14-09: Characteristics of Clostridium thermocellum Scaffoldin Knockout Mutants Grown on Cellulose

The C. thermocellum secretome is known to be extremely effective in the digestion of cellulose. This self-assembling system consists of a diversity of glycoside hydrolases that are organized through specialized binding domains onto non-catalytic scaffoldin peptides.  In order to evaluate the individual contributions of the different scaffoldin proteins to overall activity, we are systematically creating mutants in which we have knocked out the genes encoding individual scaffoldins and combinations thereof. Performance of these mutants in the degradation and utilization of Avicel have been tested, and some interesting results have been found: cellulosomal enzymes organized on scaffoldins are much more effective than a simple mixture of the same enzymes expressed as “free enzymes” (i.e., with scaffoldin genes knocked out), and the primary scaffoldin CipA appears to be more important for digestion of Avicel than are the secondary scaffoldins. Further insights are provided by comparisons of direct biochemical analyses of mixtures of cellulosomal complexes with the organism-level performance.