Design and Production of Active Cellulosome Chimeras

SELECTIVE INCORPORATION OF DOCKERIN-CONTAINING ENZYMES INTO DEFINED FUNCTIONAL COMPLEXES*

Henri-Pierre FierobeDagger , Adva Mechaly§, Chantal TardifDagger , Anne BelaichDagger , Raphael Lamed||, Yuval Shoham**, Jean-Pierre BelaichDagger , and Edward A. Bayer§DaggerDagger

From the Dagger  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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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.

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-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.

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-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.

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-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

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.


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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.

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.


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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; beta -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).

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 10-10 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.

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.


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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.

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.


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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

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.


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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).

    ACKNOWLEDGEMENTS

We are grateful to S. Champ and O. Valette for expert technical assistance.

    FOOTNOTES

* 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.

Dagger Dagger 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Felix, C. R., and Ljungdahl, L. G. (1993) Annu. Rev. Microbiol. 47, 791-819[CrossRef][Medline] [Order article via Infotrieve]
2. Béguin, P., and Lemaire, M. (1996) Crit. Rev. Biochem. Mol. Biol. 31, 201-236[Abstract]
3. Bayer, E. A., Chanzy, H., Lamed, R., and Shoham, Y. (1998) Curr. Opin. Struct. Biol. 8, 548-557[CrossRef][Medline] [Order article via Infotrieve]
4. Bayer, E. A., Shimon, L. J. W., Lamed, R., and Shoham, Y. (1998) J. Struct. Biol. 124, 221-234[CrossRef][Medline] [Order article via Infotrieve]
5. Shoham, Y., Lamed, R., and Bayer, E. A. (1999) Trends Microbiol. 7, 275-281[CrossRef][Medline] [Order article via Infotrieve]
6. Yaron, S., Morag, E., Bayer, E. A., Lamed, R., and Shoham, Y. (1995) FEBS Lett. 360, 121-124[CrossRef][Medline] [Order article via Infotrieve]
7. Lytle, B., Myers, C., Kruus, K., and Wu, J. H. D. (1996) J. Bacteriol. 178, 1200-1203[Abstract]
8. Pagès, S., Belaich, A., Belaich, J.-P., Morag, E., Lamed, R., Shoham, Y., and Bayer, E. A. (1997) Proteins 29, 517-527[CrossRef][Medline] [Order article via Infotrieve]
9. Bayer, E. A., Morag, E., and Lamed, R. (1994) Trends Biotechnol. 12, 378-386
10. Fierobe, H.-P., Gaudin, C., Belaich, A., Loutfi, M., Faure, F., Bagnara, C., Baty, D., and Belaich, J.-P. (1991) J. Bacteriol. 173, 7956-7962[Medline] [Order article via Infotrieve]
11. Mechaly, A., Yaron, S., Lamed, R., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Shoham, Y., and Bayer, E. A. (2000) Proteins 39, 170-177[CrossRef][Medline] [Order article via Infotrieve]
12. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 51-56[CrossRef][Medline] [Order article via Infotrieve]
13. Meza, R., Nufiez-Valdez, M.-E., Sanchez, J., and Bravo, A. (1996) FEMS Microbiol. Lett. 145, 333-339[CrossRef][Medline] [Order article via Infotrieve]
14. Reverbel-Leroy, C., Belaich, A., Bernadac, A., Gaudin, C., Belaich, J.-P., and Tardif, C. (1996) Microbiology 142, 1013-1023[Abstract]
15. Reverbel-Leroy, C., Pagés, S., Belaich, A., Belaich, J.-P., and Tardif, C. (1997) J. Bacteriol. 179, 46-52[Abstract]
16. Pagès, S., Belaich, A., Tardif, C., Reverbel-Leroy, C., Gaudin, C., and Belaich, J.-P. (1996) J. Bacteriol. 178, 2279-2286[Abstract]
17. Fierobe, H.-P., Pagès, S., Belaich, A., Champ, S., Lexa, D., and Belaich, J.-P. (1999) Biochemistry 38, 12822-12832[CrossRef][Medline] [Order article via Infotrieve]
18. Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975) Nature 258, 598-599[Medline] [Order article via Infotrieve]
19. Bayer, E. A., and Wilchek, M. (1990) Methods Enzymol. 184, 138-160[Medline] [Order article via Infotrieve]
20. Park, J. T., and Johnson, M. S. A. (1949) J. Biol. Chem. 181, 149-151[Free Full Text]
21. Gerngross, U. T., Romaniec, M. P. M., Kobayashi, T., Huskisson, N. S., and Demain, A. L. (1993) Mol. Microbiol. 8, 325-334[Medline] [Order article via Infotrieve]
22. Pagès, S., Belaich, A., Fierobe, H.-P., Tardif, C., Gaudin, C., and Belaich, J.-P. (1999) J. Bacteriol. 181, 1801-1810[Abstract/Free Full Text]
23. Wang, W. K., Kruus, K., and Wu, J. H. D. (1993) J. Bacteriol. 175, 1293-1302[Abstract]
24. Wu, J. H. D., Orme-Johnson, W. H., and Demain, A. L. (1988) Biochemistry 27, 1703-1709
25. Kataeva, I., Guglielmi, G., and Béguin, P. (1997) Biochem. J. 326, 617-624[Medline] [Order article via Infotrieve]
26. Ciruela, A., Gilbert, H. J., Ali, B. R. S., and Hazlewood, G. P. (1998) FEBS Lett. 422, 221-224[CrossRef][Medline] [Order article via Infotrieve]
27. Bhat, M. K. (1998) Recent Res. Dev. Biotechnol. Bioeng. 1, 59-84
28. Bhat, S., Goodenough, P. W., Bhat, M. K., and Owen, E. (1994) Int. J. Biol. Macromol. 16, 335-342[Medline] [Order article via Infotrieve]
29. Mechaly, A., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Lamed, R., Shoham, Y., and Bayer, E. A. (2001) J. Biol. Chem. 276, 9883-9888[Abstract/Free Full Text]
30. Spinelli, S., Fierobe, H. P., Belaich, A., Belaich, J. P., Henrissat, B., and Cambillau, C. (2000) J. Mol. Biol. 304, 189-200[CrossRef][Medline] [Order article via Infotrieve]
31. Tavares, G. A., Béguin, P., and Alzari, P. M. (1997) J. Mol. Biol. 273, 701-713[CrossRef][Medline] [Order article via Infotrieve]
32. Shimon, L. J. W., Bayer, E. A., Morag, E., Lamed, R., Yaron, S., Shoham, Y., and Frolow, F. (1997) Structure 5, 381-390[Medline] [Order article via Infotrieve]
33. Reverbel-Leroy, C. (1996) Ph.D. thesis, La Cellulase CelF: Un Composant Majoritaire du Cellulosome de Clostridium cellulolyticum , Université de Provence, Aix-Marseille, France


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