Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode

We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant “cellulosomized” enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.In nature, some anaerobic cellulolytic bacteria produce cellulosomes, which are organized by the action of scaffoldin subunits that usually contain a single carbohydrate-binding module (CBM) and multiple cohesin modules (2, 7, 13, 14, 28, 36). This arrangement allows the integration of several dockerin-containing enzymes into a complex, which is then targeted to the cellulosic substrate by the common CBM. The cellulosomal enzymes then exhibit enhanced synergistic activity, presumably due to their spatial proximity and coordinated interaction. In contrast, the enzyme systems of aerobic bacteria and fungi comprise free (uncomplexed) enzymes, which differ from cellulosomal systems in that many of them contain their own CBM that delivers the individual catalytic module to the surface of the substrate (39, 41, 42).In previous work, we used the designer cellulosome concept (5) to construct unique minicellulosomes of defined content (16, 32, 33). In order to construct designer cellulosomes, chimeric scaffoldins have been prepared which contained two or more cohesins that matched the dockerins of the enzymes (native cellulosomal or dockerin-fused chimeras). Enzymes that contain dockerins that match the specificity of a scaffoldin-borne cohesin can then be selectively integrated into the designer cellulosome at a specified site. Cellulosomal enzymes containing either a native dockerin or a divergent dockerin can be inserted on different sites of a chimeric scaffoldin. Alternatively, a free, noncellulosomal enzyme can be included in designer cellulosomes by replacing its native CBM with a dockerin of choice. In some cases, designer cellulosomes displayed enhanced synergistic activity over the parallel free-enzyme system (15, 17). This increased activity was shown to be a function of both a substrate-targeting effect (contributed by the CBM on the chimeric scaffoldin) and the enzyme proximity effect, thus supporting the initial hypothesis.In recent studies, we have investigated the free-cellulase system of Thermobifida fusca for use in designer cellulosome systems. This aerobic thermophilic cellulolytic bacterium contains a limited set of six free cellulases, each composed of a catalytic module and a crystalline-cellulose binding family 2 CBM (CBM2) module on either the N or C terminus of the protein. T. fusca contains three endoglucanases (Cel5A, Cel6A, and Cel9B), two exocellulases (Cel6B and Cel48A), and one processive endoglucanase (Cel9A). Previously, we converted both family 6 cellulases and the family 48 exoglucanase from the free to the cellulosomal mode of action by replacing their native CBM2s with a dockerin module (11, 12). All three chimeric enzymes exhibited cellulose-degrading activity on both soluble and crystalline substrates. The results indicated that the family 48 exoglucanase appeared to be well adapted to the cellulosomal mode of action, whereas the family 6 exoglucanase is less appropriate for inclusion into cellulosomes. Indeed, family 48 cellulases have been found to be a major component in every native cellulosome thus far described, in contrast to the family 6 cellulases, which have been identified only in free-cellulase systems.An important feature of the free-acting fungal and bacterial cellulases is that they contain a linker segment, often rich in prolines and threonines, that connects the catalytic module to the CBM (37). The role of such flexible linkers is thought to ensure independent action of the adjacent functional modules, thus allowing progressive and efficient hydrolysis of cellulose by the catalytic modules (6, 9, 10, 20, 25-27, 34, 36, 38, 40). The present communication focuses on the effect of linker length and dockerin position (relative to the catalytic module) on enzymatic activity within a designer cellulosome. For this purpose we have employed the highly active family 5 endoglucanase Cel5A from T. fusca (21, 22, 29), which was converted to the cellulosomal mode by replacement of its CBM with a dockerin module. Chimeric dockerin derivatives were prepared on either the N or C terminus of the Cel5A catalytic module, separated by linker segments of different lengths. In most cases, binary designer cellulosomes, comprising the respective Cel5A chimera together with a Cel48A chimera, were shown to be more efficient on crystalline cellulosic substrates than the combination of the wild-type free enzymes.