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

Abstract

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 cellulosebinding 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...

In Vitro Reconstitution of the Complete Clostridium thermocellum Cellulosome and Synergistic Activity on Crystalline Cellulose

“…The results do not contradict the assumption that the assembly of the fully active cellulosomes is achieved by displaying a mature CipA protein on the outside of the cell; the independently secreted cellulosomal components seem to be assembled spontaneously by cohesindockerin interaction, as was shown previously with in vitro-created components (6,9,22).…”

“…All of them used scaffoldin proteins with a limited number of cohesin-binding sites or a limited number of enzyme components (6,7,11,25,34,37). In most of these complexes, a specific dockerin-binding site was assigned to a certain enzyme component by using cohesin-dockerin pairs with different specificities.…”

Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome

“…Laccases, which facilitate the degradation of lignin, can largely contribute to current efforts to overcome limitations of enzymatic degradation of lignocellulosic biomass. In parallel, several studies have shown that highly efficient plant cell-wall degradation can be achieved by the use of the designer cellulosome complexes (20,21,37,52). Designer cellulosome technology, which harnesses the proximity effect of ordered hydrolytic enzymes to maximize their combined synergistic effects, was used in this work in combination with integration of a nonconventional enzyme that degrades lignin.…”

“…fusca enzymes involved in lignocellulose degradation have been extensively studied because they are industrially relevant due to their high thermostability, broad pH range, and high activity (19,37,42). In previous studies, the majority of these enzymes have been converted to the cellulosomal mode by fusion of a dockerin module, without adverse effects on their activity.…”

“…This Lego-like complex has been used to test the effects of enzymatic compositions, the relative positioning of the enzymes within the complex, and their spatial proximity, which, together, generate the synergistic action of the cellulosomal components (33)(34)(35). The majority of the T. fusca biomass-degrading enzymes have been converted previously to the cellulosomal mode (19,20,(36)(37)(38)(39)(40)(41), including a pair of lytic polysaccharide monooxygenases (LPMOs) involved in the oxidative cleavage of crystalline cellulose fibers (22). Interestingly, in most cases, their inclusion into designer cellulosomes proved to be more efficient than the equivalent mixture of wild-type enzymes (20-22, 37, 40, 42).…”