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reviews 44 Williams, W. V., Kieber, E. T., Weiner, D. B., P,ubin, D. H. and Greene, M. I. (1991)J. Biol. Chem. 266, 9241-9250 45 Taub, P,.. et al. (1989)J. Biol. Chem. 264, 259-265 46 Saragovi, H. U., Fitzpatrick, D., P,.aktabutr, A., Nakanishi, H., Kahn,

M. and Greene, M. I. (1991) &ience 253, 792-795 47 Chen, S. et al. (1992) Proc. Natl Acad. Sci. USA 89, 5872-5876 48 Holliger, P., Prospero, T. and Winter, G. (1993) Proc. NatlAcad. Sci. USA 90, 6444-6448

The cellulosome - a treasuretrove for biotechnology Edward A. Bayer, Ely Morag and Raphael Lamed The cellulases of many cellulotytic bacteria are organized into discrete multienzyme complexes, called cellulosomes. The multiple subunits of cellulosomes are composed of numerous functional domains, which interact with each other and with the cellulosic substrate. One of these subunits comprises a distinctive new class of noncatalytic scaffolding polypeptide, which selectively integrates the various cellulase and xylanase subunits into the cohesive complex. Intelligent application of cellulosome hybrids and chimeric constructs of cellulosomal domains should enable better use of cellulosic biomass and may offer a wide range of novel applications in research, medicine and industry. Cellulose is the most abundant renewable source of carbon and energy on the Earth. The chemical simplicity of cellulose belies its structural complexity and stability - properties that account for its contribution to modern society in the form of paper and, as cellulosic waste, as a major pollutant worldwide 1. As cellulose is a very stable polymer, effective hydrolysis of it requires the cellulolytic enzymes of several different microorganisms to act synergistically2. For the greater part of this century, cellulolytic microbes and their cellulase systems have been considered for use in the industrial conversion of cellulosic biomass. However, eventually, it became apparent that natural systems are not necessarily compatible with industry. Advanced engineering techniques alone are not sufficient to enable viable processes for solubilizing cellulosics to be designed, and it was realized that more had to be learnt about the enzymes and microbes that mediate cellulolysis. In recent years, multienzyme complexes, known as cellulosomes (see Glossary), have been identified in many cellulolytic microorganisms3,4. These complexes are dedicated to the efficient degradation of cellulose

E. A. Bayer and E. Morag are at the Department of Biophysics, The Weizmann Institute of Science, Rehovot, Israel. R. Lamed is at the Department of Molecular Microbiology and Biotechnology, University of Tel Aviv, Ramat Aviv, Israel.

© 1994, Elsevier Science Ltd

and hemicellulose. This article describes some of the structural features of cellulosomes that have been elucidated from combined biochemical and molecular biology studies and discusses the impact of an improved understanding of cellulosome structure and function on the controlled hydrolysis of lignocellulose. Other potential applications ofcellulosome technology are also discussed. The cellulosic ecosystem Cellulolytic microorganisms do not occupy an ecological niche on their own; they exist in concert with many other cellulolytic and noncellulolytic strains of bacteria and fungi 5 (Fig. 1). Nevertheless, the cellulolytic strain(s) plays a vital role in the cellulosic ecosystem as the predominant polymer-degrading species. In the plant, cellulose is usually coated by other polymers, predominantly xylan and lignin, which also hinder cellulolysis. Each of these protective polymers is characterized by a different intrinsic chemical and structural arrangement and, consequently, different groups of microorganisms and enzymes are required to degrade the different types of polymer. Xylan is degraded quite readily by xylanases6; however, hgnin poses more of a problem 7, in that the degradation process requires molecular oxygen, and the degradation products are often toxic to the cellulolytic microorganisms and inhibitory to their enzymes: TIBTECHSEPTEMBER1994 (VOL 12)

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Figure 1

Schematic description of a characteristic cellulosic ecosystem. Cellulolytic strains, aided by xylan- and lignin-degrading microbes, play a central role in solubilizing plant matter, primarily to low-molecular-weight sugars. The excess sugars and other cellular end-products are further assimilated by a host of satellite microorganisms, thus contributing to a well-balanced microenvironment.

Glossary C e l l u l o s o m e - A discrete, multienzyme protein complex responsible for the efficient degradation of cellulosic sub-

strates. C B D - Cellulose-binding domain associated with scaffoldin and/or catalytic components of the cellulosome. C o h e s i n - Subunit-binding domain of scaffoldin, which is responsible for integrating catalytic subunits into the

cellulosome complex. D o c k ~ r i n - Docking domain of catalytic subunits that

interacts with a cohesin domain of the scaffoldin subunit. Linker - Relatively short sequence that links the various domains of the cellulosomal subunits. - Protuberance-like cell-surface organelle, which contains multiple copies of the cellulosome and a fibrous connective matrix.

Protubozyme

S c a f f o l d i n - Definitive subunit of the cellulosome that organizes the catalytic subunits into a cohesive complex.

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Once the xylan and lignin have been removed, the exposed cellulose is degraded by the cellulase systems of cellulolytic bacteria and fungi 8,9, producing vast amounts of cellobiose and some glucose. Cellobiose is often inhibitory to the cellulase system, and the cellulolytic process would come to a halt rapidly, were it not for the presence of other saccharolytic microorganisms in the ecosystem. These utilize the excess cellobiose produced, thus enabling the cellulolytic strains to continue to degrade the cellulose. The saccharolytic microorganisms also assimilate excess sugars produced by the xylanolytic and lignindegrading species. The end-products produced by the various microbes include volatile fatty acids, alcohols and molecular hydrogen. Acetogenic bacteria convert a wide variety of such substrates to acetate which, together with hydrogen, serves as a substrate for the methanogens. The saccharolytic bacteria are therefore dependent on the overproduction of soluble sugars by the seemingly altruistic polymer-degrading microbes. Altruism, however, is not a normal characteristic of nature, and it can be anticipated that satellite strains reciprocate in some way.

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i!l ilii!¸,

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Figure 2 Disposition and subunit composition of the cellulosome of Clostridium thermocellum at increasing levels of resolution. (A) Scanning electron mmrograph (SEM)of a single, protubozyme-adornedbacterial cell (magnification x 20 000). (B) Transmissionelectron micrograph (TEM)of anticellulosome-labeledcells (magnification x 50 000). (C) TEM of labeled bacterium bound to cellulose, showing protracted protubozyme that connects the cell to the substrate (magnification x 50 000). (D) Negativelystained preparation of purified cellulosome (magnification × 220 000). (E) Purified cellulosomesadsorbed to cellulose fibers (magnification × 220 000). (F) Subunit profile of the isolated cellulosome separated by sodium dodecyl sulphate polyacrylamidegel electrophoresis (SDS-PAGE).

In return for the provision of comestibles, the satellite species may serve as scavengers, or produce protective agents that neutralize the toxic effects of lignin-degrading fungi. They may also provide essential vitamins and other nutrients that can be utilized by the cellulolytic strains. In any case, there is an exquisite interdependence among the polymerdegrading and satellite microbes, with respect to their contribution to, and utilization of, the pool of cellular end-products. The cellulase system of a given microbe is, thus, closely attuned to its environment. The intricate composition of the cellulase system and the nature of the enzymes that it comprises have been dictated by the processes of evolution. A specific cellulase contributes an important set of qualities to the cellulase system as a whole, within the confines of its ecosystem and, in turn, is subject to the elaborate set of controls imparted by the other members of that ecosystem. Once a cellulolytic microorganism has been segregated from its natural ecosystem for use in a biotechnological application, the delicate harmony within the organism's environment is lost. The isolated cellulase system and its individual components bring along an impressive assortment of excess molecular baggage that is relevant to its natural environment, but is alien to the purposes of the unsuspecting biotechnologist. The cellulosome Historically, fungal cellulases have been easier to study than bacterial systems, as the bacterial enzymes tend to form aggregates. However, recently, it was discovered that these aggregates have an important physiological significance. The cellulase system of the anaerobic cellulolytic bacterium, Clostridium thermocellum, was shown to consist of a discrete multifunctional, multienzyme complex, which appeared

to account for the efficient solubilization of insoluble cellulose by this organism 1°. This complex was called the cellulosome 3,4, and is considered today as one of the major molecular paradigms of bacterial cellulolysis. The cellulosome in C. thermocellum comprises numerous subunits, which are packed into polycellulosomal protuberance-like organelles 11,12, known as protubozymes (see Glossary) (Fig. 2). The cellulosomes mediate cellular adhesion to cellulose and, upon binding, the protubozymes undergo a dramatic conformational change, forming protracted contact corridors between the cell and the substrate. The process is further facilitated by noncellulosomal cellulases and, as the cell matures, celhilosomes are also released into the extracellular matrix, where they continue their cellulolytic activity. High-resolution electron micrographs of isolated cellulosomes confirm the multisubunit structure (Fig. 2d). The majority of these subunits are enzymes, i.e. cellulases and xylanases. The structure of the cellulosome is exceptionally stable 4, yet flexible, and interaction with the substrate is thought to promote conformational rearrangement 13. These data reinforce the original belief 3,10 that the organization of the various complementary enzymes (notably the endo- and exoglucanases) into a defined cellulosome complex serves to promote their synergistic action. At an early stage in cellulosome research, a multifunctional noncatalytic subunit (originally called S1) was noted 10,14. It was suggested that this subunit harbored the cellulose-binding function of the complex, that it anchored the cellulosome into the cell surface, and_that it was responsible for organizing the enzymic components into the complex. This subunit, termed scaffoldin (see Glossary), is the core of the cellulosome structure. TIBTECHSEPTEMBER1994 (VOL12)

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Figure 3 Simplified model of a typical cellulosome. The structure in yellow represents the scaffolding subunit, scaffoldin and the other catalytic (cellulolytic) subunits are shown in darker shades of blue, green and violet. Scaffoldin comprises two main types of subcomponent: a cellulose-bindingdomain (CBD) and multiple copies (numbered) of subunit-bindingdomains, cohesins, which are interconnected by linker sequences. The catalytic subunits comprise one or more catalytic domains and a docking domain, dockerin, which may also be separated by linkers. In addition, some catalytic subunits may also contain a CBD. The catalytic subunits are integrated into the cellulosome complex by the mutual interaction between their resident docking domains and the cohesins of th~ scaffoldin subunit. Both scaffoldin and some of the catalytic subunits contain other domains (not shown), the functions of which are currently unknown.

Modular structure o f cellulases and xylanases Cloning and sequencing studies have provided new insight into the interrelationship o f cellulases and xylanases ls-17. These enzymes are organized into functional domains 18, which were originally identified either by expression o f a portion o f a cloned gene encoding the enzyme, or by proteolytic treatment o f the intact enzyme; the sequence o f interest is then examined for the desired activity. With the accumulation o f a growing library o f cloned genes, the function of a newly sequenced domain can now be deduced from sequence homology 19. All cellulases and xylanases, of course, possess a catalyric domain, the sequence o f which determines the family to which a given enzyme belongs 2°. The classification o f these enzymes traverses broad evolutionary and physiological boundaries, and members of a single family group may emanate from fungi and bacteria, anaerobes and aerobes, mesophiles and thermophiles. Conversely, the various cellulases of a single microbe may belong to several family groups. In some cases, an individual cellulase may bear more than one catalytic domain, each of which belongs to a different family Some, but not all, cellulases also have a distinct cellulose-binding domain (CBD) (see Glossary) (Ref. 2•). Fungal and bacterial CBDs appear to be markedly different, with the bacterial form being much larger. There are also different types of bacterial CBDs, many TIBTECHSEPTEMBER1994 (VOL12)

of which have yet to be classified as, in some cases, cellulose-binding activity can be detected, but the sequence data cannot be aligned with the sequences of well-characterized CBDs. Further research in this area will undoubtedly lead to the discovery o f additional CBD families. Some bacteria] cellulases also contain a relatively small domain that was initially described in C. thermocellum. This domain exhibits a docking function 22, and comprises a 22-residue reiterated (or duplicated) segment, separated by a distinctive short segment o f 9-15 amino acids. This type o f domain, known as dockerin (see Glossary), mediates the attachment o f the catalytic subunit to the cellulosome in C. tkermocellum23. The dockerin domains are usually connected to one another by linking segments, known as linkers (see Glossary) TM. These segments are frequently (but not invariably) rich in proline, and threonine and/or serine residues, and are often glycosylated. The linkers bear some resemblance to cell-wall extensins in plants 24, and they may play a similar structural role in the cellulases. The domains o f scaffoldin The genes o f two similar noncatalytic subunits o f the cellulase systems o f two different cellulolytic bacteria, C. tkermocellum and C. cellulovorans, have recently been sequenced 25,26. Both genes encode large

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Figure 4 Designer cellulosomes - future potential for improving the industrial degradation of cellulosics. Heterologous constructs can be prepared, using chemical, biochemical and/or genetic means. Exogenous enzyme types can be incorporated into the intact cellulosome using bifunctional crosslinking reagents. Alternatively, enzyme-dockerin chimeras can be combined either with a purified scaffoldin subunit, or with recombinant forms of scaffoldin that contain appropriate cohesins.

polypeptides o f similar size (~1800 amino acid residues) and both proteins have a single CBD, which bears striking h o m o l o g y to the well-characterized type o f C B D found in other bacterial cellulases. Both proteins have nine distinct, but closely related, domains - the cohesins (see Glossary) - which evidently interact with the other (catalytic) subunits to form the cohesive cellulosome structure. These characteristics indicate that both polypeptides may be classified as scaffoldins. The scaffoldins from both C. thermocellum and C. cellulouoransalso have tinkers that separate the cohesin domains. T h e linkers from C. thermocellum are rich in proline and threonine residues, and are glycosylated on threonine residues with a very unusual oligosaccharide that probably belongs to a class o f oligosaccharide structures that is characteristic of bacterial cellulosomes27-29.

Apart from the similarities in their CBDs and cohesins, the scaffoldins from the two species are quite different in other respects. For example, the order o f the various domains in the two sequences is different. In addition, both polypeptides possess other domains, which bear no k n o w n similarity to each other or to other proteins. T h e corresponding functions o f these domains are also as yet unknown. The scaffoldin from C. thermocellum also contains a terminal dockerin, which has been suggested to mediate either attachm e n t to the cell, or oligomerization o f scaffoldin molecules 3°. Its real purpose is as yet unknown, but reciprocal interaction with an extraneous cohesin can be presumed.

O n the basis of the data described above, Fig. 3 illustrates the current view o f the cellulosome structure from C. thermocellum. The various functional domains o f the scaffoldin and catalytic subflnits of the cellulosome are viewed as the building blocks that govern the composite structure o f the cellulosome. The efficient degradation o f cellulosic substrates is a consequence o f the synergistic action among the different enzyme components of the structure. Ultimately, the decisive factor that distinguishes a genuine cellulosome is the presence of a scaffoldin-like polypeptide, which integrates the catalytic subunits into a discrete complex by virtue o f the very powerful cohesindockerin interactions.

Designer cellulosomes for improved processes T h e question is: can we exploit our new-found knowledge ofcellulosome structure to improve existing processes for effective waste management and commercial utilization of cellulosics? It is certainly premature for the conversion of industrial or agricultural wastes into fuels and feedstock by this route to be considered immediately. W h a t can be done at this point is to try to construct better cellulosomes, i.e. 'designer cellulosomes' (Fig. 4), and to use these improved enzyme systems in existing processes. T h e construction of improved cellulosomes can be accomplished in several ways. O n e idea is to alter the properties o f a cellulase system by chemical incorporation of an enzyme, a group o f enzymes, or another type o f component into an isolated cellulosome in order to improve its overall performance. TIBTECHSEPTEMBER1994 (VOL 12)

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reviews integrate such enzymes into a complex, and the resultant 'heterocellulosome' would now exhibit the combined catalytic properties of the new group of enzymes. In oMer to incorporate heterologous enzymes into the complex, recombinant D N A (rDNA) technology can be used to fuse a given dockerin with a given enzyme in a surrogate bacterial strain32. A desired scaffoldin can be produced separately, either from the native cellulosome, or by expressing the cloned protein in a different host. Assembly o f the enzyme-dockerin chimeras onto the scaffoldin can then be performed in vitro, by virtue of the specific interactions with the intrinsic cohesins. In this manner, the set of enzymes in the cellulosome can be manipulated to suit the nature of the substrate. For example, hyperactive cellulases and xylanases from different organisms, or recombinant organisms can be bolstered with selected ligninases, pectinases, etc., which can be incorporated into the same enzyme complex in order to degrade a specific type o f cellulosic substrate efficiently. In the future, this notion can be further extended by modifying the nature of the native scaffoldin by r D N A technology. Thus, a CBD can be fused to exogenous types o f cohesins (for example, from different microbial sources) to produce a chimeric scaffoldin. Likewise, appropriate dockerin counterparts can be fused to desired enzymes. The resultant chimeras will form a combined enzyme system - a chimeric cellulosome - that will be targeted to the substrate via its CBD. The simple example in Fig. 4 illustrates the selective combination of an endoglucanase and an exoglucanase. Figure 5 Future applications of cellulosomal domains in research, medicine and industry. Selected domains, which exhibit a desired activity, specificity or fun(:tion, can be incorporated by crosslinking or by fusion into the components of unrelated affinity systems. The resultant hybrid biomolecules can then be used to mediate between the molecular counterparts for a variety of applications.

The cellulosomal CBD would serve to target the resultant 'supercellulosome' complex to the surface of the substrate, where the exogenously added component(s) would facilitate the hydrolytic action o f the resident enzymes. For example, historically, one of the major problems in using cell-free cellulase preparations for industrial degradation o f cellulosics has been feedback inhibition by the end-product, cellobiose. In the natural ecosystem, cellobiose accumulation is prevented by its uptake by the parent or satellite cells. For commercial processes, this problem can be countered by chemical or biochemical crosslinking of purified [3-glucosidase to an intact cellulosome 1,31. The resultant supercellulosome would convert the cellobiose to the noninhibitory glucose, and the enzyme complex would continue to hydrolyze the substrate. A more ambitious approach would be to substitute the resident cellulosomal components with heterologous enzymes that would be more suitable for a desired substrate. In this case, native scaffoldin could be used to TIBTECHSEPTEMBER1994 (VOL 12)

Hybrid affinity systems for unconventional applications The clastic view o f cellulosomal structure can be extended further to provide tools that may eventually be universally apphcable to other fields o f the biological sciences. Indeed, genes encoding CBDs are easily cloned in high-expression vectors, and their application as affinity tags for immobilization an d purification has already been demonstrated 21. Various heterologous enzymes and other biologically active materials have been fused to CBDs in oMer to immobilize them to cellulose33-3s. Similarly, dockerins and cohesins also appear to act in an independent manner, and can be overexpressed in surrogate host bacteria 32,39. They can conceivably be fused or crosslinked to a variety o f enzymes or components of other affinity systems, such as binding proteins, nucleic acids and other biologically active materials. The linkers also seem to be appropriate for application in heterologous systems4°. The strong cohesin-dockerin interaction forms a tenacious affinity system, perhaps analogous to the avidin-biotin complex 41. In both cases, the presence o f denaturants at elevated temperatures are required for dissociation. Cellulosomal domains may provide an alternative or auxiliary affinity system for a surprising variety of applications (Fig. 5). Towards this goal, dockerins or cohesins can be fused or conjugated

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reviews Box 1. Examples of cellulolytic microorganisms that appear to produce cellulosome-like multienzyme complexes Microbe

Refs

Acetivibrio cellulolyticus Bacillus circulans Bacteroides cellulosolvens Butyrivibrio fibrisolvens Cellulomonas sp. Clostridium cellobioparum Clostridium cellulolyticum Clostridium cellulovorans Clostridium josui Clostridium papyrosolvens Clostridiurn thermocellum Fibrobacter succinogenes Neocallimastix frontalis Ruminococcus albus Ruminococcus flavefaciens Thermomonospora curvata

27 42 27 43 27 27 44 26,27 45 46 3 47 48 27 49 50

to protein A, antibodies, lectins, DNA, etc., to form hybrid biomolecules. For example, a dockerin-antibody hybrid can be mixed with a cohesin-lectin hybrid in order to couple a desired antigen and a glycoprotein. The antigen and glycoprotein can, of course, also be part of other types of coupled systems; for example, two different cell types. It is already clear, however, that this interaction is characterized by a different set of principles. On the one hand, dockerins are much larger than biotin, and their incorporation into a desired affinity system by genetic or chemical means is more complicated. On the other hand, unlike the tetrameric avidin, which binds strongly to four molecules of biotin, a single cohesin will presumably bind to a single dockerin. This can indeed be advantageous, as it should enable better control of the coupling of affinity components for a given application. If higher-order systems are required for a given application, discrete multicomponent complexes can be produced by constructing a scaffoldin chimera that contains a series of different cohesins in succession. Individual fusion proteins that contain appropriate dockerins a n d desired affinity components can then be combined selectively to form complexes. One of the exciting aspects of this approach is that thousands of cellulolytic strains have already been described. A growing list of strains are already suspected of expressing cellulosomes or related entities (see Box 1), all of which will be at the disposition of the prudent biotechnologist. However, we still have much to learn. We are only at the beginning of deciphering the intricacies of the component parts of the cellulosomes and in understanding their potential. In any case, we foresee a broad application of cellulo-

somal domains, which will add a new dimension to our arsenal of immobilization techniques, nonradioactive detection methods, diagnostics, and other tools of medical or industrial importance. In fact, the situation today is quite similar to that which precipitated the development of the avidinbiotin system as a generally applicable technologysl. Therefore, we would like to end this review by paraphrasing anotherS2: we cannot, of course, foresee all possible applications, given our own limited imagination and/or restricted knowledge. It seems that the potential ofcellulosomal domains in biotechnological processes is unlimited, and that their successful implementation will be directly dependent on the needs and imagination of the user.

Acknowledgements This article is dedicated to Maria A. Rudzinska of the Rockefeller University (New York, NY, USA). We thank P. Btguin and J-P. Aubert of the Pasteur Institute (Paris, France), G. E Hazlewood of the A F R C Institute of Animal Physiology and Genetics Research (Cambridge, UK) and H.J. Gilbert of the University of Newcastle upon Tyne (Newcasde, UK), for providing pre-publication data.

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• New genes in old sequence: a strategy for finding genes in the bacterial genome by Mark Borodovsky, Eugene V. Koonin and Kenneth E. Rudd Trends in Biochemica/Sciences t 9, 309-3 t 3 (t 994) IBTECHSEPTEMBER1994 {VOL 12)