Abstract
The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display seven distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G, and Cel9M). In this study, the 10 uncharacterized GH9 enzymes were overproduced in Escherichia coli and purified, and their activity pattern was investigated in the free state or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in mini-cellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole noncellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous mini-cellulosomes that exhibit more activity on crystalline cellulose than the best homogeneous tri-functional complex. Altogether, our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerization.
Highlights
Family-9 glycoside hydrolases displaying various organizations are plethoric in cellulosome-producing bacteria
Sequence Homologies and Domain Organization of GH9 Enzymes from C. cellulolyticum—The annotation of the genome of C. cellulolyticum revealed the presence of 13 genes that putatively encode GH9 enzymes
The profusion of GH9 enzyme-encoding genes in cellulosome-producing microorganisms suggests a pivotal role for this family of enzymes during plant cell wall degradation by the cellulolytic complexes
Summary
Family-9 glycoside hydrolases displaying various organizations are plethoric in cellulosome-producing bacteria. Many GH9 cellulases display side activities on related polysaccharides such as 1,3–1,4-glycans [5], xylan [6], or xyloglucan [7], but their favorite substrate is either soluble (carboxymethylcellulose and cellodextrins) or insoluble (amorphous) cellulose In addition to their catalytic domains, a large proportion of known GH9 enzymes contain ancillary modules/domains like carbohy-. The cellulosomes are heterogeneous large extracellular complexes that efficiently degrade cellulose and related plant cell wall polysaccharides The simplest cellulosomes such as those produced by mesophilic and cellulolytic clostridia are composed of a single major scaffolding protein hosting a CBM displaying affinity for cellulose and a series of cohesin modules that strongly interact with a complementary module, the dockerin, borne by the catalytic subunits. The sole noncellulosomal GH9 of C. cellulolyticum was studied, as well as an engineered form of this enzyme displaying a dockerin module
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