AIR1-CT92-0321 Cellulose Degradation by Clostridium thermocellum

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AIR Cluster III - Bioconversion : Biological Conversion : Biotechnology : Detergents : Fibre

Electron micrograph showing a cross-section of two C. thermocellum cells adhering to a cellulose fibre. Bar = 100mm

INTRODUCTION

Plant cell walls contain three main components: cellulose which is insoluble and forms crystalline, compact fibres which are highly resistant to degradation; hemicellulose which is amorphous and more easily degraded; and lignin. The thermophilic, anaerobic bacterium Clostridium thermocellum produces a cellulase system having a very high specific activity against crystalline cellulose. Thus, it provides a good model system in order to understand the essential features required for the degradation of natural cellulosic substrates. A particular feature of the C. thermocellum cellulase system is that the various cellulolytic components are associated in a multienzyme complex termed the cellulosome. Activity against crystalline cellulose appears to be dependent upon the integrity of the cellulosome, suggesting that the topological organisation of the complex allows optimal synergistic interactions between the components. Below, the main aspects of the project are highlighted.

SUMMARY

This item is based on the final report of project 0321, which was subtitled Investigation of the Structure and Mechanism of Individual Enzymes and Study of the Organization of the Cellulase Complex and the second annual report from project 1271. The objective of the proposed work was to improve the feasibility of enzymatic conversion of cellulosic materials into sugars by gaining a better understanding of the factors that effect the efficiency of conversion by the enzymes responsible. The released sugars can be used as a carbon source for fermentations generating chemicals, pharmaceuticals and fuels, as well as in modification of animal feed. Such plant cell wall degrading enzymes also find applications in fibre processing, vegetable oil extraction, flavour release, textiles, detergent and chemicals. The main problems with current commercial enzymes relate to heat-stability, activity and pH profile. In both projects reported here thermophilic (heat loving) organisms are used since these provide a source of more stable enzymes. However, the approaches differ, reflecting differences between the bacterial system (in which the enzymes are organised into a complex known as the cellulosome) and the fungal system in which a wide range of enzymes are released into the growth medium.

OBJECTIVE

The cellulose system of Clostridium thermocellum is composed of at least 14 different components that are tightly associated in a multienzyme complex termed cellulosome. The high specific activity of the cellulosome toward crystalline cellulose is probably due to the spatial organization of the subunits within the complex, which allows optimal synergistic interactions.

The project comprises three aspects :

• The structure and mechanism of action of individual enzymes synthesized from their respective genes cloned in Escherichia.Coli will be investigated in detail. The structure of one such enzyme, endoglucanase CelD, has already been determined by X-ray crystallography. Further experiments will be performed to refine the topology of the active center and to study interactions between the enzyme and cocrystallized substrates and inhibitors. In addition, the crystallization and 3-D structure determination of endoglucanase CelC will be attempted. CelC is an endoglucanase whose sequence is unrelated to CelD, and which could serve as a structural paradigm for a vast family of cellulases including more than 25 endoglucanases from various organisms.

• The organization of the various subunits within the cellulosome will be investigated. Previous work has indicated that the catalytic subunits are bound to a large scaffolding glycoprotein termed S1 by means of a conserved, non-catalytic domain which consists of two highly similar segments of 22 aminoacids each. The 3-D structure of this region will be determined. The incorporation into the complex of chimeric proteins bearing the conserved, reiterated region will be attempted. The gene encoding the scaffolding protein will be cloned and its sequence will be searched for reiterated anchoring sites to which the catalytic subunits bind. The spatial organization of the subunits will be investigated by using reversible cross-linking reagents.

• Tools for the genetic manipulation of C. thermocellum will be developed.

RESULTS

Three endoglucanases and one xylanase have been characterised in detail, including the determination of their three dimensional structure. The results can now be utilised to model other enzymes belonging to the same families, as determined by primary sequence analysis. In addition, it has been shown that enzymes displaying very little sequence similarity may nonetheless share very similar structures and mechanisms. The basic building principle of the cellulosome has been elucidated. Catalytic subunits of the complex contain a conserved, non catalytic region acting as a docking domain. This region, which has been termed dockerin domain, mediates attachment of the catalytic components to a large glycoprotein termed CipA, which acts as a scaffolding component and as a cellulose binding factor. The sequence of CipA contains a cellulose-binding domain and nine highly conserved, reiterated segments. These segments correspond to a series of receptors, termed cohesin domains, which bind the dockerin domains borne by the catalytic subunits. A class of cell surface proteins involved in anchoring cellulases to the surface of C. thermocellum cells has been identified. These proteins contain cohesin domains, which can bind either the dockerin domains of catalytic subunits, or the dockerin domain of the scaffolding protein CipA. The latter is specifically bound by a distinct type of cohesin domain, which does not recognise the dockerin domains borne by catalytic subunits.

EXPLOITATION

A patent has been applied for to cover potential applications of the cohesin and dockerin domains of type II for the construction of artificial multiprotein complexes. By combining two types of cohesin domains, it should be possible to engineer new scaffolding proteins on which polypeptides can be targeted to bind at specific sites depending on their dockerin domain. Genencor Intl, Inc has expressed interest in exploring the feasibility to develop the technology for commercial applications.

PARTICIPANTS

The project was carried out by the Cell Physiology Unit and Structural Immunology Unit at the Pasteur Institute together with the University of Ghent.