title: A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates.
abstract: BACKGROUND
  Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture.
  
  
  RESULTS
  Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27-35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins.
  
  
  CONCLUSIONS
  The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.
authors.forename: Yael
authors.initials: Y
authors.lastname: Vazana
authors.forename: Yoav
authors.initials: Y
authors.lastname: Barak
authors.forename: Tamar
authors.initials: T
authors.lastname: Unger
authors.forename: Yoav
authors.initials: Y
authors.lastname: Peleg
authors.forename: Melina
authors.initials: M
authors.lastname: Shamshoum
authors.forename: Tuval
authors.initials: T
authors.lastname: Ben-Yehezkel
authors.forename: Yair
authors.initials: Y
authors.lastname: Mazor
authors.forename: Ehud
authors.initials: E
authors.lastname: Shapiro
authors.forename: Raphael
authors.initials: R
authors.lastname: Lamed
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. ed.bayer@weizmann.ac.il.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer