Abstract
BackgroundClostridium thermocellum is a thermophilic anaerobic bacterium that degrades cellulose by using a highly effective cellulosome, a macromolecular complex consisting of multiple cellulose degrading enzymes organized and attached to the cell surface by non-catalytic scaffoldins. However, due largely to lack of efficient methods for genetic manipulation of C. thermocellum, it is still unclear how the different scaffoldins and their functional modules contribute to cellulose hydrolysis.ResultsWe constructed C. thermocellum mutants with the primary scaffoldin CipA (cellulosome-integrating protein A) truncated at different positions or lacking four different secondary scaffoldins by using a newly developed thermotargetron system, and we analyzed cellulose hydrolysis, cellulosome formation, and cellulose binding of the mutants. A CipA truncation that deletes six type I cohesin modules, which bind cellulolytic enzymes, decreased cellulose hydrolysis rates by 46%, and slightly longer truncations that also delete the carbohydrate binding module decreased rates by 89 to 92%, indicating strong cellulosome-substrate synergy. By contrast, a small CipA truncation that deletes only the C-terminal type II dockerin (XDocII) module detached cellulosomes from the cells, but decreased cellulose hydrolysis rates by only 9%, suggesting a relatively small contribution of cellulosome-cell synergy. Disruptants lacking any of four different secondary scaffoldins (OlpB, 7CohII, Orf2p, or SdbA) showed moderately decreased cellulose hydrolysis rates, suggesting additive contributions. Surprisingly, the CipA-ΔXDocII mutant, which lacks cell-associated polycellulosomes, adheres to cellulose almost as strongly as wild-type cells, revealing an alternate, previously unknown cellulose-binding mechanism.ConclusionsOur results emphasize the important role of cellulosome-substrate synergy in cellulose degradation, demonstrate a contribution of secondary scaffoldins, and suggest a previously unknown, non-cellulosomal system for binding insoluble cellulose. Our findings provide new insights into cellulosome function and impact genetic engineering of microorganisms to enhance bioconversions of cellulose substrates.
Highlights
Clostridium thermocellum is a thermophilic anaerobic bacterium that degrades cellulose by using a highly effective cellulosome, a macromolecular complex consisting of multiple cellulose degrading enzymes organized and attached to the cell surface by non-catalytic scaffoldins
We found that the primary scaffoldin cellulosome-integrating protein A (CipA) and its carbohydratebinding module (CBM) are most critical and that all four secondary scaffoldins analyzed are required for maximum rates of cellulose hydrolysis
Construction of C. thermocellum mutant strains In C. thermocellum DSM1313, the strain used in this study, the major cellulosome scaffoldin CipA harbors eight repeated CohI modules, a CBM, and a C-terminal XDocII module (Figure 1, Additional file 1)
Summary
Clostridium thermocellum is a thermophilic anaerobic bacterium that degrades cellulose by using a highly effective cellulosome, a macromolecular complex consisting of multiple cellulose degrading enzymes organized and attached to the cell surface by non-catalytic scaffoldins. The cellulosome is a supermolecular machine that is secreted into the growth medium or attached to the cell wall of celluloytic bacteria and consists of multiple structural scaffoldins and enzymatic subunits that interact with each other to efficiently degrade lignocellulose substrates [1,2]. Among cellulosome-producing bacteria, the thermophilic anaerobic bacterium Clostridium thermocellum contains one of the most efficient cellulosome systems for the hydrolysis of lignocellulose substrates. The most critical structural component of the C. thermocellum cellulosome is the primary scaffoldin CipA (cellulosome-integrating protein A), which binds insoluble cellulose and serves as a scaffold for multiple cellulolytic enzymes [9]. CipA interacts via its C-terminus with secondary scaffoldins, which anchor CipA and its associated cellulolytic enzymes to the cell wall and lead to the formation of more complex polycellulosome structures [10]
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