REVIEWS
CELLULOSOMES: PLANT-CELL-WALLDEGRADING ENZYME COMPLEXES
Roy H. Doi* and Akihiko Kosugi‡
Cellulose, the main structural component of plant cell walls, is the most abundant carbohydrate
polymer in nature. Although abundant, it is extremely difficult to degrade, as it is insoluble and is
present as hydrogen-bonded crystalline fibres. Anaerobic microorganisms have evolved a
system to break down plant cell walls that involves the formation of a large extracellular enzyme
complex called the cellulosome,which consists of a scaffolding protein and many bound
cellulases. Cellulosomes have many potential biotechnological applications as the conversion of
cellulosic biomass into sugars by cellulosomes could result in the production of high-value
products such as ethanol or organic acids from inexpensive renewable resources. Rapid
advances in cellulosome research are providing basic information for the development of both
in vitro and in vivo systems to achieve such goals.
CELLULOSE
The most abundant plant
polysaccharide consisting of
(1→4)β-D-glucan chains
hydrogen-bonded to one
another along their length.
HEMICELLULOSE
Cross-linking glycans that
comprise up to about 30% of
plant cell walls; the two major
hemicelluloses are xyloglucans
and glucuronoarabinoxylans.
*Section of Molecular &
Cellular Biology, University
of California, Davis,
California, USA.
‡
Fine Chemicals Division,
Kaneka Corporation,
1-8, Miyamae-machi,
Takasago-cho Takasago,
Hyogo, 676-8688, Japan.
Correspondence to R.H.D.
e-mail: [email protected]
doi:10.1038/nrmicro925
The degradation of plant cell walls by microorganisms
has an important role in the carbon cycle of the earth.
Most plant cell walls are composed of approximately
15–40% CELLULOSE, 30–40% HEMICELLULOSE and pectin,
and 20% lignin. These components are degraded enzymatically to yield smaller oligomers and eventually glucose, pentoses and other small carbon compounds,
which are metabolized to CO2.
Although abundant in nature, cellulose is a particularly difficult polymer to degrade, as it is insoluble
and is present as hydrogen-bonded crystalline fibres1.
Hemicellulose, pectin and lignin are generally easier to
degrade than cellulose. Two types of enzyme systems
for the degradation of plant cell walls have been
observed in microorganisms. In the case of aerobic
fungi and bacteria, several individual endoglucanases,
exoglucanases and ancillary enzymes are secreted that
can act synergistically to attack plant cell walls. The
best studied of these enzymes are the glycosyl hydrolases of Trichoderma reesei. These free enzyme systems
have been well documented and reviewed2,3 and will
not be discussed further in this article. In anaerobic
microorganisms, a different type of system has evolved
that involves the formation of a large, extracellular
enzyme complex called the CELLULOSOME, which consists
of a scaffolding protein and many bound enzymes4.
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Several recent reviews have discussed the structure
and function of cellulosomes5–12. In this review, we will
focus on the most recent findings that have added to
our knowledge of cellulosome structure, regulation
and genetics, and the synergism between CELLULASES
and hemicellulases.
Cellulosome-producing microorganisms
Experimental evidence of the presence of cellulosomes
has only been obtained for anaerobic bacteria.
Although there is growing evidence that anaerobic
fungi also produce cellulosomes13–18, the presence of a
6
SCAFFOLDIN gene, which encodes an important component of the cellulosome, has not yet been documented
for the anaerobic fungi. A list of cellulosome-producing
microorganisms is presented in TABLE 1. This list is
expected to grow rapidly in the next few years as
more cellulosome-producing microorganisms are
discovered.
It should be noted that the presence of cellulosomelike structures does not always confer cellulolytic activity to a microorganism. Clostridium acetobutylicum
synthesizes a 650-kDa cellulosome19, which has low
degradative activity on CMC (carboxymethyl cellulose,
a soluble form of cellulose), but cannot grow on cellulose and cannot hydrolyse crystalline cellulose20. This
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CELLULOSOME
An extracellular enzyme
complex consisting of a
scaffoldin and cellulosomal
enzymes that are capable of
degrading plant cell walls.
Cellulosomes are produced by
anaerobic microorganisms.
CELLULASE
Glycosyl hydrolases that degrade
cellulose.
SCAFFOLDIN
A scaffolding protein found in
cellulosomes containing cohesin
domains that bind cellulosomal
enzymes.
COHESIN
Domains in the scaffoldin to
which cellulosomal enzymes are
bound. There are at least three
types of cohesins, which vary in
amino acid sequence.
DOCKERIN
Duplicated sequences present in
cellulosomal enzymes that bind
to cohesins. There are at least
three types of dockerins, which
vary in amino acid sequence.
AVICEL
A commercially available
microcrystalline cellulose.
species, which is used industrially to produce acetone and
butanol, cannot currently be grown on less-expensive
forms of carbon such as cellulosic biomass.
Cellulosome composition
Electron-microscopy studies have shown that extracellular protuberances are associated with cellulosome-producing bacteria7 and it is believed that these
protuberances contain cellulosomes. Cellulosomes
consist of a fibrillar protein (the scaffolding protein)
with masses (enzyme subunits) positioned periodically along the fibrils21. The non-enzymatic scaffolding protein or scaffoldin 6 contains binding sites
6
(COHESINS) for the cellulosomal enzyme subunits,
which have various different functions and invariably
contain a cohesin-binding site called a DOCKERIN6
(FIG. 1).
The cohesin–dockerin interaction is an important
factor in cellulosome assembly. It is now apparent that
the cellulosome fraction is not homogeneous in composition. The main reasons for cellulosome heterogeneity are species-specific variation in scaffoldin
properties, which allows the assembly and the composition of the cellulosome to differ between bacterial
species7,10, and the fact that most scaffoldins contain
between six and nine different cohesins, which can
bind up to 26 different cellulosomal enzymes (TABLE 2).
Depending on which enzymes are bound to the scaffoldin, there is the potential to make many cellulosomes
with many different compositions within a single
microorganism.
Table 1 | Cellulosome-producing anaerobic bacteria and fungi
Microorganism
M/T*
Source
References
Acetivibrio cellulolyticus
M
Sewage
34
Bacteroides cellulosolvens
M
Sewage
86
Butyrivibrio fibrisolvens
M
Rumen
87
Clostridium acetobutylicum
M
Soil
49
Clostridium cellobioparum
M
Rumen
88
Clostridium cellulolyticum
M
Compost
39
Clostridium cellulovorans
M
Wood fermenter
89
Clostridium josui
M
Compost
33
Clostridium papyrosolvens
M
Paper mill
23
Clostridium thermocellum
T
Sewage soil
53
Ruminococcus albus
M
Rumen
90
Ruminococcus flavefaciens
M
Rumen
35
Neocallimastix patriciarum
M
Rumen
91
Orpinomyces joyonii
M
Rumen
92
Orpinomyces PC-2
M
Rumen
93
Piromyces equi
M
Rumen
94
Piromyces E2
M
Faeces
94
Anaerobic bacteria
Anaerobic fungi ‡
*M/T indicates the optimum growth temperature; M, mesophilic; T, thermophilic (above 50°C).
‡
The anaerobic fungi have been postulated to possess cellulosomes, as their enzymes contain non-catalytic
dockerin domains (NCDD)17. The genes encoding scaffoldins from these anaerobic bacteria have been
sequenced; however, no scaffoldin-encoding gene has been isolated from the anaerobic fungi so far.
Modified with permission from REF. 10 © (2003) American Society for Microbiology.
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| JULY 2004 | VOLUME 2
So, there are interspecies differences among cellulosomes owing to the variations in the properties of scaffoldins and intraspecies variations depending on the
type of enzymes that bind to the scaffoldin itself. The
composition of cellulosomes is complicated further by
the fact that, although some bacterial species that synthesize cellulosomes seem to contain only one type of
scaffoldin, other species have been shown to have multiple scaffoldins22,95, which could also potentially yield
different types of cellulosomes.
Experimentally, the heterogeneity in the cellulosome population has been demonstrated particularly
well with Clostridium papyrosolvens. The cellulosome
population from this species was separated into seven
distinct subpopulations by anion-exchange chromatography, and each subpopulation had morphological differences as well as functional differences23,24.
Electron photomicrographs of C. papyrosolvens cellulosomes are particularly distinctive as the fractions not
only contain different cellulosomes but, within each
fraction, the morphology of the cellulosomes seems to
be homogeneous. Anion-exchange chromatography
has also been used to obtain homogeneous subpopulations of Clostridium thermocellum cellulosomes that
differ with respect to subunit composition and enzyme
activities such as avicelase (the activity that is capable
of degrading microcrystalline cellulose or AVICEL) and
xylanase activity25. Subpopulations of cellulosomes
from Clostridium cellulovorans in which the function
and subunit composition of the cellulosomes were
clearly different have also been observed26. One subpopulation had much more plant-cell-wall-degrading
activity than the other. This mixture of cellulosomes
could allow the bacterial cell to attack various substrates more efficiently, as greater functional display is
possible and could facilitate synergism between the
enzymes.
Cellulosome assembly
The observation of heterogeneous populations of
cellulosomes raises an important question: do cellulosomes assemble by the random assembly of the cellulosomal enzyme subunits on the scaffoldins or is
the assembly nonrandom, with the scaffoldins binding the cellulosomal enzymes in a more organized
and specific manner? The fractionation of cellulosomes into relatively homogeneous fractions indicates that some form of organized assembly process is
occurring. In nature and during billion of years of
evolution, most natural events are not random; it is
most likely therefore that the extracellular assembly
of cellulosomes is nonrandom. Exactly how this
occurs is still far from being understood. As the scaffoldin and cellulosomal enzymes are secreted from
the cell, there must be mechanisms to fold them into
mature forms and to determine which enzymes
assemble on a particular scaffoldin. If this were not the
case, there would be an almost infinite number of compositionally different cellulosomes and it is unlikely
that relatively homogeneous fractions of cellulosomes
would be observed24.
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Plant cell wall
EngE
CbpA, contains
cellulose-binding
domain (CBD)
Dockerin
domain
Cohesin
domain
Cellulosomal
enzymes
Hydrophilic
domain
Bacterial cell surface
Surface layer
homology
(SLH) domain
Figure 1 | A schematic model of a Clostridium cellulovorans cellulosome. The scaffoldin
protein is shown with its cellulose-binding domain (CBD), nine cohesins, four hydrophilic domains
and nine cellulosomal enzymes bound to the scaffoldin through their dockerins. EngE is believed
to tether the C. cellulovorans cellulosome to the cell surface10.
Polycellulosomes can have a mass of more than
100 MDa and consist of large numbers of cellulosomes. They appear as protuberances on the surface of
cells. How are polycellulosomes assembled? There is one
report of a recombinant cohesin that can bind to a scaffoldin27. This indicates that it might be possible for
interactions to occur between scaffoldins that could lead
to the formation of polycellulosomes that are linked
through scaffoldin–scaffoldin interactions. The other,
more complex structure that could lead to the assembly
of polycellulosomes is represented by the interaction of
the scaffoldin of Acetivibrio cellulolyticus with adaptor
proteins22. This latter observation will be discussed in
more detail below.
Scaffoldins in different microorganisms
The scaffoldin is a major part of any cellulosome, as
its ternary functions include binding cellulosomal
enzymes, binding the substrate, cellulose, and binding
cell-surface-associated proteins. FIGURE 2 illustrates the
types of scaffoldins that have been characterized so far.
Each scaffoldin usually contains cohesins and a cellulose-binding domain (CBD) or carbohydrate-binding
module (CBM)28. However, there are significant variations among the scaffoldins that have been characterized: scaffoldins can contain different types of cohesin, a
dockerin29, hydrophilic domains of unknown function30–33, a glycosyl hydrolase domain34, various uncharacterized domains and one scaffoldin that has been
found even lacks a CBD35 (FIG. 2). With the characterization of more scaffoldins, other unique features will
undoubtedly be identified.
When scaffoldins were first characterized, the
cohesins that were found were called type I cohesins
and were involved in binding cellulosomal enzymes
containing dockerins known as type I dockerins.
NATURE REVIEWS | MICROBIOLOGY
Further studies showed that there were other cohesins
that were not homologous to the type I cohesins, and
these are known as type II29 and type III35 cohesins.
Type I cohesins bind to type I dockerins. A dockerin
has been found that does not bind type I cohesins,
but binds to type II cohesins that are found on cellsurface-binding proteins; this dockerin is known as a
type II dockerin29. So, in C. thermocellum, the type II
dockerin of the scaffoldin is linked to the type II cohesins
of cell-surface-binding proteins that bind to the cell
surface via a particular motif known as surface layer
homology (SLH) domains36 (BOX 1).
A more complex organization has been observed in
A. cellulolyticus cellulosomes22. In this system, there are
two primary scaffoldins, ScaA and ScaC, and an adaptor scaffoldin, ScaB. ScaA is the primary scaffoldin that
binds the cellulosomal enzymes and contains a CBM,
type I cohesins and a type II carboxy-terminal dockerin domain. It is linked to ScaC by the adaptor protein, ScaB. ScaB binds to the C-terminal dockerin in
ScaA through a type II cohesin and binds to a cohesin
in the primary anchoring scaffoldin, ScaC, through
its C-terminal dockerin. Given that ScaA has seven
cohesins and a C-terminal dockerin, ScaB has four
type II cohesins and a C-terminal dockerin, and ScaC
has three cohesins and a C-terminal SLH domain, it is
possible that this complex could bind at least 96 cellulosomal enzymes to form a large enzyme complex that
is bound to the cell surface by ScaC22 (FIG. 3). This complex system found in A. cellulolyticus for binding the
cellulosome to the cell surface is somewhat similar to
the SdbA and OlpB anchoring system that is found in
C. thermocellum7.
As only a limited number of scaffoldins have been
characterized, it is likely that many variations are yet to
be discovered. One possibility that has been proposed
is the presence of more than one primary scaffoldin,
each of which can bind specifically to a different set of
enzymes37. It is interesting that more examples of multiple scaffoldins in a species have not been reported, as
the presence of two or more different scaffoldins
would increase the types and functions of cellulosomes
available to the cell.
Cohesin–dockerin specificity
The cohesin–dockerin interaction is crucial for cellulosome assembly. All cellulosomal enzymes contain dockerin domains that interact with the cohesins that are
present on the scaffoldins38. There is species specificity in this interaction; for example, the dockerins
that are found in Clostridium cellulolyticum cellulosomal enzymes do not interact with the cohesins that
are found in C. thermocellum scaffoldins and viceversa39. This specific interaction has been analysed by
mutational studies of the cohesin40 and dockerin41–43
domains, and X-ray crystallographic studies of the
cohesin domain42,44,45 and the cohesin–dockerin complex46. The interaction between cohesins and dockerins
was postulated to reside in four specific amino acid
residues in the dockerin domain39,40. This prediction was
validated by the fact that mutating these four residues
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Table 2 | Cellulosomal subunits of clostridia
Cellulosomal enzymes Function
Clostridium acetobutylicum
CelA
Endoglucanase
CelE
Endoglucanase
CelF
Exoglucanase
CelG
Endoglucanase
CelH
Exoglucanase
CelL
Endoglucanase
EngA
Endoglucanase
ManA
Mannanase
CAC0919
Sialidase
CAC3469
Endoglucanase
Clostridium cellulolyticum
CelA
Endoglucanase
CelC
Endoglucanase
CelD
Endoglucanase
CelE
Endoglucanase
CelF
Exoglucanase
CelG
Endoglucanase
CelH
Endoglucanase
CelJ
Endoglucanase
CelM
Endoglucanase
ManK
Mannanase
RglY
Rhamnogalacturonan lyase
Clostridium cellulovorans
EngB
Endoglucanase
EngE
Endoglucanase
EngH
Endoglucanase
EngK
Endoglucanase
EngL
Endoglucanase
EngM
Endoglucanase
EngY
Endoglucanase
ExgS
Exoglucanase
ManA
Mannanase
PelA
Pectate lyase
XynA
Xylanase/acetyl xylan esterase
Clostridium josui
CelB
Endoglucanase
CelE
Endoglucanase
CelD
Exoglucanase
AgaA
α-Galactosidase
Clostridium thermocellum
CbhA
Cellobiohydrolase
CelA
Endoglucanase
CelB
Endoglucanase
CelD
Endoglucanase
CelE
Endoglucanase
CelF
Endoglucanase
CelG
Endoglucanase
CelH
Endoglucanase
CelJ
Endoglucanase
CelK
Endoglucanase
CelN
Endoglucanase
CelO
Endoglucanase (cellobiohydrolase)
CelP
Endoglucanase
CelQ
Endoglucanase
CelS
Exoglucanase
CelT
Exoglucanase
CseP
Unknown
ChiA
Chitinase
LicB
Lichenase
ManA
Mannanase
XynA (XynU)
Xylanase/acetyl xylan esterase
XynB (XynV)
Xylanase
XynC
Xylanase
XynD
Xylanase
XynY
Xylanase/feruloyl esterase
XynZ
Xylanase/feruloyl esterase
Molecular mass (kDa) Modular structure*
54
96
81
77
80
60
67
47
91
110
GH5-DS1
CBD3-Ig-GH9-DS1
GH48-DS1
GH9-CBD3-DS1
GH9-CBD3-DS1
GH9-DS1
GH44-DS1
GH5-DS1
GH74-DS1
(SLH)3-GH5-X-DS1
50
51
63
97
78
80
83
85
58
48
75
GH5-DS1
GH8-DS1
GH5-DS1
CBD4-Ig-GH9-DS1
GH48-DS1
GH9-CBD3-DS1
GH9-CBD3-DS1
GH9-CBD3-DS1
GH9-DS1
DS1-GH5
GPL11-DS1
49
110
79
97
58
96
80
80
47
94
57
GH5-DS1
(SLH)3-GH5-X-DS1
GH9-CBD3-DS1
CBD4-Ig-GH9-DS1
GH9-DS1
CBD4-Ig-GH9-DS1
CBD2-GH9-DS1
GH48-DS1
DS1-GH5
X-CBD2-GPL9-DS1
GH11-DS1-CE4
51
81
80
52
GH8-DS1
GH9-CBD3-DS1
GH48-DS1
GH27-DS1
138
53
64
72
90
82
63
102
178
101
82
75
58
80
83
65
62
55
38
67
74
50
70
70
120
92
CBD4-Ig-GH9-X-X-CBD3-DS1
GH8-DS1
GH5-DS1
Ig-GH9-DS1
GH5-DS1-CE2
GH9-CBD3-DS1
GH5-DS1
GH26-GH5-CBD11-DS1
X-Ig-GH9-GH44-DS-X
CBD4-Ig-GH9-DS1
GH9-CBD3-DS1
CBD3-PT-GH5-DS1
GH9-DS1
GH9-CBD3-DS1
GH48-DS1
GH9-DS1
UN-DS1
GH18-DS1
GH16-DS1
CBD4-GH26-PT-DS1
GH11-CBD4-DS1-CE4
GH11-CBD6-DS1
X-GH10-DS1
CBD22-GH10-DS1
CBD22-GH10-CBD22-DS1-CE1
CE1-CBD6-DS1-GH10
*The modular structures of cellulosomal subunits are indicated by abbreviations: CBD, cellulose-binding domain; CE, carbohydrate esterase
family; DS1, dockerin domain type I; GH, glycosyl hydrolase; GPL, polysaccharide lyase family 9 (pectate lyase); Ig, immunoglobulin-like module;
PT, proline-rich linker; SLH, surface layer homology domain; UN, unknown domain; X, unknown domain containing a hydrophilic domain. Modified
with permission from REF. 10 © (2003) American Society for Microbiology.
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| JULY 2004 | VOLUME 2
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resulted in a change in the cohesin–dockerin recognition specificity43. The mutagenesis studies indicated
that the specificity of this interaction was strongly
affected by a single amino acid change (threonine to
leucine) at a given position in the dockerin43, although
it is thought that other subtle interactions are also
involved. The association between dockerin and
cohesin domains was shown to be largely dependent
on hydrophobic interactions41. The three-dimensional
structure of the dockerin–cohesin complex indicates
that the cohesin–dockerin interaction is mediated
mainly by hydrophobic interactions between one of
the ‘faces’ of the cohesin and α-helices 1 and 3 of the
dockerin.
Acetivibrio cellulolyticus
ScaA (CipV) (199 kDa)
ScaB (100 kDa)
ScaC (124 kDa)
Bacteroides cellulosolvens CipBc (245 kDa)
Clostridium acetobutylicum CipA (154 kDa)
Cellulosomal enzymes
Clostridium cellulolyticum CipC (155 kDa)
Clostridium cellulovorans CbpA (189 kDa)
Clostridium josui CipC (120 kDa)
Clostridium thermocellum
CipA (196 kDa)
OlpA (49 kDa)
OlpB (178 kDa)
Orf2p (75 kDa)
SdbA (69 kDa)
Ruminococcus flavefaciens
ScaA (93 kDa)
ScaB (181 kDa)
Signal peptide
Cellulose-binding
domain (family 3)
Type I cohesin
Type II cohesin
Type III cohesin
Glycosyl hydrolase
(family 9)
Type II dockerin
Hydrophilic
domain
Unknown
domain
T, P, S, D, E or
K-rich linking segments
Surface layer homology
(SLH) domain
Figure 2 | The modular structure of scaffoldins from various microorganisms. The nonenzymatic scaffoldin protein contains binding sites (cohesins) for the cellulosomal enzyme
subunits6. Most scaffoldins contain between six and nine different cohesins. The species-specific
variations in scaffoldin properties allow the composition of the cellulosome to differ between
bacterial species7,10. Some bacterial species that synthesize cellulosomes contain only one type
of scaffoldin, however, other species have been shown to contain multiple scaffoldins22,95, which
could also potentially yield different types of cellulosomes.
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The cellulosomal enzymes include cellulases, hemicellulases, pectinase, chitinase and many ancillary enzymes
that can degrade plant cell wall materials. Cellulases are
part of a large group of glycosyl hydrolases that have
been categorized into several families on the basis of
their amino acid homology. Hemicellulases are able to
degrade hemicelluloses, a class of polysaccharides that
can form hydrogen bonds with cellulose fibrils and
form a network in plant cell walls. Xylans and mannans
are examples of hemicelluloses. A list of cellulosomal
enzymes representing several glycosyl hydrolase families
is presented in TABLE 2. Some 26 cellulosomal enzymes
have been identified for C. thermocellum7. Some of the
enzymes work in concert to facilitate the degradation of
the main polymers, for example, xylans and mannans.
These include both cellulosomal and non-cellulosomal
enzymes that remove various groups from the xylan and
mannan backbones.
The endoglucanases, which cleave cellulose internally, primarily belong to glycosyl hydrolase families 5
and 9, and the exoglucanases, which can attack cellulose
from either the reducing or non-reducing ends, belong
to family 48. The family 9 endoglucanases are versatile
as they not only cleave cellulose molecules internally,
but also proceed in a processive manner along the chain
from the cleavage site, and could be important enzymes
in the cellulosome47.
C. cellulovorans48, C. acetobutylicum49 and C. cellulolyticum50 contain large gene clusters as well as unlinked
genes that encode cellulosomal enzymes. The organization of the gene clusters in these species is related and
the clusters might have evolved from a common set of
genes. In the case of the C. cellulovorans gene cluster, a
transposase gene is located at its 3′ end, indicating that
lateral gene transfer might have occurred48. By contrast,
the genes encoding the C. thermocellum cellulosomal
enzymes are scattered throughout the chromosome51
and no clusters of cellulosomal enzyme genes have been
observed, except for several genes involved in binding
the cellulosome to the cell surface52.
Regulation of cellulosomal genes
What are the factors that regulate the expression of cellulosomal genes? The regulation of cellulosomal gene
expression has been examined at the microscopic,
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Box 1 | Cellulosomes and SLH domains
Many Gram-positive bacteria have a surface-layer protein (SLP) that surrounds the
exterior cell wall. This layer of proteins is attached to secondary cell-wall polymers in
the rigid cell-wall layer. The surface layer homology (SLH) domains of several
extracellular enzymes are homologous to regions of the SLP and it is believed that,
like the SLP, SLH domains also attach to secondary cell-wall polymers and bind these
SLH-containing enzymes to the cell surface. As the cellulosomes of Clostridium
thermocellum are attached to surface layer SLH- and cohesin-containing proteins
through dockerin–cohesin interactions, it is believed that this interaction tethers the
C. thermocellum cellulosomes to the cell surface. In the case of Clostridium
cellulovorans, a cellulosomal enzyme, EngE, contains both a dockerin and three
tandem SLHs. EngE can bind to the scaffoldin protein through its dockerin as well as
to the cell surface through its tandem SLHs. So, the C. cellulovorans cellulosome is
bound to the cell surface through EngE, which binds to both the scaffoldin protein
and the cell surface.
physiological and transcription levels. The earliest
microscopic studies demonstrated the presence
of protuberances, which contain polycellulosomes53.
In a study using scanning electron microscopy, the protuberances were observed from cellulose-grown cells,
but not glucose-, fructose-, CELLOBIOSE- or CMC-grown
cells54. The formation of protuberances took about
4 hours when C. cellulovorans cells were grown on cellulose. Within 5 minutes of the addition of the soluble
sugars glucose, cellobiose or methylglucose, the protuberances could no longer be detected. This indicated
that the dissociation of the protuberances was rapid
and that the presence of soluble sugars was responsible.
An early transcription study on a cellulosomal gene,
engB, indicated that it was transcribed as a single transcription unit and that the relative amount of engB
mRNA was much higher in cellulose-grown cells than
in cellobiose-grown cells55.
CELLOBIOSE
An individual unit of cellulose.
SIGMA-A
Sigma factors are variable
protein components of the
bacterial RNA polymerase that
influence transcription by
determining where the
polymerase binds to DNA. In
Bacillus, σA is a housekeeping
sigma factor, σB an alternative
sigma factor that responds to
stress and σL the Bacillus subtilis
homologue of σ54, the major
variant sigma factor in E. coli.
Plant cell wall
Unknown
domain
ScaA
Glycosyl hydrolase
domain (Family 9)
CBD
Dockerin
domain
(Type II)
ScaB
ScaB dockerin
domain
ScaC
SLH
domain
Bacterial cell surface
Cohesin domain
(Type I)
Cohesin domain
(Type II)
Cohesin domain
(new group)
Cellulosomal
subunits
Figure 3 | A model of the Acetivibrio cellulolyticus cellulosome. This cellulosome has two
primary scaffoldins, ScaA and ScaC, and an adaptor scaffoldin, ScaB22. The proteins are
connected via dockerin–cohesin interactions.
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The growth medium has been shown to affect both
subunit structure and function of the cellulosome.
When cells are grown on different substrates, such as
glucose, cellobiose, xylan, mannan or pectin, and their
cellulosomes are fractionated by anion-exchange chromatography, fractions are obtained that differ in subunit composition and enzymatic activity4,56,57. This
implies that the cell responds to different substrates by
expressing cellulosomal genes, which results in a population of cellulosomes with activities that are directed
towards the available substrate. So, the growth substrate has a significant effect on cellulosome synthesis
and subunit composition.
As the cellulosome comprises a scaffoldin protein
and a large number of enzymatic subunits, it is of
interest to determine how their genes are regulated,
whether there is coordinate expression of the genes to
form this multisubunit enzyme complex, and what
type of promoter region controls their expression.
Transcription studies of the C. cellulovorans large cellulosomal gene cluster indicated that there are several
operons within the gene cluster and that there is coordinate expression of several of the operons58. The promoters for the genes were similar to those found for
SIGMA-A RNA polymerases of Gram-positive bacteria.
When cells were grown on different substrates such as
glucose, xylan, mannan or pectin and their mRNA
analysed, abundant expression was observed for most
of the genes in the cellulosomal gene cluster as well as
for cellulosomal genes unlinked to the cluster, and
moderate or low levels of expression were observed
when various monosaccharides were the substrates.
The xylanase and pectate lyase genes were specifically
induced in the presence of xylan and pectin, respectively. The results indicated that cellulases and hemicellulases were coordinately expressed, that cellulase
expression was regulated by a CATABOLITE-REPRESSION-like
mechanism, and that the presence of hemicelluloses
influenced cellulose utilization by the cell58. Analysis of
the transcription of cellulosomal genes that were
unlinked to the large cellulosomal gene cluster indicated that most were monocistronic and could be
expressed coordinately with the genes in the large gene
cluster56. Previous studies with C. thermocellum also
concluded that a catabolite-repression-like mechanism
was controlling the expression of cellulosomal genes59.
One of the main enzymes of the C. thermocellum
cellulosome is the CelS exoglucanase. The level of
expression of CelS is much higher when cells are
grown on cellulose than on cellobiose. When the
transcriptional level of celS mRNA was determined, it
was found that transcriptional activity was inversely
proportional to the growth rate. Two transcriptional
start points were observed upstream of the translational start point that exhibited homology to the
σA and σB promoter sites in Bacillus subtilis. The relative
activity of the promoters remained constant under
the conditions studied. celS is therefore regulated at
the transcriptional level and its expression is modulated by the growth rate under conditions of nitrogen
and cellobiose limitation60.
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REVIEWS
Cell wall polymer
EngH
Catalytic
domain
ExgS
EngK
Ig-like
domain
CBD
CBM
Dockerin
domain
1
2
3
Cohesin
domain
CBD
Mini CbpA
Hydrophilic
domain
Host cell surface
Figure 4 | A model of a designer mini-cellulosome. Mini-cellulosomes contain ‘miniscaffoldins’, which can either contain one particular cohesin, and thus bind one particular
enzyme, or a variety of cohesins and thus bind a variety of enzymes. This mini-scaffoldin has
a cellulose-binding domain (CBD), one hydrophilic domain, and three cohesin domains
(labelled 1, 2 and 3) to which three different cellulosomal enzymes are attached through their
dockerin domains.
30%. The antisense strain also overproduced two noncellulosomal proteins, P105 and P98. These results show
that Cel48F has an important role in the degradation of
crystalline cellulose and that the translational inhibition
of the synthesis of Cel48F could reduce the production
of another transcriptionally linked protein, CipC.
Further analysis of cellulolysis has been obtained by
disrupting the cipC gene of C. cellulolyticum66. This
mutant was severely impaired in its ability to degrade
crystalline cellulose and it produced a small amount of a
truncated CipC (P120), which could complex with cellulosomal enzymes. However, none of the enzymes
associated with P120 was encoded by the genes distal to
cipC in the large cipC gene cluster. The complex formed
with P120 contained three important proteins designated P98, P105 and P125, and 12 other dockerincontaining enzymes encoded by genes outside the cipC
gene cluster. These results indicate that a mutation in
the cipC gene has a strong polar effect and that the
enzymes encoded downstream in the cipC gene cluster
are essential for the efficient degradation of crystalline
cellulose by the cellulosome.
Without question, our relatively recent ability to use
direct genetic analysis techniques to analyse cellulosomal genes will lead to a much better understanding of
cellulosomal structure and function.
Biotechnological uses of cellulosomes
The scaffoldin CipA has an important role in the
C. thermocellum cellulosome and cipA expression was
analysed together with the tandemly located olpB and
orf2 genes61. As with the celS gene, it was found that
expression of cipA, olpB and orf2 was regulated by the
growth rate, with higher expression at a low growth
rate and lower expression at a high growth rate. Two
important promoters with homology to σA and σL of
B. subtilis were observed upstream of the cipA gene.
Interestingly, the σL-like promoter was expressed under
all growth conditions, whereas transcription from the
σA-like promoter was significant only under carbonlimiting conditions. By contrast, only a single σA-like
promoter was observed upstream of the cbpA gene of
C. cellulovorans under all conditions that were tested56.
The understanding of the regulation of expression of the
scaffoldin protein gene is a fundamental aspect of the
study of cellulosome synthesis and assembly, and should
be thoroughly analysed.
Direct genetic analysis of cellulosomes
CATABOLITE REPRESSION
Transcriptional repression of a
prokaryotic operon by the
metabolic products of the
enzymes that are encoded by the
operon.
With our increasing ability to use direct genetic analysis
techniques with clostridia62–64, the modification of cellulosomal subunits in vivo should become a reality.
Antisense RNA technology has been exploited to study
the effect of reducing the synthesis of Cel48F, a major
component of C. cellulolyticum cellulosomes65. An antisense RNA was directed against the ribosome-binding
site and the beginning of the coding region of Cel48F.
The strain containing this antisense RNA had a much
lower Cel48F content, fewer cellulosomes, and the activity of the cellulosome against avicel was reduced by
NATURE REVIEWS | MICROBIOLOGY
There is much interest in exploiting the properties of
cellulosomes for practical purposes6. The specific
cohesin–dockerin interaction, the strong cellulosebinding property of the CBD domain, the potential for
transforming non-cellulose degraders to cellulose
degraders and the construction and use of ‘designer’
cellulosomes for specific degradative activities are
important properties of the cellulosome that can be
used in biotechnology.
Exploiting the specific cohesin–dockerin interaction. The
sequence specificity in cohesin–dockerin interactions
has been exploited to construct ‘mini-cellulosomes’,
which contain ‘mini-scaffoldins’ with either speciesspecific cohesins or cohesins from different species39. A
mini-scaffoldin with species-specific cohesins will bind
enzymes only from that species. Mini-scaffoldins that
are constructed to contain cohesins from two or more
different species will bind cognate enzymes from
those species. These mini-cellulosomes have been
used to study phenomena such as cohesin–dockerin
interactions67–69, cellulosomal enzyme synergy70–72, the
synergy with neighbouring enzymes69, the effect of
the CBM on enzyme activity69 and the potential for
metabolic pathway engineering (K. Ohmiya, personal
communication). An example of a mini-cellulosome
constructed from a mini-scaffoldin and enzymes is
illustrated in FIG. 4.
Mini-cellulosomes from C. thermocellum have been
used to demonstrate the one-to-one stoichiometric relationship between a cohesin and a dockerin-containing
endoglucanase and the enhancing effect of the CBD on
cellulosome activity71. Mini-cellulosomes constructed
VOLUME 2 | JULY 2004 | 5 4 7
REVIEWS
from only C. cellulovorans components were used to
investigate the synergy between cellulases70, between cellulases and hemicellulases72, and between a cellulosomal
enzyme and non-cellulosomal enzymes73,74. In all cases,
synergy was observed, indicating that the synergy
between the enzymes in cellulosomes makes the cellulosome structure more effective in attacking the substrate.
The synergy observed between cellulosomes and noncellulosomal enzymes also suggests that the maximum
effectiveness in degrading natural substrates requires the
interaction of cellulosomes and non-cellulosomal
degradative enzymes73,74.
A scaffoldin with three different cohesins that can
bind three enzymes by their cognate dockerins has been
constructed (K. Ohmiya, personal communication).
These researchers hope to show that this designer minicellulosome can organize three tandem enzymes into a
metabolic pathway that is capable of converting a substrate into a desired product by ‘enzyme channelling’ in
a similar manner to that found in vivo. This could lead
to the future development of artificial metabolic pathways with controlled activities for the synthesis of any
desired product.
Practical applications of the CBD. The presence of
CBDs in many scaffoldin proteins has led to the development of some practical applications for the CBD
domain75,76. In a series of recent papers, Shoseyov and
colleagues have shown that fusion proteins containing a
CBD and various other proteins are capable of binding
to a cellulose matrix. These fusion proteins can be used
as a bioreactor77, a plant-growth modulator78,79 and a
protein-purification system80. The advantages of using
a CBD system are that a relatively inexpensive, inert cellulose matrix with excellent physical properties can be
used, a large-scale, safe, affinity-purification procedure
is possible and it can be used to modify physical and
chemical properties in agro-biotechnology, both in vitro
and in vivo.
The Clostridium acetobutylicum cellulosome. When
the C. acetobutylicum genome was sequenced49, it was
observed that several cellulosomal genes were present in
a gene cluster similar to that found in C. cellulolyticum
and C. cellulovorans. However, as mentioned above,
C. acetobutylicum, which is an important species for the
production of acetone and butanol, produces a cellulosome with a molecular mass of about 665 kDa 19
but cannot grow on cellulose20. This is due to either
extremely low production of cellulosomes or a deficiency in the cellulosome that prevents it from degrading crystalline cellulose. At present, much research is
focused on characterizing the C. acetobutylicum cellulosome, with the aim of converting this microorganism
into an active user of cellulosic biomass.
One approach to this problem is to insert the cellulosomal genes from an active cellulosome producer, such
as C. cellulolyticum, into C. acetobutylicum to produce
an active heterologous cellulosome that is capable of
degrading crystalline cellulose81. For this purpose, a
mini scaffoldin gene (CipC1c) from C. cellulolyticum
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| JULY 2004 | VOLUME 2
containing a cellulose-binding motif (CBM3a), one X2
module (a hydrophilic domain of unknown function)
and a cohesin (cohesin 1), and a chimeric scaffoldin
Scaf3 gene containing a cohesin from C. thermocellum
(cohesin 3) fused to the C-terminus of CipC1c (CipC1c–
Coh3t) were transformed into C. acetobutylicum. Both
mini-scaffoldins were produced and secreted by
C. acetobutylicum and both were able to bind their cognate enzymes, indicating that the synthesis of active
mini cellulosomes by C. acetobutylicum is possible.
Another approach has been to overexpress a
homologous mini-cellulosome containing a CBD, two
cohesin domains and a cognate enzyme in C. acetobutylicum82. This approach produced a mini-cellulosome of 250 kDa, with two major subunits of 122 kDa
and 84 kDa, as opposed to the normal 665 kDa. The
mini-cellulosome was unable to degrade avicel or bacterial cellulose, but did show low activity on CMC and
phosphoric-acid-swollen cellulose, as shown previously. These experiments were the first to demonstrate
the in vivo expression of mini-cellulosomes and indicate that C. acetobutylicum and other species can be
transformed with cellulosomal genes to form active
cellulosomes in vivo. This finding will allow the development of many commercially important microorganisms that use cellulosic biomass as an inexpensive
growth substrate.
Heterologous expression of cellulosomal genes. If microorganisms could use cellulose as a growth substrate,
inexpensive biomass such as corn stover, rice straw, sawdust and wheat straw could be used to produce ‘valueadded’ products such as ethanol, amino acids and
organic acids. One approach would be to transform
microorganisms with cellulosomal genes so that cellulosic biomass could be directly converted to valuable
products. The expression of C. cellulovorans EngB in
C. acetobutylicum has been reported83. In another
study, the genes for mini-scaffoldins derived from
C. cellulolyticum and C. thermocellum were introduced
into C. acetobutylicum and expressed as active miniscaffoldins81. With further refinements, it is likely that
cellulosomal genes will be expressed in several different species to allow these organisms to utilize biomass
and agricultural wastes to produce products such as
ethanol, amino acid and organic acids. This will
require the transfer of a scaffoldin gene and a number
of exoglucanase, endoglucanase and hemicellulase
genes into the microorganism of interest.
Improving cellulosomal enzyme properties: DNA
shuffling. A full understanding of the function of the
cellulosome should lead to the synthesis of a maximally
efficient cellulosome with specified properties. As well as
maximizing the synergy both between the cellulosomal
enzymes and between cellulosomes and non-cellulosomal enzymes, another approach would be to modify and
improve the properties of the enzymes. The creation of a
recombinant cellulase with greater heat stability has been
reported84. In this case, DNA shuffling was carried out
between two highly homologous endoglucanases from
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REVIEWS
Future areas for investigation
Box 2 | DNA shuffling
A variety of protein-engineering approaches can be used
to modify the properties of cellulosomal enzymes.
In vitro recombination, or DNA shuffling, is one such
technique that can be used to exchange functional
domains in proteins. The figure shows a schematic
example of a DNA shuffling experiment to create a
recombinant C. cellulovorans endoglucanase with greater
heat stability than the parental enzymes (EngB, a
cellulosomal enzyme and EngD, a non-cellulosomal
enzyme)84. The recombinant clones obtained had
improved stability at 55°C compared with the parental
enzymes, which were only stable up to 45°C.
engB
engD
Cleave with DNase,
then PCR
In vitro recombination
with DNA ligase
The current status of cellulosomal research indicates
that advances in the following areas are possible.
Cellulosome genetics. Although biochemical studies of
cellulosomes have progressed well, the most limiting
aspect of cellulosome research is the lack of genetic
analysis of the cellulosome. The increasing use of, and
improvements in, genetic manipulations in clostridia
will facilitate further studies on the function and regulation of cellulosomal genes, the structure of the cellulosome, and the secretion and extracellular assembly of
the cellulosome.
Assembly of cellulosomes and polycellulosomes. The
assembly process is one of the most intriguing aspects of
cellulosome and polycellulosome synthesis. How are
cellulosomes assembled extracellularly? Are there extracellular chaperones or are chaperone-like functions
incorporated into the cellulosomal proteins? What
determines which enzymes are going to bind to a particular scaffoldin molecule? Are there built-in factors that
determine cohesin–dockerin interactions? How are
polycellulosomes assembled? One approach to answering these questions is to determine whether specific cellulosomal enzymes are bound to specific cohesins present
in the scaffoldin in preference to other cellulosomal
enzymes.
Clone fragments in E. coli
Screen for endoglucanase
activity
Screen for thermal stability
3 clones containing temperature-stable
recombinant endoglucanase
C. cellulovorans. One of the enzymes, EngB, is a cellulosomal enzyme whereas the other enzyme, EngD, is noncellulosomal. Three recombinants were obtained that
had improved stability at 55ºC compared with the
parental enzymes, which were stable up to 45ºC. Not
only were the recombinant enzymes more stable at the
higher temperature, but they were as active as the
parental EngB. So, by using DNA shuffling, it would
seem that further improvements or modifications of
cellulosomal enzymes are possible85 (BOX 2).
NATURE REVIEWS | MICROBIOLOGY
DNA shuffling to improve cellulosomal enzyme properties. DNA-shuffling technology can be used to modify
the properties of cellulosomal enzymes and to improve
the function and specificity of cellulosomes. By using
targeted selection methods, almost any desired enzyme
characteristic can be obtained. It seems likely that continued use of this technology will result in more active
heat-stable enzymes that can function at low and high
pH values and under high and low salt concentrations.
Designer cellulosomes — construction of functionspecific cellulosomes. Cellulosomes with specific dockerin-binding capacities can be created by using cohesins
from various scaffoldins. By creating chimeric scaffoldin
proteins with tandem dockerin-specific cohesins, it
should be possible to construct designer cellulosomes
and mini-cellulosomes with enzymatic pathways in
which efficient substrate channelling occurs. This could
lead to the development of complex and sophisticated
bioreactors and biosensors.
Transformation of cellulosomal genes — conversion of
non-cellulolytic to cellulolytic organisms. By creating
recombinant plasmids containing cellulosomal genes
and by using transformation techniques, it should
become possible to convert several microorganisms into
cellulose degraders. These microorganisms will be able
to use cellulosic biomass and agricultural wastes and
convert them directly to useful products, such as
ethanol, butanol, amino acids and organic acids, that are
currently synthesized from expensive substrates by these
microorganisms.
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REVIEWS
Synergy between cellulosomal enzymes and cellulosomal
and non-cellulosomal enzymes. To efficiently degrade
cellulosic biomass, synergy studies should be carried
out between cellulosomal enzymes from purified cellulosomes with defined enzymatic activities. The
combination of specific enzymes should result in
activity that is much greater than the activity of the
individual enzymes. In addition, synergy studies
between mixtures of cellulosomes and non-cellulosomal
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Acknowledgements
The research reported from our laboratory was supported in part by
a grant from the US Department of Energy.
Competing interests statement
The authors declare that they have no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez: http://www.ncbi.nlm.nih.gov/Entrez/
Clostridium acetobutylicum | Clostridium thermocellum
SwissProt: http://www.ca.expasy.org/sprot/
CipA
FURTHER INFORMATION
Roy Doi’s laboratory: http://biosci.ucdavis.edu/BioSci/Faculty
AndResearch/DisplayFacultyProfile.efm?ResearcherID=1334
Access to this links box is available online.
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