Why did some cellulolytic anaerobes evolve to produce such a complicated mechanism in the production of an intricate multi-component conglomerate of enzymes, CBMs and other functional modules, complexed into a discrete type of complex, which is located in

bundle-like organelles over the cell surface? Such an expense in metabolic energy would imply that the bacterium would gain a significant compensation for the effort. Abiochemical rationale for enhanced cellulolytic activity of cellulosomes on recalcitrant forms of cellulose was originally proposed already upon its discovery (88, 120):

The (cellulosome) complex apparently comprises various different forms of cellulases, each of which may bear separate specificities toward different quaternary structures on the complex cellulose substrate. The major organizational role of this complex might be designed for effective delivery to the substrate as well as to bring into proximity the various complementary enzymes (e. g., exo and endocellulases). In addition, the complex may be structured in such a way as to enable the protection of various product intermediates and to facilitate their transfer to other cellulase components for further hydrolysis. In any event, the cellulase subunits seem to be arranged within the CBF (cellulosome) complex in a defined supramolecular fashion designed for highly efficient cellulose degradation.

Proof of the targeting and proximity effects was eventually realized through the use of arti­ficial “designer cellulosomes,” which enabled controlled incorporation of selected cellulases into a cellulosome-like complex (144, 145). For this purpose, a chimeric scaffoldin that contains divergent cohesins and matching dockerin-bearing enzymes can be mixed in vitro to form minicellulosomes of defined composition and spatial arrangement (Figure 13.10). The capacity to prepare cellulosomes of uniform composition and to control the types of enzymes included therein has contributed a better understanding of the factors important for efficient cellulosome action. Thus, the two major factors that serve to enhance decon­struction of recalcitrant forms of cellulose are indeed targeting to the substrate surface by the scaffoldin-borne cellulose-binding module (CBM), and the consequent proximity of the enzyme components.

The incorporation of enzymes into cellulosomes would ensure that different complemen­tary activities are all contained in the same microscopic area of the cellulose substrate rather than being dispersed statistically over its surface. Separation of relatively small amounts of enzymes by large distances would thwart the synergistic action of complementary en­zymes, each of which bears a CBM that prevents its free diffusion as in the case of the free (non-cellulosomal) enzymes. The free systems are limited by the fact that such enzymes are statistically isolated and hence do not benefit of the full complementary action of the other enzymes. The “proximity effect” of the cellulosomes counteracts this problem. The “remedy” for the free systems is the production of very large quantities of enzymes to over­come their solitude, which is the strategy taken by aerobic bacterial and fungal systems. In fact, this maybe the reason why cellulose-binding CBMs are appended to non-cellulolytic enzymes: both cellulosomes and the free enzyme systems use CBMs to prevent the useless dispersion of their enzymes (whether cellulolytic or not). Disruption of the sites of the cellulose-hemicellulose interface is a key to potent digestion of the plant cell wall. It is im­perative that the different enzymes, the cellulases and hemicellulases, are in close proximity to each other. Because of its large size (relative to bacteria), the fungi probably deliver their cellulase mixture in a localized specific area (tip of hypha perhaps) and then the enzymes remain together in that area as a function of their respective CBMs. In contrast, due to its smaller size, it is C. thermocellum’s best interest to remain attached to a multienzyme system capable of accommodating almost any type of enzyme needed to breakdown completely a piece of plant cell wall without having to move. This may be the reason that cellulosomes appear to be so superior to the free enzyme systems.

With the exception of the cellulosome cluster in C. acetobutylicum, which probably pro­duces a crippled complex (83,84), it can be seen from the few known genomes of cellulosome — producing bacteria that there is a much larger number of dockerin-containing enzymes that can be attached to a scaffoldin than there are slots (cohesins) on the scaffoldin. In these bac­teria, there are more hemicellulose-degrading enzymes than there are cellulases. This infers that cellulosome organization is beneficial not only for crystalline cellulose digestion, but
equally for the digestion of hemicelluloses, pectins, and other plant cell wall polysaccharides. This holds true even for C. thermocellum, a bacterium capable of assimilating only cellulose and its degradation products. Nevertheless, it appears to serve as the major polysaccharide­degrading bacterium in its ecosystem, passing on the surplus (e. g., cellobiose) and/or su­perfluous (e. g., xylose, etc.) sugars to its companion (saccharolytic) strains that share the same locale. Particular types of plant cell wall-derived biomass require very precise enzyme mixtures. In this context, previous attempts to “benchmark” one cellulase “system” against another did not particularly reflect an intrinsic ability but the “fit” of the enzyme mixture with respect to the actual substrate used. In other words, it is hard to compare one complex mixture to another.

For many decades, it was believed that the solution to efficient degradation of recalcitrant cellulose would be found in conventional engineering approaches by employing a set of sol­uble enzymes, such as the then-known endo — and exo-glucanases. The amount of financial resources versus the expectations of society resulted in great disappointment that served to minimize further scientific activity in this type of research. Very little has been achieved on the scientific front during the interim period. However, some positive results in this area have been recorded, including the discovery of the cellulosome (the topic of this chapter) and the realization that the bacterial cellulases are multi-modular entities that exhibit distinct functionalities. It is hoped that in the future these novel contributions will encourage a new burst of serious scientific and applied efforts in this area. The resolution and harnessing of the cellulosome (146) may thus provide a renewed opportunity for combating the diffi­culties encountered in digesting recalcitrant cellulosic biomass in an effective, cost-efficient manner.

Acknowledgments

The authors are grateful to Claire Boisset, Henri Chanzy, Pedro M. Coutinho, the late Martin Schulein, Ely Morag, Yuval Shoham, Ilya Borovok, Henri-Pierre Fierobe, Jean-Pierre and Anne Belaich, Marco Rincon and Harry Flint for their input, discussions, and collaboration, past and present. This work was fundedby Research Grants 394/03 and 422/05 from the Israel Science Foundation (Jerusalem), a grant from the Alternative Energy Research Initiative, and by grants from the United States-Israel Bi-national Science Foundation (BSF), Jerusalem, Israel. EAB is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry at The Weizmann Institute of Science.