of the cellulosomal scaffoldins in Clostridium thermocel-
lum and Clostridium cellulovorans [34,35], other clostridial
scaffoldins have been sequenced [36,37]. These, in turn,
were followed by the sequencing of
various other scaffoldins from non-
clostridial species [38–43]. A list of the
diverse nature of known scaffoldins
is provided in Table2.
Recent genome sequencing proj-
ects of several true cellulosome-
producing bacte ria , includ ing
Clostridium thermocellum [2 02] ,
Clostridium acetobutylicum [203],
Clostridium cellulolyticum [20 4] ,
Clostridium papyrosolvens [2 05] ,
Ruminococcus albus [44] and Rumino-
coccus flavefaciens [45], have further
enriched our knowledge of the bac-
terial cellulosomes and the informa-
tion achieved has allowed us to grasp
their sheer complexity. In addition
to the intricate scaffoldins, these
genomes encode large numbers of
dockerin-containing proteins (from
several dozen in some cellulosome-
producing bacteria to over 200 in
R. flavefaciens, ranging in size from
~40 to ~170 kD) – much larger
than what a single scaffoldin can
accommodate. The cellulosomal
enzymes include numerous cel-
lulases from divergent glycoside
hydrolase families, but the majority
of the enzymes are noncellulolytic
and specialize in the degradation
of pectin and hemicelluloses. Each
genome also reveals the presence
of dockerin modules attached to
proteins that are not carbohydrate-
acting enzymes (including putative
proteases and protease inhibitors) as
well as dockerin-containing proteins
of currently unknown function.
Polysaccharide-cleaving enzymes (glycoside hydrolases
and polysaccharide lyases) and accessory carbohydrate
esterases are classified in a large number of families
based on amino acid sequence similarities [9], accessible
online via the carbohydrate-active enzyme (CAZy) data-
base [201]. Preliminary examination of the occurrence of
members of these families in cellulosomes shows that in
each of the fully sequenced (public) genomes mentioned
above [46,47], there is only one cellulosomal GH48 cello-
biohydrolase, in sharp contrast to the occurrence of much
larger numbers of GH9 endoglucanases. In C. thermocel-
lum, the dockerin-containing GH48 enzyme is believed
to be a major and decisive component of the cellulosome,
when grown on microcrystalline cellulose [48,49]; growth
Table3. Selected bacterial multifunctional enzymes .
Modular structure‡Mode (f,c)§Accession
code
Source
Cellulase– cellulase
GH9-CBM3-CBM3-CBM3-GH48 fCAB06786 Anaerocellum thermophilum
GH5-CBM10-GH6 fABS72374 Teredinibacter turnerae
T7902
GH6-CBM3-GH12-CBM2 fABK52388 Acidothermus cellulolyticus
11B
Cellulase–hemicellulase
GH10-CBM3-CBM3-GH48 fACM60945 Anaerocellum thermophilum
DSM 6725
CBM30-Ig-GH9-GH44-Doc-CBM44 cBAA12070 Clostridium thermocellum
Hemicellulase–hemicellulase
CBM22-CBM22-GH10-CBM3-CBM3-CBM3-
GH43-CBM6
fAAB95326 Caldicellulosiruptor sp.
Rt69B.1
GH5-CBM3-CBM3-GH44 fABP66691. Caldicellulosiruptor
saccharolyticus
GH43-CBM6-CBM2-CBM22-GH10 fABD82867 Saccharophagus degradans
2–40
GH44-Fn3-GH26-CBM3 fABC88431 Paenibacillus polymyxa
GH30-GH54-GH43-Doc cZP_00510825 Clostridium thermocellum
GH11-CBM22-GH10-Doc-GH11 cORF00468 Ruminococcus avefaciens
UNK-CBM22-GH10-CBM22-Doc-GH43-
CBM6
cORF03865 Ruminococcus avefaciens
Hemicellulase–carbohydrate esterase
CE1-CBM6-Doc-GH10 cABN53181 Clostridium thermocellum
GH5-Doc-CE2 cAAA23224 Clostridium thermocellum
GH11-CBM6-Doc-CE4 cAAC04579 Clostridium thermocellum
GH11-CBM22-GH10-Doc-CBM22-CE4 cORF01222 Ruminococcus avefaciens
GH11-CBM22-GH10-Doc-GH11-CE4 cORF03896 Ruminococcus avefaciens
GH43-CBM6-CBM22-Doc-CE1 cORF00764 Ruminococcus avefaciens
GH53-CE3-Doc cORF01739 Ruminococcus avefaciens
‡ Family numbers follow the GH, CE and CBM abbrev iations
§ f: Free (dockerin- containing) enzyme; c: Ce llulosomal (dockerin-conta ining) enzyme.
CBM: Cellulose-binding modul e; CE: Carbohydrate esterase; Doc: Dockerin; GH: Glycoside hydrolase; Fn3: Fibron ectin 3
domain; Ig: Immuno globulin-like domain; UNK : Unknown domain/module

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332
Review Himmel, Xu, Luo, Ding, Lamed & Bayer
of the bacterium on cellobiose results in reduced amounts
of this component in the cellulosome [26,50] . Moreover,
this bacterium also produces a CBM-containing noncel-
lulosomal GH48 [51], the only microorganism known to
produce two of these intriguing exoglucanases. The GH9
enzymes include several different modular themes and
consequent alterations in activity patterns [52]. The versa-
tility in the repertoire of the cellulosomal GH9 enzymes
may be a necessary and advantageous adaptation of cel-
lulosomes [53,54]. In addition to the latter cellulosomal
enzymes, additional components, both enzymatic and
structural, seem to be affected by the carbon source
on which the bacterium is grown [43, 48,50,55–58]. More
recently, genome sequencing combined with metabolic
profiling have further revealed in much greater detail the
striking dependence of growth substrate on cellulosome
content [26,45,59].
The concept of producing artificial multienzyme
complexes based on recombinant DNA technology
was initiated long ago [60] . After a lengthy ‘learning
process’, prototype ‘designer cellulosomes’ were indeed
reported [61–63], whereby chimeric cohesin-containing
scaffoldins and complementary dockerin-containing
enzymes could be produced. Enhanced synergistic
activities of exoglucanases and endoglucanases, selec-
tively incorporated into discrete designer cellulosomes,
as well as cellulase–hemicellulase complexes, served to
demonstrate unambiguously both the CBM-mediated
targeting effect and the enzyme-proximity effect for
the first time.
In subsequent studies, mixed fungal and bacterial
enzymes were shown to be appropriate in the cellulo-
some mode [64], as well as the incorporation of distinctly
foreign enzymes (e.g., noncellulosomal enzymes, includ-
ing GH6 cellulases) into designer cellulosomes [64–67].
Intriguingly, a GH6 endoglucanase was recently shown
to perform well in designer cellulosomes, whereas a
GH6 exoglucanase exhibited measurable but markedly
reduced activities in the cellulosome mode [68]. A partic-
ularly ingenious report [69] demonstrated extraordinary
flexibility in the possible ‘geometries’ of designer cellu-
losomes, indicating the utility of their modular compo-
nents (e.g., cohesins, dockerins and CBMs) as building
blocks for future synthetic cellulosomes.
It is hoped that this synthetic biology approach may
someday alleviate the problem of limited production
capacity inherent in the anaerobic setting, since the rather
few cellulosome-producing organisms thus far identified
all appear to be strict anaerobes. In this context, designer
cellulosomal components can be produced independently
in a prolific aerobic host cell system. Alternatively, the
genes encoding these components can be introduced
directly into host bacteria [70–72], fungi [73] or yeasts [74,75],
thereby providing novel cellulolytic microbes for efficient
degradation of cellulosic biomass.
Multifunctional enzyme systems
Enzymes that are composed of two or more catalytic
modules related to the degradation of plant cell walls are
termed ‘multifunctional enzymes’. The significant feature
of their modular architectures is that they are composed
of more than one catalytic module and distinct CBM(s)
and these enzymes are usually of very high molecular
weight (Figure3). The presence of two different enzymes
in the same polypeptide chain would seem to indicate that
the forced proximity of the designated catalytic modules
necessitates concerted action on a given portion of the
lignocellulosic substrate. Naturally occurring multifunc-
tional enzymes exist in both free enzyme systems and
cellulosomal systems. Some multi functional enzymes and
their modular architectures are listed in Table3. Based on
their primary catalytic modules, multifunctional enzymes
can be classified into four types as described below.
Cellulase–cellulase systems
This type of multifunctional enzyme may include two
or more cellulases, such as the catalytic GH5, GH6,
GH9, GH48 and other ancillary modules or domains,
such as CBMs, X modules, fibronectin domains and
Ig-like domains, in their molecular architecture (Table3).
These very large enzymes should, in theory, be capable
of hydrolyzing microcrystalline cellulose, since some of
them contain both an endoglucanase and an exogluca-
nase module in the same polypeptide chain, along with
one or more CBMs. Such enzymes would presumably
act with enhanced synergy, since their proximity to one
another would allow the newly formed free chain ends in
the midst of the cellulose chain, created by the endoglu-
canases, to be exposed to processive action by the exoglu-
canase. Interestingly, a multifunctional cellulase, CelA,
of the hypothermophile, Anaerocellum thermophilum [76]
(now renamed Caldicellulosiruptor bescii), carries three
consecutive CBM3s, which raises the question of why so
many? One of these is a CBM3c fused to the neighboring
GH9, which would seemingly modify its activity from a
simple to a processive endoglucanase (as described in an
earlier section). The presence of the other two, presumably
conventional cellulose-binding CBM3s, may be linked to
the very high temperatures (75–80°C) of the natural envi-
ronment in which the bacterium thrives; the CBM3 dyad
may be necessary to ensure tight binding to the substrate
under such extreme conditions. Interestingly, this bacte-
rium is reportedly capable of efficiently degrading various
types of lignocellulosic biomass without a pretreatment
step [7 7], although additional research will be required to
corroborate this claim.
Hemicellulase–hemicellulase systems

Cellulose
22 22
10
3 3 3
43
6
Hemicellulose 1
Hemicellulose 2
Catalytic modules
CBMs CBM
CBMs
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Microbial enzyme systems for biomass conversion: emergingparadigms Review
future science group www.future -science.com 333
These multifunctional enzymes are
comprised primarily of two or more
modules of hemicellulases as well as
CBMs related to the binding to vari-
ous hemicelluloses. These hemicel-
lulases include GH10, GH26, GH43
and GH54, whereas the CBMs con-
sist primarily of hemicellulose-bind-
ing CBMs, such as CBM6, CBM22
and CBM30 (Table3). CBMs related
to the binding of hemicelluloses usu-
ally bind to single polysaccharide
chains and may serve to direct one
or both of the associated enzymes to
relevant portions of the substrate,
either before or during the process of
degradation, as newly exposed sites
on the substrate become accessible.
The structures of their binding sites
are extended clefts of various depths
in which aromatic residues, appro-
priately oriented, recognize and bind
to ligands [78,79]. It is interesting that
distinctive cellulose-binding CBMs
have also been found in these mul-
tifunctional enzymes. For example,
GH26 usually exhibits mannanase
activity, GH10 is one of the major
xylanase families and GH43 and GH44 have xylosi-
dase and xyloglucan activity, respectively. Nevertheless,
multifunctional enzymes, which contain these catalytic
modules also include CBM3s that are thus far associated
almost exclusively with the property of cellulose binding.
Again, multifunctional hemicellulase–hemicellulase
enzymes of the hyperthemophile, Caldicellulosiruptor
saccharolyticus, a close relative of A. thermophilum [76],
carry a CBM3 dyad, presumably owing to the very high
temperatures at which the bacterium thrives.
Cellulase–hemicellulase systems
Mixtures of cellulase catalytic modules (GH9 and
GH48) and hemicellulase catalytic modules (GH10
and GH44) have also been found as multifunctional
enzymes. CBMs with the ability to bind to cellulose
(CBM3) and hemicellulose (CBM30), as well as other
modules, such as Fn3 and Ig-like, were also identified
in these enzymes (Table3). One would be tempted to
speculate that such mixed types of multienzyme systems
would infer that the enzymes act at areas of the substrate
interface where cellulose and certain hemicelluloses
meet within the plant cell wall.
Hemicellulase–carbohydrate esterase systems
These complex enzymes consist of hemicellulase
catalytic modules (GH5, GH10, GH11, GH43 and
GH53) and carbohydrate esterase modules (CE1, CE2,
CE3 and CE4), as well as selected CBMs (CBM3, 6 and
22) (Table3) . Members of CE1 through C4 are known
to exhibit acetyl xylan esterase activity (CE2 and CE3
thus far show such activity exclusively). The utility of
coupling a xylanase with a xylan esterase is clear, since
the two would simultaneously sever the acetyl substitu-
ent and hydrolyze the main-chain glycosidic bond, thus
effecting a particularly efficient cleavage at this common
type of site on the xylan polymer. Even more remark-
able is the combination of a xylanase and CE1 feru-
loyl esterase on the same polypeptide chain, as in one
of the C. thermocellum cellulosomal enzymes. In this
case, effective cleavage of the xylan main chain together
with severing of the xylan–lignin junction would be
especially beneficial to the parent bacterium.
Biochemical signicance
The biochemical characteristics of some multifunctional
glycoside hydrolases have been examined in detail.
Some of these modules displayed enzymatic activities
on various substrates, including Avicel, xylan, man-
nan, lichenin, chitin and carboxymethylcellulose [80,81].
Remarkably, as mentioned earlier, an especially large cel-
lulase gene, coding CelA with the modular architecture
Figure3. Interaction of multifunctional enzyme systems with the plant cell wall. The
enzyme shown schematically in the gure is based on xylanase C from the hyperthermophile,
Caldicellulosiruptor sp. Rt69B.1 [12 6]. In this case, the enzyme is attached to the crystalline cellulosic
substrate by the strong interaction of adjacent family-3 CBMs and the catalytic modules are
then directed secondarily to two dierent hemicellulosic portions of the plant cell wall by
dierent types of hemicellulose-specic CBMs (from families 6 and 22, respectively, as depicted
in the gure).
CBM: Cellulose binding module.

Biofuels (2010) 1(2) future science group
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Review Himmel, Xu, Luo, Ding, Lamed & Bayer
GH9-CBM3-CBM3-CBM3-GH48, was isolated from
the extremely thermophilic, cellulolytic bacterium A.
thermophilum. The GH9 module at its N-terminus
exhibited endoglucanase activity; however, removal
of the C-terminal GH48 module led to a significant
reduction of the activity of the truncated CelA towards
Avicel. The enhanced Avicelase activity of the intact
CelA presumably resulted from intramolecular syner-
gism between the GH9 endoglucanase and the GH48
exoglucanase [82]. This work provided direct experimen-
tal evidence to support the concept that intramolecular
synergy of various catalytic modules can be obtained in
a single multifunctional enzyme.
The formation of multifunctional enzymes could be
regarded as the naturally occurring fusion of various
catalytic and other modules to perform their respective
functions in the hydrolysis of plant cell walls. Similar
to the designer cellulosomes described above, we can
adopt this ‘natural’ strategy to design and construct
artificial multifunctional enzymes (chimeras) based
on the structures and functions of the modular com-
ponents, according to the requirement of a particular
substrate configuration, in order to
attain optimal levels of degrada-
tion. Compared with the mixture
of individual enzymes, the chimeras
possess the following advantages:
Efficient degradation due to
intramolecular synergy;
Reduced requirement for protein
purification;
Simplified protein immobilization;
Easier optimization of physical
characterization, including pH and
tolerance to higher temperature.
Like designer cellulosomes, some
multifunctiona l enzyme chime-
ras have been created successfully
and have shown promising func-
tions [83 –85]. In this context, a fusion
protein of a xylanase (Xyln) and
arabinofuranosidase (Ara) was con-
structed via a flexible linker peptide.
Compared with the Xyln–Ara free
enzyme mixture, the Xyln–Ara chi-
mera yielded 30% higher activity on
wheat arabinoxylan. This result sup-
ports the concept of intramolecular
synergy of these modules and dem-
onstrates the feasibility of generating
effective multifunctional enzymes for
the improvement of xylan bioconver-
sion [86]. Therefore, the creation of artificial, multifunc-
tional, lignocellulosic hydrolases is a realistic and practical
approach for the improvement of biomass conversion.
A key consideration in artificial construction of mul-
tifunctional enzymes is the preservation or improve-
ment of the protein and enzymatic characteristics of
the individual components. It is a challenge to create
a chimera that possesses optimal enzyme functions for
one or more parental enzymes. The following factors
may need to be considered for this purpose:
The order of the connected enzymatic modules;
The composition of the CBMs;
The types and lengths of intermodular linker peptides.
Cell-anchored enzyme systems
Free enzyme systems of aerobic microorganisms are
characterized by enzymes, which are usually secreted
directly into the extracellular milieu in relatively large
quantities and with essentially no residence time on
the microbial cell surface. In contrast, the cellulo-
some resides on the cell surface and is released into the
Table4. Cell-surface plant cell wall-degrading enzymes from dierent bacteria.
Enzyme Enzyme activity Mode of cell
attachment
Bacterium Ref. or
source
XynX Xylanase SLH Clostridium thermocellum M67438.1,
GI:144775
LicA Lichenase SLH C. thermocellum X89732.2,
GI:20152505
AapT a-amylase-pullulanase SLH B acillus sp. D28467.1,
GI:460686
AlkA Cellulase SLH Bacillus sp. M27420.1,
GI:142664
XynB Xylanase SLH Caldicellulosiruptor sp. AF036923.1,
GI:2760904
Man26A Mannanase SLH Cellulomonas mi AF126471.1,
GI:5359709
EgA Endoglucanase SLH Clostridium josui D85526.2,
GI:109715768
ManA Mannanase SLH Anaerocellum thermphilum AF126471.1,
GI:5359709
PglA Polygalacturonase SLH Thermoanaerobacterium
thermosulfurigenes
U50951.1,
GI:1542972
GH10 Xylanase Ssm Ruminococcus avefaciens ORF01899
CsxA Exo-b-
d-
glucosaminidase CBM35 Amycolatopsis orientalis [125]
Cel48A Cellobiohydrolase CBM37 Ruminococcus albus [97]
Cel9B Endoglucanase CBM37 R. albus [97]
Cel9C Endoglucanase CBM37 R . albus [98]
Cel5G Endoglucanase CBM37 R. albus [98]
Xyn11C Xylanase CBM37 R. albus [98]
CBM35, CBM37, CBMs from familie s 35 a nd 37, respectively
CBM: Cellulose binding module; SLH: S- layer homology module; Ssm: So rtase-signal motif (LPXTG)

Microbial enzyme systems for biomass conversion: emergingparadigms Review
future science group www.future -science.com 335
extracellular medium only at later stages in the growth
cycle. Consequently, the cellulosome is essentially a cell-
surface entity. Indeed, as described above, the presence
of a tenacious, cellulose-binding CBM on the scaffoldin
subunit also serves to mediate binding of the entire bac-
terial cell to its cellulosic substrates prior to their enzy-
matic hydrolysis. In fact, the presence of this CBM and
the fact that the cellulosome is attached to the cell are
the major reasons that led to the initial discovery of the
cellulosome [87–89] .
Both cellulosomal and noncellulosomal enzymes
can be attached to the cell surface, using several pos-
sible mechanisms (Figure4). In C. thermocellum, the
cellulosome is attached to the bacterial cell surface via
a special class of anchoring proteins (Figure4B), which
can be defined as scaffoldins by virtue of a second,
divergent type of cohesin that they possess in various
copies (1 to 7 in C. thermocellum) at their N-terminus.
At their C-terminus, these anchoring scaffoldins con-
tain an S-layer homology (SLH) module that mediates
attachment to the bacterial cell surface (Table2) [90–95] .
Interestingly, at least two C. thermocellum enzymes,
a xylanase and a lichenase, also contain a C-terminal
SLH module, indicating their individual attachment to
the cell surface (Table4 & Figure4A). Likewise, SLH mod-
ules are components of glycoside hydrolases from several
other plant cell wall-degrading bacteria. In R. flavefa-
ciens, however, the cellulosome is attached covalently
to the cell surface via the ScaE scaffoldin [43], which
contains a sortase signal motif at its C-terminus (Table2
& Figure4C) . Genome sequencing of this bacterium has
revealed several other putative structural proteins that
include a similar sortase signal motif, indicating that
the host proteins are potential cell surface components.
At least one of these proteins includes a module whose
sequence is consistent with a GH10 xylanase.
More recently, an alternative mechanism of attach-
ment of plant cell wall-degrading enzymes to the
surfaces of their parent bacterial cells has been docu-
mented. In this context, numerous CBMs were discov-
ered recently in the rumen bacterium, R. albus. This
novel type of CBM, eventually classified as family
37, is found exclusively in this bacterium. Indeed, R.
albus remains an intriguing case. In the draft genome
sequence of R. albus strain 8 (~90% coverage), approxi-
mately 40 proteins contain at least one CBM37 mod-
ule and half of these proteins are classified as putative
carbohydrate-acting enzymes (glycoside hydrolases,
pectate lyases and carbohydrate esterases) [44]. Likewise,
a similar number of dockerin-containing proteins have
been detected. However, to date, no cohesin module
has been sequenced or unambiguously identified in
R. albus. This would either indicate that there may be
cohesin sequences in the residual (~10%) unsequenced
portion of the genome or that there are, in fact, no
cohesins in this strain and hence no scaffoldins. If the
latter case is true, then why would the bacterium pro-
duce dockerin-containing proteins? The answer may
lie in the nature of the rumen environment in which
this bacterium inhabits. We have learned recently that
the related rumen bacterium, R. flavefaciens, populates
the bovine rumen in multiple strains, which may each
produce different types of cellulosomal components [96].
If R. albus also follows this pattern, then the possibility
exists that this particular strain (strain 8) may produce
and secrete various dockerin-bearing proteins, while
another (or other) strain(s) may produce complemen-
tar y cohesin-containing scaffoldins. Future genome
sequencing of other R. albus strains should provide
insights into whether this bacterium can be considered
a true cellulosome-producing microorganism.
The CBM37s were initially discovered in R. albus
on the basis of adhesion-defective strains that lacked
specific surface proteins [97]. Surprisingly, these pro-
teins were subsequently identified as the critical GH48
enzyme and a GH9 enzyme, both of which were non-
cellulosomal but carried a module of unknown func-
tion on their C-terminus. Subsequent studies revealed
that the latter type of module was a CBM [98], which
was then classified in a new CBM family (family 37).
The CBM37s were collectively shown to exhibit a broad
specificity pattern, which indicated a mechanism for
binding the parent enzymes to cellulosic substrates.
The anchoring of the CBM37-bearing enzymes to the
bacterial cell surface was later demonstrated [99] – pre-
sumably to cell envelope polysaccharides (Figure 4D) ,
thus indicating a multiplicity of roles (combined sub-
strate and cell-surface binding) for this fascinating type
of CBM. It seems that the cell-attachment role is not
confined to the CBM37s, since a similar role has also
been demonstrated for at least one member of another
CBM family (family 35) [100]. Moreover, the status of
a conserved type of module from anaerobic fungi, long
being considered a ‘dockerin’ [101–105], has recently been
demonstrated to bind to saccharide components of a
surface-attached GH3 b-glucosidase [106]. On the one
hand, the attachment of a multiplicity of cellulolytic
enzymes onto a b-glucosidase is very logical, since the
concerted breakdown of cellulose into soluble cellodex-
trins in the presence of such an enzyme would presum-
ably counteract product inhibition of the cellulases and
promote efficient hydrolysis of cellulosic substrates. On
the other hand, categorizing such an enzyme complex
as a cellulosome is probably invalid. If the b-glucosidase
is a glycoprotein and the alleged dockerins recognize its
saccharide moieties and not a bona fide cohesin, then
the said dockerin would be a CBM and not a dockerin
per se. Consequently, the fungal ‘cellulosomes’ cannot be

Cell
membrane
Peptido-
glycan
Cell surface
polysaccharides
Cell
interior
1010
Enzymes
Scaffoldin subunit
CBM
CohesinDockerin
Type II
CBM
48
537
37
11
Extracellular
matrix
Anchoring
protein
Sortase
signal motif
A
B
C
D
9922 10
SLH
X
SLH
37
Biofuels © Future Science Group (2010)
Biofuels (2010) 1(2) future science group
336
Review Himmel, Xu, Luo, Ding, Lamed & Bayer
considered authentic cellulosomes but would represent a
completely novel type of CBM-mediated multienzyme
complex and unique type of paradigm for biomass
degradation.
Mixed, exotic & undiscovered paradigms
It is clear from the above sections that the division into
distinct paradigms is not necessarily strict. Microbes
capable of using lignocellulosic substrates and perhaps
the bacteria in particular, must employ particularly
‘intelligent’ strategies to survive in often extreme envi-
ronments. To this end, the cellulolytic microbes exploit
to their advantage every possible weapon and scheme
they possess in their molecular and cellular arsenal.
As we have observed, the anaerobic thermophilic cel-
lulolytic bacterium, C. thermocellum, is highly reputed
for its intricate cellulosome system – the first to have
been described. However, the bacterium also produces
Figure4. Modes of attachment
of glycoside hydrolases and
cellulosomes to the bacterial
cell surface. (A) Some single
enzymes bear SLH modules,
which can interact noncovalently
either with the peptidoglycan
layer[93,95] or with secondary cell
wall polymers [94] and the enzymes
are thereby attached individually
to the cell surface. The cell-surface
enzyme shown schematically in
the gure is based on Clostridium
thermocellum Xyn10X that also
contains family 9 and 22CBMs.
(B)Several cellulosomes are known
to be attached to the cell wall via a
special type of cohesin-containing
anchoring scaoldin that also
carries an SLH module [93,9 5].
The type-II anchoring cohesins
interact with a complementary
type-II dockerin of the enzyme-
bearing scaoldin subunit.
(C)The Ruminococcus avefaciens
cellulosome is attached to the cell
surface via covalent linkage to the
peptidoglycan layer via an enzyme-
mediated process. At least one
enzyme from the same bacterium
is also known to contain a similar
sortase-signal motif, which may thus
represent a more common mode of
enzyme attachment to the bacterial
cell surface. (D) An alternative
mode of enzyme attachment to
the cell surface involves special
types of CBM, such as family-37
CBMs from Ruminococcus albus, that
mediate individual noncovalent
attachment of dierent enzymes to
cell-surfacepolysaccharides.
CBM: Cellulose-binding module;
SLH: S-layer homology.

Microbial enzyme systems for biomass conversion: emergingparadigms Review
future science group www.future -science.com 337
several free cellulases, both with and without an ancillary
substrate-targeting CBM. Likewise, its mesophilic rela-
tive, C. cellulovorans, also produces both a well-charac-
terized cellulosome and noncellulosomal enzymes, which
are believed to work together in a synergistic manner.
In addition, C. thermocellum can produce several cell-
anchored hemicellulases, attached to the cell surface via
an SLH module. Finally, many of the C. thermocellum
and R. flavefaciens cellulosomal enzymes are multifunc-
tional, some having two or three different catalytic mod-
ules as well as several ancillary modules in addition to
the dockerin – up to a total of seven different modular
components in at least one case. Therefore, some cel-
lulolytic bacteria and C. thermocellum in particular, can
apparently exploit mixed strategies in order to decompose
cellulosic substrates in an efficient manner.
The above-described strategies for microbial attack
on lignocellulosic materials are not the only ones that
can be used in nature. As we proceed in our study of
microbial cellulolytic systems, it becomes clear that
some bacteria and fungi fail to conform to our current
dogma. For example, a completely different strategy and
newly emerging paradigm is evident in the xylanolytic
system of Geobacillus stearothermophilus T-6, in which
the relevant genes are arranged on a large (~40 kb) chro-
mosome cluster. When grown on xylan, the bacterium
secretes a lone extracellular endoxylanase that cleaves
the main chain of the substrate, thereby producing
short, branched xylodextrin units of two or more sug-
ars. These unusual branched xylosaccharides are then
taken up into the cell via highly specialized ABC sugar
transporter systems, which are lacking in other com-
peting bacteria [107]. Once inside the cell, the branched
xylosaccharides are subjected to further degradation by
a cadre of intracellular enzymes, which include a GH10
xylanase and associated enzymes from GH39, GH43,
GH51, GH52, GH67 and CE4. The imported carbo-
hydrates are thus converted to monosaccharides, which
are then assimilated by the usual metabolic pathways.
In this manner, the bacterium competes with other
microbes successfully for its fair share of the xylan in
its ecosystem. The connection between the specificities)
of the hydrolytic enzymes and characteristics of their
products, to those of the sugar-binding lipoprotein com-
ponents of the transporter systems, appears to be a more
general phenomenon in the lignocellulolytic bacteria. In
C. thermocellum, for example, the sugar specificities of
the different binding lipoproteins are consistent with
the observed substrate preference, whereby the larger
cellodextrins are assimilated faster than cellobiose [108].
In truth, we may only be at the beginning of our
renewed quest to understand the different lignocellu-
losic-degrading paradigms in nature. It seems that none
of the above-described strategies are consistent with the
highly efficient cellulose-degrading
systems of some other bacteria,
suc h a s Cytophaga hutchinsonii
and Fibrobacter succinogenes [109] ,
both of which have been subjects
of recent genome sequencing proj-
ects. Both of these bacteria appear
to lack known processive cellulases,
most of their cellulases appear to
lack CBMs and none of them bear
dockerins. Further analyses of the
different cellulolytic bacteria and
fungi and intensive programs for
characterization of their enzymes
will be necessary for discovery of
additional mechanisms of micro-
bial degradation of lignocellulosic
substrates. Future genome sequenc-
ing programs that focus on plant
cell wall degrading microorganisms will undoubtedly
reveal new and exotic paradigms.
Biomass conversion schemes
Today, biomass conversion is based on the fermentation
of biomass sugars to biofuels, especially ethanol. The pri-
mary unit operations for processing lignocellulose bio-
mass include biomass pretreatment, hydrolysis of cellulose
and hemicelluloses to monosaccharides and fermentation
of these sugars to liquid fuels products [110]. The follow-
ing general strategies have been used for the conversion of
biomass to ethanol by fermentation: hybrid hydrolysis and
fermentation (HHF), simultaneous saccharication and
fermentation (SSF), simultaneous saccharification and
co-fermentation (SSCF) and consolidated bioprocessing
(CBP). HHF allows saccharification to proceed at differ-
ent temperatures or for longer times, for example, than
fermentation. SSCF was introduced more recently and
accommodates new and primarily engineered fermenta-
tive stains able to convert all biomass sugars to ethanol
with equal efficiency. HHF, SSF and SSCF currently
require extensive pretreatment strategies of the cellulosic
feedstock and the addition of exogenous cellulolytic
enzymes. The capital cost of pretreatment and utilization
of cellulase enzymes are still the main barriers to enabling
cost-effective bioethanol production. Potential single-step
conversion of biomass to biofuels, afforded by CBP strat-
egies [111–113], would thus provide an ideal approach for
solving the biomass conversion cost problem. Emerging
evidence today is beginning to demonstrate the feasibility
of CBP at the industrial scale [114].
Why are better cellulases needed?
Regardless of the biomass conversion scheme eventually
employed in industrial biofuels plants, the catalytic
Key terms
Hybrid hydrolysis and fermentation:
Process that permits staging of the
saccharication and fermentation steps,
often designed to allow a high
temperature enzyme treatment
followed by a lower temperature
fermentation step
Simultaneous saccharication and
fermentation: Process that combines
the hydrolysis of cellulose and other
plant cell wall polysaccharides with the
fermentation of the sugars released
Consolidated bioprocessing:
Combines cellulase production,
substrate hydrolysis and fermentation
into a single step, by employing a single
microorganism capable of both
expressing cellulolytic enzymes and
converting soluble sugars to ethanol

Biofuels (2010) 1(2) future science group
338
Review Himmel, Xu, Luo, Ding, Lamed & Bayer
efficiency of the glycoside hydrolases utilized, also
known as specific activity, must be near theoretical val-
ues if the process is to be cost effective and robust. This
consideration is especially true of the cellulases. As long
as deconstruction remains a biochemical-based process,
the cost of cellulose-degrading enzymes will remain
a major process cost [110,115,116] . The reason for this
outcome is based in the very nature of heterogeneous
catalysis. Enzymes that act upon insoluble polymeric
substrates (e.g., cellulose, chitin and protein) typically
display lower catalytic rates (kca t) than enzymes acting
upon soluble substrates [117,118]. This situation results
in very high protein-based loadings for cellulases rela-
tive to other glycoside hydrolases. For example, 25 mg
of a Trichoderma reesei cellulase preparation (Spezyme
or GC220) converts 80% of a 1-g sample of Avicel to
glucose in 5 days at 50°C. Whether the cellulases are
free (noncomplexed) or complexed (cellulosomal), the
impact on process cost is important. Free cellulases
are expected to be produced ‘off site’ at special enzyme
production facilities and will be priced based on per-
formance, whereas cellulosomal enzymes or other
enzymes produced during CBP have a carbon-demand
cost, which taxes both metabolic flux and product for-
mation. Regarding sales margins of free enzymes, prof-
its from sales of these enzymes can be enhanced if the
specific performance of the enzymes is increased while
keeping the cost of production the same; consequently,
pricing will be based on performance. Optimizing the
performance of the molecular catalysts will permit
other unit operations in the plant to undergo excur-
sions of inefficiency, a very likely scenario when one
considers the uncertainty in feedstock type and quality
that the processing plant will encounter.
Future perspective
To date, only glimpses of the picture regarding the
molecular mechanisms of plant cell wall-degrading
enzymes have been accomplished. It is expected that
new enzymes and new paradigms will be found,
based on aggressive genome sequencing and analysis
of cell wall-degrading microorganisms. Metagenomic
sequencing will also help in this regard, especially
when teamed with new strategies for understanding
the roles of participating microbes in masses of decay-
ing biomass, including those strains that are today
‘uncultureable’ [119,120]. A lthough many hydrolytic
enzymes have been found, all indications point to a
vast reservoir of yet-to-be-discovered analogs in the
biosphere. Building on this concept and even more
important than discovery of another ten examples of
GH10, for example, would be the discovery of entirely
new paradigms of biological cell wall hydrolysis or
molecular fragmentation. Of course, we need to know
more about the structure–function relationships of
glycoside hydrolases in general and in the case of cel-
lulases these studies are further hampered by a lack of
good structural data for cellulose. We can also expect
continued utilization of diverse disciplines in science
to be brought to the problem of cell wall hydrolysis.
One outstanding example of this is the recent link-
age between computational science and classical bio-
chemistry in solving cellulase mechanistic problems
[121,122]. The advent of more accessible supercomput-
ers running thousands of processors, improved (more
scalable) codes for molecular dynamics and quantum
mechanics/molecular mechanics and better force fields
for biological problems (CMAP for proteins [123] and
TeamSugar for carbohydrates [124]), makes the acqui-
sition of biologically relevant simulations of cellulose
hydrolysis more promising. When teamed with muta-
tional biochemistry and high-throughput robotics
enzyme assays, the aggressive time line for improv-
ing existing enzymes and even developing biomimetic
counterparts seems imminently possible.
It has been 60 years since the A rmy Natick
Laboratories first isolated T. reesei from the rotting
cotton military accruements sent back from the war
in the south Pacific and it remains remarkable that the
precise molecular mechanisms of action of the enzymes
produced by this and related lignocellulose degrading
fungi are as yet unknown. In many ways, the diversity
and probable extensive bandwidth of plant cell wall-
degrading enzymatic and microbial systems harbored by
the biosphere remains a mystery. However, this mystery
we must solve. Terrestrial plant ecosystems produce car-
bonaceous compounds from CO2 in a highly oxygen-
ated form, the poly saccharides, and store these polymers
in both metabolically accessible (storage) and inacces-
sible (structural) forms. In many ecosystems, dead plant
matter takes years if not centuries to fully degrade; an
indicator that the biosphere can tolerate removal of some
fraction of carbon with no ill effects. Lignocellulose,
therefore, is the best feedstock for a sustainable and
reliable source of liquid fuels. We just need a fully cost
effective and robust conversion scheme to produce fer-
mentable sugars from biomass, such as corn stover and
other crop residues, hard and soft woods, construc-
tion waste and perhaps even municipal solid waste. A
glimpse at the recent literature in this field confirms
that progress towards gaining a deep understanding of
these processes is building momentum. With continued
focus on biomass conversion science, we should enjoy
new sources of biofuels for centuries to come.
Financial & competing interests disclosure
This work was supported by the US Department of Energy Of fice of
Science, Of fice of the Biological and Environmental Research,

Microbial enzyme systems for biomass conversion: emergingparadigms Review
future science group www.future -science.com 339
through the BioEnergy Science Center (BESC), a DOE Bioenergy
Research Center, by grants from the Israel Science Foundation (grant
nos 966/ 09 and 159/07) and from the United States–Israel
Binational Science Foundation (BSF), Jerusalem, Israel. Edward A
Bayer is the incumbent of The Maynard I. and Elaine Wishner Chair
of Bio-organic Chemistry at the Weizmann Institute of Science. The
authors have no other relevant af filiations or financial involvement
with any organization or entity with a financial interest in or finan-
cial conflict with the subject matter or materials discussed in the
manuscript. This includes employment, consultancies, honoraria,
stock ownership or options, expert t estimony, grants or patents
received or pending, or royalties.
Executive summary
Lignocellulosic biomass
Most abundant source of carbon and energy on Earth.
Attractive alternative to current petroleum-based fossil fuels.
Resistant to chemical and enzymatic conversion: high cost of conversion the major deterrent for commercialization.
Lack of knowledge of molecular architecture of the plant cell wall polymers.
Free enzyme systems
Carbohydrate-active enzymes include cellulases, hemicellulases (e.g., xylanases, mannanases and arabinofuranases), pectate lyases and
carbohydrate esterases that together deconstruct plant cell wall polysaccharides in an ecient manner.
Cellulases and hemicellulases are classied as glycoside hydrolases: multimodular enzymes from 115 dierent families, which contain a
catalytic module that cleaves the glycoside bond and (usually) a carbohydrate-binding module (CBM) that targets the enzyme to the
(poly)saccharide substrate.
Cellulases include endo - and exo-acting enzymes, together with b-glucosidases, which work synergistically to hydrolyze the particularly
resistant crystalline cellulose brils.
Cellulosomes
Noncatalytic scaoldins contain a CBM for substrate targeting and multiple cohesin modules for integrating the enzymatic subunits into a
multicomponent complex.
Cellulosomal enzymes contain a dockerin module that binds strongly to the scaoldin-borne cohesins.
Synergistic action is achieved by concentrating the enzymes together at dened sites on the lignocellulosic substrate.
Articial ‘designer cellulosomes’ may provide a future solution for ecient conversion of cellulosic biomass to soluble sugars.
Multifunctional enzyme systems
Composed of two or more catalytic modules on the same polypeptide chain.
May be cellulosomal (dockerin-containing) or noncellulosomal (CBM-containing).
Some cellulolytic hyperthermophiles contain numerous multifunctional enzymes with multiple CBMs.
This ‘natural’ strategy may serve as a concept for articial chimeric multifunctional enz ymes.
Cell-anchored enzyme systems
May be cellulosomal (scaoldin-attached) or noncellulosomal (direct attachment of enzymes to the microbial cell surface).
Cell-surface attachment may be mediated by noncovalent or covalent interaction.
Mixed, exotic & undiscovered paradigms
Lignocellulolytic microbes that have evolved employ a diverse set of strategies for degradation of plant cell wall biomass.
It is logical to expect that the biosphere harbors new paradigms for biomass deconstruction not yet discovered and the lessons learned
from their ana lysis will greatly benet biomass conversion science.
Biomass conversion schemes
In order to make biomass conversion schemes cost eective, sustainable and attractive to industr y, the deconstruction of plant cell walls
must be fully understood at the molecular level.
New biomass-processing schemes, based on past experience and future innovation, will optimize biocatalyst performance, both
enzymatic and microbial.
Why are better cellulases needed?
Capture of fermentable sugars from plants, either crop residues or plants grown for energy purposes, is essential to maintain our thriving
world energy economy.
Dramatic progress in the basic sciences of plant recalcitrance can be traced to new programs supporting multidisciplinar y research with
long-term goals.
Ultimately, mankind will have the knowledge to manipulate the biosphere with the skills needed to safely withdraw plant biomass and
convert it into much needed fuels and chemicals.

Biofuels (2010) 1(2) future science group
340
Review Himmel, Xu, Luo, Ding, Lamed & Bayer
No writing assistance was utilized in the produc-
tion of this manuscript.
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