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Chapter 6 : Cellulosome-Enhanced Conversion of Biomass: On the Road to Bioethanol
Category: Applied and Industrial Microbiology
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The cellulosome was first isolated on the basis of the cellulose-binding function of the anaerobic thermophilic bacterium Clostridium thermocellum. Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose. The cellulose utilization systems in cellulosome-producing bacteria include over 100 different genes that must be orchestrated and timely expressed. The concept of directly converting biomass to ethanol by a mixed clostridial fermentation was fashionable some 30 years ago when it was found that the product pattern of C. thermocellum in favor of ethanol could become almost quantitative in stable coculture with another ethanol-producing anaerobe. The action of the exocellular protuberance-bound cellulosome may serve to delay or limit diffusional loss of the hydrolyzed sugar to the environment and/or competing bacteria. Consolidated bioprocessing (CBP) was recently extended for direct production of ethanol in yeast by cloning an endoglucanase and a ß-glucosidase in Saccharomyces cerevisiae. At present, C. thermocellum, as a very potent cellulolytic, anaerobic thermophile, still seems to be the microorganism of choice for future bioethanol production from biomass. Theoretically, C. thermocellum can be engineered metabolically to produce better yields of ethanol or other products. Eventually, yeast cell surfaces may be modified to contain designer cellulosomes for direct ethanol conversion. The combination of CBP with the designer cellulosome concept may ultimately provide optimized degradation of specific cellulosic feedstocks for bioethanol production.
Cellulosome architecture of C. thermocellum. The cellulosomal enzymes are attached via their type I dockerin modules to the type I cohesins of the CipA scaffoldin. In turn, the CipA scaffoldin is attached, via its divergent type II dockerin, to a series of anchoring scaffoldins—SdbA, Orf2p, and OlpB—which bear one, two, and four type II cohesins, respectively. The anchoring scaffoldins carry at their C terminus an SLH module that anchors the cellulosome to the bacterial cell surface. A single CBM on the CipA scaffoldin binds the cellulosome and the entire cell to the cellulose substrate.
Scaffoldins of different cellulosome-producing bacteria. Micrographs of the bacteria are also included. The four scaffoldins of the C. thermocellum paradigm are shown. The type II cohesin-dockerin pairs are shown in a darker shade of gray, and the anchoring component, SLH module or sortase signal, is designated by the adjacent symbol of an anchor. The other clostridial species are characterized with a single scaffoldin. The four scaffoldins of the A. cellulolyticus system are more cross-interactive than that of the C. thermocellum paradigm (see the text for details). The reversed types of cohesindockerin pairings are evident in the B. cellulosolvens system, as are the two exceptionally large scaffoldins. The R. flavefaciens system is especially elaborate, with the single-cohesin ScaC “adaptor” scaffoldin providing the means with which to modify the repertoire of cellulosomal components.
Schematic representation of the designer cellulosome concept. In the native complex (left) the specificity of the cohesin-dockerin interaction is usually identical. If the native components are used, we would expect random incorporation of the enzymes, resulting in a large collection of artificial cellulosomes heterogeneous in their content. In designer cellulosomes (right) composed of matching chimeric components, the specificity of the different cohesin-dockerin pairs is divergent, thus facilitating the controlled incorporation of enzyme components.
Engineering of potent cellulolytic microorganisms for production of biofuels. Bacterial, mold, or yeast host cell systems can be converted into a cellulosome producer by cloning appropriate genes that code for cellulases and/or designer cellulosome components (i.e., a chimeric scaffoldin and desired dockerin-containing hybrid enzymes). The resultant cellulolytic cells can be grown directly on cellulosic biomass to produce the desired end product, e.g., sugar intermediates or final bio-fuel.
Dockerin-containing genes and/or gene products of the C. thermocellu m genome a