From the Bioénergétique et
Ingéniérie des Protéines, Centre National de la
Recherche Scientifique, Institut de Biologie Structurale et
Microbiologie-Institut Fédératif de Recherche 1, 13402 Marseille, France, the § Department of Biological Chemistry,
The Weizmann Institute of Science, Rehovot 76100, Israel, the
¶ Université de Provence, 13331 Marseille, France, the
Department of Molecular Microbiology and Biotechnology, Tel Aviv
University, Ramat Aviv 69978, Israel, and the ** Department of Food
Engineering and Biotechnology and Institute of Catalysis Science and
Technology, Technion-Israel Institute of Technology, Haifa 32000, Israel
Received for publication, March 8, 2001, and in revised form, April 3, 2001
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ABSTRACT |
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Defined chimeric cellulosomes were produced in
which selected enzymes were incorporated in specific locations within a
multicomponent complex. The molecular building blocks of this approach
are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellum and Clostridium
cellulolyticum, wherein cellulolytic enzymes are incorporated
into the complexes by means of high-affinity species-specific
cohesin-dockerin interactions. To construct the desired complexes, a
series of chimeric scaffoldins was prepared by recombinant means. The
scaffoldin chimeras were designed to include two cohesin modules from
the different species, optionally connected to a cellulose-binding
domain. The two divergent cohesins exhibited distinct specificities
such that each recognized selectively and bound strongly to its
dockerin counterpart. Using this strategy, appropriate
dockerin-containing enzymes could be assembled precisely and by design
into a desired complex. Compared with the mixture of free cellulases,
the resultant cellulosome chimeras exhibited enhanced synergistic
action on crystalline cellulose.
Cellulosomes are macromolecular complexes produced in cellulolytic
microorganisms and designed for efficient degradation of cellulose and
associated plant cell wall polysaccharides (1-5). Cellulosomes are
composed of a collection of subunits each of which comprises a set of
interacting functional modules or domains. A typical cellulosome
contains a cellulose-binding scaffoldin subunit that organizes the
other enzymatic components into the complex by virtue of a
high-affinity interaction among complementary domains. Thus, scaffoldin
contains a cellulose-binding domain (CBD)1 and multiple copies of
cohesin domains. Each cohesin interacts tenaciously with a dockerin
domain on an enzyme subunit, thereby incorporating the enzymes into the complex.
Various reports have indicated that the cohesins of a given
cellulosome appear to recognize all of the dockerin-containing enzymes
within the same species (6, 7), suggesting that the intra-species
cohesin-dockerin interaction is relatively nonspecific. On the other
hand, at least for two clostridial species, Clostridium thermocellum and Clostridium cellulolyticum, the
recognition between their cohesins and dockerins was shown to be
species-specific (8).
In an earlier review (9), we suggested the use of hybrid forms
of cellulosomal components for improved hydrolysis of cellulosic substrates. We now provide experimental evidence demonstrating that
increased synergistic action among cellulolytic enzymes can be achieved
by selective incorporation into cellulosome-like complexes. To this
end, several chimeric scaffoldins and hybrid enzymes were designed. The
chimeric scaffoldins comprised an optional CBD and two cohesin domains
of unlike specificity, one from each clostridial species. Recombinant
enzyme constructs contained a catalytic module together in the same
polypeptide chain with a dockerin domain from either species. The
cellulosome chimeras were assembled in vitro, simply by
combining, in equimolar amounts, three desired components,
i.e. the chimeric scaffoldin and two enzymes. The resultant
cellulosome chimeras exhibited enhanced synergy on a microcrystalline
cellulose substrate. This approach is appropriate for incorporating
other types of enzyme and non-enzyme components into complexes for
biotechnological application.
Plasmids--
Plasmids and encoded proteins are summarized in
Fig. 1. Positive clones were verified by DNA sequencing. BL21(DE3)
(Novagen, Madison, WI) was used as production host for pET derivatives.
pJFAc, encoding for a His tag-containing construct of
the native dockerin-bearing CelA of C. cellulolyticum, was
obtained by inserting the primer
5'-AGCTAGAACACCACCACCACCACCACTAATA-3' into a
HindIII site (the His tag is underlined in all sequences) located at the 3' extremity of the coding region of pA2 (10).
To exchange the native C. cellulolyticum CelA
dockerin, the dockerin-encoding region of C. thermocellum
CelS was amplified from pQE30-docS (11) using the primers
5'-GCCGCCTTAGAAGCCAAGACAAGCCCTAGCCCATCTACTAAATTATAC-3' (AsuII site in bold) and
5'-CCCCCCAAGCTTTTAGTGGTGGTGGTGGTGGTGGTTCTTGTACGCCAATGT-3' (HindIII site in bold). The amplified fragment was
ligated into AsuII-HindIII-linearized pJFAc
resulting in pJFAt.
pETFt was obtained using the overlap-extension polymerase
chain reaction method (12, 13). The DNA encoding the catalytic domain of celF was amplified from pETFc (14, 15) using the forward 5'-GACCTAGGTTGTGCTTCTTCACT-3' (unique
AvrII site in bold) and reverse
5'-TACTTTATATGTCATGCTCGGGAAGAGTATTGCATAAACTC-3' primers. The DNA encoding a C. thermocellum dockerin-domain
was amplified from pQE30-docS using the forward
5'-TTCCCGAGCATGACATATAAAGTACCTGGTACTCCTTCTACTAAATTATACGGCGACGTC-3' and reverse 5'-GTGCTCGAGGTTCTTGTACGGCAATGTATCTAT-3'
primers, introducing a XhoI site (bold) at the 3'
extremity. The two resultant overlapping fragments (overlapping regions
in italics) were mixed, and a combined fragment was synthesized using
the external primers. The fragment was cloned into
AvrII-XhoI-linearized pETFc, thereby generating pETFt.
pETscaf1 was constructed by polymerase chain reaction
amplification of two fragments: the Ct-CBDt-encoding DNA was amplified from p2CBD3 (6) using 5'-GGAATACCATGGTTCCGTCAGACGGTGTG-3' (NcoI, bold) and
5'-GATCCTTAAGAGAATCTGACGGCGGTA-3' (AflIII, bold).
The Cc-encoding region was amplified from pETCipC1 (16) using primers 5'-GATTCTCTTAAGGTTACAGTAGG-3'
(AflIII, bold) and 5'-CGGGATCCTTATTGAGTACCAGG-3'
(BamHI in bold). The fragments were ligated into
NcoI-BamHI-linearized pET9d.
pETscaf2 was constructed similarly. The DNA coding for
Cc was amplified from pETCipC1 using primers
5'-CATGCCATGGGCGATTCTCTTAAAG-3' (NcoI in
bold) and 5'-CCAGGATCGATCGTTACACTACC-3' (PvuI in
bold) and the CBDt-Ct region from p2CBD3 using primers
5'-TGGCACGATCGATCCGACCAAGGGAGC-3' (PvuI in bold)
and 5'-CGCGGATCCTAATCTCCAACATTTAC-3' (BamHI, bold).
pETscaf3 was constructed from pETCipC1 and pCBD3.
The stop codon in the former plasmid was eliminated using primers
5'-TGCAGGAAGTCTTCCAGCTGGAGG-3' (located upstream of a unique
BamHI site) and
5'-CCGCCCCTCGAGTTCCTTTGTAGGTTGAGTACC-3' (XhoI
site in bold, located immediately downstream of the gene). The
amplified fragment was ligated into the original plasmid using the
BamHI-XhoI sites, resulting in pETCip1X.
Ct-encoding DNA was amplified from pCBD3 using primers
5'-GGGCGGCTCGAGCCATCAACACAGCTTGTAACA-3' and
5'-GGGCGGCTCGAGGATCCTATCTCCAACATTTAC-3', thus
introducing a XhoI site (bold) at both extremities. The
resulting fragment was then ligated into the XhoI site of pETCip1X.
pETscaf4 was constructed by amplifying the Ct-encoding region
from p2CBD (6) using NdeI-containing primers
5'-GGGCGGCATATGGTTCCGTCAGACGGT-3' and
5'-GGGCGGCATATGCGGTGTGTTTGTCGGTGT-3' and ligating the polymerase chain reaction product into NdeI-linearized
pETcoh1B (17).
Production and Purification of Recombinant
Proteins--
Escherichia coli was grown at 37 °C to
A600 = 1.5 in Luria-Bertani medium
supplemented with 1.2% glycerol (w/v) and the appropriate antibiotic.
The culture was then cooled to 25 °C (Ac, At, all chimeric
scaffoldins) or 18 °C (Fc and Ft), and isopropyl
thio- Nondenaturing PAGE--
Samples (7.5 µM final
concentration) were mixed at room temperature in 10 mM
Tris-HCl, pH 8, 250 mM NaCl, and 10 mM
CaCl2, and 4 µl were subjected to PAGE (4-15% gradient)
using a PhastSystem apparatus (Amersham Pharmacia Biotech, Uppsala, Sweden).
Gel Filtration--
Gel filtration was performed by HPLC using a
calibrated Zorbax G-250 (Interchim, Montlucon, France) column
equilibrated with 50 mM Tris-HCl, pH 8, 0.4 M
KCl, 10 mM CaCl2, and 0.01% surfactant P20
(BIAcore AB, Uppsala, Sweden) at a flow rate of 1 ml
min Surface Plasmon Resonance (SPR)--
Experiments were performed
using a BIAcore system as described earlier (17) with 10 mM
Tris-maleate, pH 6.5, 2.5 mM CaCl2, and 0.005%
P20 as running buffer (flow rate 25 µl
min Enzyme Activity--
Samples (8 µM in 20 mM Tris-maleate, pH 6.0, 0.1 M NaCl, 10 mM CaCl2, 50 µl) were incubated at 37 °C
with 4 ml of Avicel (8 g liter Design and Preparation of Recombinant Components--
Four
different chimeric scaffoldins were engineered (Fig.
1), each containing two different cohesin
species that exhibit divergent specificities (8). Scaf1 and
Scaf2 are based on the cellulosomal scaffoldin from C. thermocellum in which the two cohesins are separated by an
internal CBD (21). Scaf3 is based on the C. cellulolyticum
cellulosome and contains an N-terminal CBD (22). Scaf4, which lacks a
CBD, was designed to determine whether simple complexation of enzymes
would also promote synergistic activity.
The enzyme components were all based on two established
cellulosomal enzymes: the family-5 CelA (10) and the family-48 CelF (15), herein referred to as Ac and Fc, respectively. To complement the
native enzymes, two hybrid constructs (termed At and Ft) were designed
in which the intrinsic dockerin domain (designated "c") of the
respective C. cellulolyticum enzyme was replaced by a
dockerin domain (designated "t") of differing specificity from the
corresponding family-48 enzyme, CelS (23), from C. thermocellum (Fig. 1). Thus, four different enzyme pairs can be
incorporated onto each chimeric scaffoldin: Ac + At, Ac + Ft, Fc + At,
and Fc + Ft.
The engineered proteins were produced in E. coli
and affinity-purified in one step on either cellulose or
nickel-nitrilotriacetic acid according to the presence of a CBD
or His tag, respectively. The chimeric scaffoldins were found to be
very stable upon storage for several days at 4 °C, whereas low
levels of spontaneous cleavage between the catalytic and the dockerin
modules were detectable for both wild-type and hybrid enzymes.
Analysis of Chimeric Cellulosome Complexes--
Complex formation
in the presence of calcium was verified using three different
techniques: nondenaturing PAGE, gel filtration HPLC, and SPR.
Nondenaturing PAGE clearly demonstrated that near complete complex
formation could be achieved simply by mixing the desired components
in vitro (Fig. 2). Binary or
ternary mixtures of the free proteins resulted in a single major band
of altered electrophoretic mobility. These results were confirmed by
gel filtration HPLC of stoichiometric mixtures of the same components (Fig. 3). The peaks corresponding to the
free components disappeared and were replaced by a major peak of higher
molecular mass. Apparent masses of 150 and 200 kDa were found for
binary and ternary complexes, respectively. The results are in good
agreement with the expected sizes of the desired cellulosome chimeras.
A minor peak (~400 kDa) was also observed, which may suggest that low
levels of oligomerization may also occur.
SPR-based estimates of affinity parameters for the interaction
of Ac and Fc with the various chimeric scaffoldins revealed similar
Kd values (1-2.5 10
The interaction between the chimeric scaffoldins and different
combinations of the enzyme components is presented in Fig. 4A. In this set of
experiments, enzymes bearing C. cellulolyticum dockerins
were introduced prior to those from C. thermocellum. Under
saturating or near saturating conditions, the amounts of enzyme, Ac or
At (85-95 RU) and Fc or Ft (130-140 RU), that were bound to the
immobilized chimeric scaffoldin were in close agreement with the
calculated Rmax (maximum binding capacity of
analyte in RU), thus confirming a stoichiometry of 1:1:1 for the
ternary complexes. The results indicate that complexation between the enzymes and the C. cellulolyticum cohesin does not disturb
subsequent binding of the second enzyme to the C. thermocellum cohesin.
In another set of experiments, Scaf4 was employed to examine
whether assembly of the chimeric cellulosomes is affected by the mode
of interaction among the different components. For this purpose, the
desired enzymes were either introduced simultaneously or in reverse
order (Fig. 4B). The resultant sensograms clearly demonstrated that each type of cohesin binds in an independent manner
to the appropriate dockerin-containing enzyme irrespective of the order
of incorporation.
Enhanced Synergy of Cellulosome Chimeras--
Cellulosome chimeras
were generally found to be more active than simple mixtures of the free
enzyme pairs (Fig. 5). Bringing two
cellulolytic modules into close proximity clearly enhances the
catalytic efficiency on crystalline cellulose. The observed enhancement of activity increased with incubation time (data not shown)
and reached a maximum after 24 h. The cellulolytic activity of the
enzyme complexes was further improved when the scaffoldin contained a
CBD. When attached to the CBD-containing scaffoldins (Scaf1,
Scaf2, and Scaf3), the heterogeneous enzyme mixtures (Ac + Ft or
Fc + At) exhibited an enhanced synergy of about 2-3-fold. In the
absence of a CBD (Scaf4), enhanced levels of synergy (~1.5-fold) were
also observed. The most effective combinations of components were Scaf1
and Scaf2 together with enzymes Fc and At. Interestingly, the
homogeneous mixture of family-5 enzymes (Ac and At) also displayed levels of enhanced synergy roughly equivalent to those of the heterogeneous mixture of Ac and Ft. In contrast, chimeric complexes containing only the family-48 enzymes (Fc and Ft) showed little if any
synergistic action.
The results of the present work provide experimental verification
of earlier proposals whereby cellulosome chimeras can be constructed by
combining appropriate dockerin-containing enzymes and recombinant
cohesin-containing scaffoldins (9). The concept is shown schematically
in Fig. 6. The construction of the
desired cellulosome chimeras is generated by the tenacious binding
interaction between complementary modules, each located on
complementary interacting components, i.e. cohesins on
chimeric scaffoldins and dockerins on enzyme subunits. The resultant
multicomponent protein complexes assume some or all of the functional
characteristics of the parent components, such that their proximity
within the same complex leads to enhanced synergistic activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactoside was added to a final
concentration of 400 µM (Ac, At, all chimeric scaffoldins) or 40 µM (Fc and Ft). After 16 h, the
cells were centrifuged (10 min, 3000 × g), resuspended
in 30 mM Tris-HCl, pH 8, and broken in a French press. The
purification of His-tagged proteins (see Fig. 1) was performed on
nickel-nitrilotriacetic acid resin (18). Scaf1 and Scaf2
were purified on Avicel as described previously (6). The concentration
of purified proteins was estimated by absorbance (280 nm) in 6 M guanidine hydrochloride based on the known amino acid
composition of the desired protein.
1. Samples were diluted in the same buffer
(14 µM final concentration), and 10 µl were loaded onto
the column. Chromatographic data were recorded at 280 nm.
1). Biotinylated chimeric scaffoldins
(19), 95-100 RU of Scaf1 and Scaf2, 110-120 RU of Scaf3, and
65-70 RU of Scaf4, were coupled to streptavidin-bearing sensor chips.
The cellulases were diluted to 5 nM in the same buffer and
allowed to interact with the immobilized chimeric scaffoldin by
injections of 600 s.
1, Fluka,
Buchs, Switzerland). At predetermined time intervals, 1-ml aliquots
were centrifuged and examined for reducing sugars (20). The final
protein concentration during the assay was 0.1 µM, and
glucose was used as the standard for reducing sugar analysis.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the recombinant
proteins used in this study. White (C. thermocellum) and gray (C. cellulolyticum)
symbols denote the source of the respective domains (see
"Key to symbols"). The cohesin domains are
numbered according to their original position in the
respective native cellulosomal scaffoldin. A hydrophilic domain
(X) of unknown function is part of the C. cellulolyticum scaffoldin. In the shorthand notation for the
enzymes, A and F represent the catalytic domains
from C. cellulolyticum cellulosomal family-5 CelA and
family-48 CelF, respectively; c and t refer to
the dockerin domains derived from C. cellulolyticum or
C. thermocellum, respectively.
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Fig. 2.
Electrophoretic mobility of components and
assembled complexes on nondenaturing gels. A,
Scaf1-based cellulosome chimeras with Fc and At: lane 1,
Scaf1 alone; lane 2, Fc alone; lane 3, At alone;
lane 4, binary mixture of Scaf1 and Fc; lane 5,
binary mixture of Scaf1 and At; lane 6, ternary mixture of
Scaf1, Fc and At. B, Scaf3-based chimeras with the same
enzyme components: the lanes are the same as in A
except for the substitution of the designated chimeric scaffoldin. In
each lane, equimolar concentrations (7.5 µM) of the
indicated proteins were used, except in the control in lane
3, where 30 µM At was applied because of the
characteristic diffuse banding pattern of the free protein. Similar
quality gels were obtained for Scaf2- and Scaf4-based systems.
Note that the minor bands observed in several of the lanes do not
reflect significant proteolytic cleavage of the dockerin domain as
indicated in separate SDS-PAGE experiments (data not shown).
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Fig. 3.
Gel filtration HPLC analysis of Scaf3-based
components and assembled chimeric cellulosome complexes. Injected
proteins are indicated on each chromatogram. Vertical lines
indicate the positions of molecular mass markers: blue dextran,
V0 > 2 MDa; -amylase, 200 kDa; alcohol
dehydrogenase, 150 kDa; bovine serum albumin, 66 kDa; ovalbumin, 44 kDa; and carbonic anhydrase, 29 kDa. Data of similar quality were also
obtained for chimeric cellulosomes based on Scaf1, Scaf2, and
Scaf4. Note that the chimeric scaffoldin elutes at a significantly
higher position than its expected molecular mass, an effect possibly
related to an extended arrangement of its modules or the known tendency
of uncomplexed cohesins to dimerize (30-32). On the other hand, the
elution of both dockerin-containing enzymes At and Fc is retarded. This
effect appears to reflect the presence of the dockerin domain because
the recombinant forms of the dockerin-free (truncated) enzymes elute
more rapidly, in agreement with the expected molecular mass (data not
shown).
10
M). These results were in accordance with previously
determined values for the cohesin-dockerin interaction in C. cellulolyticum (17). The affinity of At and Ft, however, was too
high (>1011 M
1) to
be determined using the BIAcore system, suggesting a much stronger
interaction in the C. thermocellum cellulosome.
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Fig. 4.
SPR sensograms showing sequential binding of
cellulases onto immobilized chimeric scaffoldins. A,
Scaf1-, Scaf2-, and Scaf3-based systems in which the order of
injection is the same, where the designated C. cellulolyticum enzymes are followed by those of C. thermocellum; and B, Scaf4-based systems, in which the
order of injection is altered, where c,t = C. cellulolyticum enzymes followed by C. thermocellum,
t,c = C. thermocellum followed by C. cellulolyticum, and (c+t) = simultaneous injection
of the designated enzymes. In all graphs, curve 1 = injection of Ac and At (solid lines); curve
2 = injection of Ac and Ft (dotted lines);
curve 3 = injection of Fc and At
(dashed lines); and curve 4 = injection of Fc and Ft (dot-dashed lines). The black
bars above the sensograms indicate the duration of the
injection of the desired cellulase (5 nM each). Note the
characteristic broad curve of enzymes containing the C. cellulolyticum-derived dockerins (first injection) compared with
the sharp curves of those from C. thermocellum (second
injection), reflecting the remarkably high affinity of the latter
cohesin-dockerin interaction.
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Fig. 5.
Activity of chimeric cellulosomes on
microcrystalline cellulose. The designated pairs of enzymes were
mixed in stoichiometric amounts with the indicated chimeric scaffoldin.
The activity on Avicel of the resultant chimeric cellulosome complexes
was compared with that of equimolar concentrations of the free enzymes
alone or in the presence of free CBD from CipC. Microcrystalline
cellulase activity represents the amount of soluble sugars released
(µM), measured after 24 h of incubation at 37 °C,
to a total level of solubilization estimated at about 2% of the
original amount of substrate. Glucose was used as a standard. The data
show the mean and standard deviation of three independent
experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Schematic representation of the
approach. A chimeric scaffoldin is produced containing an optional
carbohydrate-binding module (CBM) and multiple
(n) cohesin modules of different dockerin specificities. The
dockerin counterparts comprise distinct modules as part of the
polypeptide chains of the desired protein component (e.g.
enzymes A, B, C, and N). The chimeric cellulosome
complex is constructed by simply mixing in solution the chimeric
scaffoldin and dockerin-containing components. The resultant complex
exhibits enhanced synergistic functions due to the close proximity of
the interacting components. (See "Key to symbols" on
Fig. 1).
In previous works, single enzyme components of the C. thermocellum cellulosome have been shown individually to exhibit enhanced activity on insoluble cellulose substrates upon incorporation via a suitable scaffoldin into a cellulosome-like complex. In an early study, Wu et al. (24) reported that a purified cellulosomal cellulase (CelS) can be combined with the native scaffoldin, leading to an increase in hydrolytic activity of the complex on crystalline cellulose. More recently, Kataeva et al. (25) showed that a different cellulosomal enzyme (endoglucanase CelD) interacts stoichiometrically with scaffoldin constructs, and the resultant complexes were found to degrade cellulose in a synergistic manner. Yet another cellulosomal enzyme (endoglucanase CelE) was shown by Ciruela et al. (26) to exhibit enhanced crystalline cellulase activity upon prior interaction with the full-length recombinant scaffoldin. In each of these latter studies, only one enzyme type was incorporated into the given complexes, and the observed enhancement of activity was attributed mainly to targeting of the enzyme to the solid substrate by the scaffoldin-borne CBD. Finally, Bhat and colleagues (27, 28), reconstituted a simplified cellulosome by combining purified preparations of native cellulosomal components, including the full-size scaffoldin with selected enzymatic subunits. The resultant reconstituted complex exhibited enhanced synergy on cellulose compared with the activity of the mixture of free enzymes.
In this study, we investigated the synergistic interaction of a heterogeneous system wherein two different recombinant cellulosomal enzymes were incorporated selectively into discrete artificial cellulosome complexes by virtue of their vectorial interaction with defined chimeric scaffoldins. Each of the chimeric scaffoldins contained two cohesins of divergent specificities. The scaffoldins were designed to examine the contribution of location of the designated modules therein. Thus, Scaf1 and Scaf2 both contain cohesins from the two species, but their position vis-à-vis the internal CBD is reversed. The content of Scaf3 is very similar to that of Scaf2, except its CBD is at an N-terminal rather than an internal position. Finally, the cohesins of Scaf4 are identical to Scaf1, except Scaf4 lacks a CBD. The data indicate that the activity levels of the chimeric cellulosomes were significantly higher than those of the combined free enzyme systems, thereby demonstrating that proximity of the different enzymes within the complex indeed appears critical to the observed enhancement of synergistic action. The presence of a targeting CBD in the chimeric scaffoldin conferred an additional contribution toward the final level of enzyme activity displayed by a given complex.
Since the enzymes from C. thermocellum are thermophilic, whereas those from C. cellulolyticum are mesophilic, enzymes from the two bacteria would be incompatible in the same complex. Thus, the mesophilic C. cellulolyticum cellulases (family-5 CelA and family-48 CelF) were selected for this work because their recombinant forms have already been shown to act synergistically in the free state on crystalline cellulose (33). The incorporation of the enzymes into defined chimeric cellulosomes provided a further enhancement of 2-3-fold.
It is interesting to note that some enzyme combinations proved better than others. It is currently unknown why complexes composed of the homogeneous mixture of Ac and At resulted in enhanced synergy whereas those of Fc and Ft displayed no synergy. It is also unclear why those containing the combination of Fc and At consistently showed heightened levels of synergy over those containing Ac and Ft. A possible stabilizing effect of the cohesin-dockerin interaction on the Fc and/or At constructs could account for the observed differences. In any case, improved levels of synergy may eventually be expected by using higher degree systems that contain additional or other combinations of enzymes.
An analysis of complex formation by three complementary methods revealed that the selectivity and stoichiometry of the cohesin-dockerin interaction are strictly maintained. In this context, the individual dockerin-containing enzymes were incorporated into the desired chimeric scaffoldins via binding to the matching cohesins, in an independent manner, irrespective of the order of application. The enzymes could be added sequentially or mixed together and applied concurrently with the same effect, complete and selective incorporation of the desired components.
This work represents an initial demonstration of the approach
to and use of the cohesin-dockerin interaction as a selective type of
molecular adapter for incorporating desired proteins into multicomponent complexes. In doing so, we have used the components of
two well characterized cellulosome systems, which exhibit divergent cohesin-dockerin specificities. The present system can be refined and
elaborated in several ways. To extend the system, cohesin and dockerin
pairs can be used from other cellulosome species (3) to selectively
incorporate additional enzyme components into higher order complexes.
Alternatively, mutagenesis experiments may provide tailor-made
specificities for this purpose (8, 11, 29). Moreover, the genetic
approach can be applied to increase the tenacity of the
cohesin-dockerin interaction, thereby reinforcing the stability of the
resultant chimeric cellulosomes. In any event, the capacity to control
the specific incorporation of enzymatic and non-enzymatic components
into defined chimeric cellulosome complexes should have considerable
biotechnological value for a broad variety of applications (9).
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ACKNOWLEDGEMENTS |
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We are grateful to S. Champ and O. Valette for expert technical assistance.
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FOOTNOTES |
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* This work was supported by a contract from the European Commission (Fourth Framework, Biotechnology Programme, BIO4-97-2303) and by grants from the Israel Science Foundation (administered by the Israel Academy of Sciences and Humanities, Jerusalem). Additional support was provided by the Otto Meyerhof Center for Biotechnology, established by the Minerva Foundation (Munich, Germany), and by funds from the Technion-Niedersachsen Cooperation (Hannover, Germany).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: +972-8-934-2373; Fax: +972-8-946-8256; E-mail:
bfbayer@wicc.weizmann.ac.il.
Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M102082200
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ABBREVIATIONS |
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The abbreviations used are: CBD, cellulose-binding domain; HPLC, high-performance liquid chromatography; PAGE (nondenaturing), polyacrylamide gel electrophoresis (in the absence of detergent); RU, resonance units; SPR, surface plasmon resonance.
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REFERENCES |
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