I n recent years, the concept of designer cellulosomes has become a popular notion for a prospective solution to improving cellulase action, an approach first proposed over a decade ago (Bayer and Lamed 1992; Bayer et al. 1994; Ohmiya et al. 2003). But what is wrong with the native cellulosome systems? Indeed, they are considered to be the most efficient natural enzyme systems for cellulosic biomass, but the cellulosome-producing bacteria produce them in relatively small quantities, compared to the free cellulase systems produced by the aerobic fungi and bacteria. The current idea, therefore, would be to produce large quantities of arti­ficial cellulosomes or cellulosomal components in an appropriate host cell system, which would then be cost-effective for industrial use.

Designer cellulosomes are artificial cellulosome complexes of defined composition, con­taining different recombinant enzyme components (Figure 5.5). The exact position of a given enzyme in the cellulosome can be predetermined by producing chimaeric scaffoldins that contain divergent cohesins of defined specificity and by attaching to the enzymes dockerins of matching specificity. Using this approach, the desired cellulosomes will self — assemble according to our initial design. In this manner, we can control the enzyme content

Chimaeric Scaffoldin Dockerin-containing Enzymes

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Designer Cellulosomes

Figure 5.5. Construction of a designer cellulosome. A chimeric scaffoldin is designed containing a substrate-targeting CBM and multiple divergent cohesins (enumerated) for selective incorporation of the enzymes (A, B, and C). The recombinant enzyme hybrids contain a dockerin module selected for its matching specificity with one of the cohesins of the chimeric scaffoldin.

and position of the enzyme within the complex. Thus, any given complement of enzymes can be incorporated into such cellulosome complexes. These designer cellulosomes can be utilized as tools for both understanding cellulosome action and for future application in waste management (Bayer et al. 2007) and for production of biofuels (Bayer et al. 2008a, c).

The production of designer cellulosomes has been shown to be experimentally feasible. The first proof of concept involved the preparation of an exhaustive array of small bi­functional artificial cellulosomes, whose activity on specific substrates was examined (Fierobe et al. 2001, 2002). Small chimaeric scaffoldins were prepared that contained two divergent cohesins and matching dockerin-bearing cellulases, and their synergistic action on recalcitrant cellulosic substrates was demonstrated. Additional studies involved the action on crude native substrates, such as wheat straw, whereby designer cellulosomes containing a xylanase together with potent cellulases were demonstrated to dramatically enhance the degradation of complex lignocellulosic substrates (Fierobe et al. 2005) . Later studies demonstrated that enzymes foreign to the native cellulosomes can also be included in the active state into designer cel- lulosomes (Caspi et al. 2006, 2008; Mingardon et al. 2007a), and radical designer cellulosome architectures can be produced at will, as long as the appropriate pre-design and experimental expertise are applied (Mingardon et al. 2007b).

To date, designer cellulosomes have been fabricated on a small scale using only two or three enzymes in complex and with only very modest gains in synergistic activity. Future work with designer cellulosomes should determine whether more impressive results can be obtained with larger numbers of enzymes within the individual designer cellulo — somes, as we extend the size of designer cellulosomes to approach that of the native cellulosome systems. Another distinctive difference between the artificial and natural systems is that designer cellulosomes are uniform in composition, containing stoichiometric ratios of the desired enzymes whereas the native cellulosomes are heterogeneous in content and dispersion of its enzymes. The possible consequences of these differences vis-a-vis efficiency of deconstruction of cellulosic biomass are yet to be determined experimentally.