Interplay between Clostridium thermocellum Family 48 and Family 9 Cellulases in Cellulosomal versus Noncellulosomal States

Abstract

The anaerobic, thermophilic cellulolytic bacterium Clostridium thermocellum is known for its elaborate cellulosome complex, but it also produces a separate free cellulase system. Among the free enzymes, the noncellulosomal enzyme Cel9I is a processive endoglucanase whose sequence and architecture are very similar to those of the cellulosomal enzyme Cel9R; likewise, the noncellulosomal exoglucanase Cel48Y is analogous to the principal cellulosomal enzyme Cel48S. In this study we used the designer cellulosome approach to examine the interplay of prominent cellulosomal and noncellulosomal cellulases from C. thermocellum. Toward this end, we converted the cellulosomal enzymes to noncellulosomal chimeras by swapping the dockerin module of the cellulosomal enzymes with a carbohydrate-binding module from the free enzyme analogues and vice versa. This enabled us to study the importance of the targeting effect of the free enzymes due to their carbohydrate-binding module and the proximity effect for cellulases on the designer cellulosome. C. thermocellum is the only cellulosome-producing bacterium known to express two different glycoside hydrolase family 48 enzymes and thus the only bacterial system that can currently be used for such studies. The different activities with crystalline cellulose were examined, and the results demonstrated that the individual chimeric cellulases were essentially equivalent to the corresponding wild-type analogues. The wild-type cellulases displayed a synergism of about 1.5-fold; the cellulosomal pair acted synergistically when they were converted into free enzymes, whereas the free enzymes acted synergistically mainly in the wild-type state. The targeting effect was found to be the major factor responsible for the elevated activity observed for these specific enzyme combinations, whereas the proximity effect appeared to play a negligible role.

A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure‐guided recombination

“…Chimeric genes were assembled from 24 gene fragments, representing the eight blocks from each of the three parents, with the sequence-independent sitedirected chimeragenesis method [23] to generate a gene library of 3 8 (6561) different sequences (Table S1; Fig. S4).…”

“…Glycoside hydrolase family 48 cellulases (Cel48; EC 3.2.1.176) are ideal candidates for designer cellulosomes. As one of the most important families of bacterial cellulases [7,8], they are usually a major constituent of bacterial cellulosomes [9,10]. Of the 116 bacterial Cel48 genes currently predicted in the CAZy database (http://www.…”

Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome

“…Expression of the proteins was achieved by adding isopropyl β-D-thiogalactopyranoside (0.1 mM final concentration) to midexponential phase cultures of E. coli BL21(DE3) harboring target plasmids with incubation for a further 3 h at 37°C. The His-tagged recombinant proteins were purified from cell-free extracts by immobilized metal ion affinity chromatography as described previously (47). Yields for WT BglA are typically between 70 and 90 mg per liter culture medium, and the yield of purified BglA-CohII was 25.2 mg per liter of culture medium.…”

Bioprospecting thermostable cellulosomes for efficient biofuel production from lignocellulosic biomass

“…Construction of cohesin domains in the designer cellulosome from various species allows incorporation of the desired dockerin-bearing enzymes, which could interact synergistically with efficient hydrolysis of the complex substrate Caspi et al 2008;Vazana et al 2010).…”