Abstract
ABSTRACTDesigner cellulosomes consist of chimeric cohesin-bearing scaffoldins for the controlled incorporation of recombinant dockerin-containing enzymes. The largest designer cellulosome reported to date is a chimeric scaffoldin that contains 6 cohesins. This scaffoldin represented a technical limit of sorts, since adding another cohesin proved problematic, owing to resultant low expression levels, instability (cleavage) of the scaffoldin polypeptide, and limited numbers of available cohesin-dockerin specificities—the hallmark of designer cellulosomes. Nevertheless, increasing the number of enzymes integrated into designer cellulosomes is critical, in order to further enhance degradation of plant cell wall material. Adaptor scaffoldins comprise an intermediate type of scaffoldin that can both incorporate various enzymes and attach to an additional scaffoldin. Using this strategy, we constructed an efficient form of adaptor scaffoldin that possesses three type I cohesins for enzyme integration, a single type II dockerin for interaction with an additional scaffoldin, and a carbohydrate-binding module for targeting to the cellulosic substrate. In parallel, we designed a hexavalent scaffoldin capable of connecting to the adaptor scaffoldin by the incorporation of an appropriate type II cohesin. The resultant extended designer cellulosome comprised 8 recombinant enzymes—4 xylanases and 4 cellulases—thereby representing a potent enzymatic cocktail for solubilization of natural lignocellulosic substrates. The contribution of the adaptor scaffoldin clearly demonstrated that proximity between the two scaffoldins and their composite set of enzymes is crucial for optimized degradation. After 72 h of incubation, the performance of the extended designer cellulosome was determined to be approximately 70% compared to that of native cellulosomes.
Highlights
Designer cellulosomes consist of chimeric cohesin-bearing scaffoldins for the controlled incorporation of recombinant dockerin-containing enzymes
It presents an elementary structure based on a primary scaffoldin subunit that attaches to the substrate via a carbohydratebinding module (CBM) and incorporates different enzymes via specific high-affinity type I cohesin-dockerin interactions
The cellulosome is attached to the bacterial cell surface via a second type of cohesin-dockerin interaction between the primary scaffoldin and an anchoring scaffoldin, which connects to the cell via an S layer homology (SLH) module [3]
Summary
Designer cellulosomes consist of chimeric cohesin-bearing scaffoldins for the controlled incorporation of recombinant dockerin-containing enzymes. The cellulosome was first discovered in Clostridium thermocellum as a multisubunit entity of complicated quaternary structure with a size of about 18 nm [2] It presents an elementary structure based on a primary scaffoldin subunit that attaches to the substrate via a carbohydratebinding module (CBM) and incorporates different enzymes via specific high-affinity type I cohesin-dockerin interactions. The fabrication of designer cellulosomes was first proposed in 1994 and is based on the very high affinities [1, 13] and specific interactions [14, 15] between matching cohesin and dockerin modules, enabling self-assembly of their components For this purpose, a chimeric scaffoldin is produced that, unlike native cellulosomal systems, contains cohesins of divergent specificities together with a collection of enzymes that contain matching types of dockerins. A few studies have attempted to design more complex structural composites [19, 20]
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