Thermobifida fusca exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A

Abstract

Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein-protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes' CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerincontaining family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.

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

“…Interestingly, members of the family-6 glycoside hydrolase enzymes have never been found to be part of any known cellulosome system. However, endocellulase Tfu_1074 (Cel6A) was demonstrated to confer enhanced cellulose-degrading activity in simple designer cellulosomes (38,53). In contrast, Tfu_0620 (Cel6B) is an exoglucanase, which was shown to cause an antiproximity effect in designer cellulosomes, and was therefore deemed incompatible with the cellulosomal mode (38).…”

“…However, endocellulase Tfu_1074 (Cel6A) was demonstrated to confer enhanced cellulose-degrading activity in simple designer cellulosomes (38,53). In contrast, Tfu_0620 (Cel6B) is an exoglucanase, which was shown to cause an antiproximity effect in designer cellulosomes, and was therefore deemed incompatible with the cellulosomal mode (38). Therefore, in this study, we chose to work with the well-characterized family-48 exocellulase Cel48A, with GH family-48 enzymes being a major component in the natural secreted cellulosomes (54-57) together with the family-5 endocellulase Cel5A, whose synergistic effect with Cel48A has been demonstrated previously and which were extensively used in their dockerin-bearing chimeric forms as successful components of designer cellulosomes (20-22, 37, 58).…”

“…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).…”

Integration of bacterial lytic polysaccharide monooxygenases into designer cellulosomes promotes enhanced cellulose degradation

“…To do so, we have chosen to study two LPMOs from the bacterium T. fusca, because our group has already performed the cellulosomal conversion of a dozen biomass-degrading enzymes from this model organism (21,27,30,31). The LPMOs E7 and E8 were initially described in an earlier study (32) but were then considered to be CBMs able of slightly enhancing the activity of other T. fusca cellulases, because their classification as LPMOs was not yet evident.…”

“…Furthermore, obtaining a functional chimeric enzyme does not necessarily warrant that it will be beneficial when incorporated into a cellulosome complex. For instance, our group reported that the exoglucanase Cel6B from T. fusca could be successfully converted to the cellulosome mode but, upon insertion in a complex, would cause a strong antiproximity effect (31).…”