A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

Yael Vazana,Yoav Barak,Yair Mazor,Melina Shamshoum,Raphael Lamed,Tuval Ben-Yehezkel,Ehud Shapiro,Yoav Peleg,Tamar Unger,Edward A Bayer

doi:10.1186/1754-6834-6-182

Yael Vazana, Yoav Barak + Show 8 more

Open Access

https://doi.org/10.1186/1754-6834-6-182

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Abstract

BackgroundSelect 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.ResultsUsing 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.ConclusionsThe 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.

Highlights

Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation
In order to study the importance of the spatial organization of the cellulases in the scaffoldin we used different intermodular linker lengths between the cohesin and the carbohydratebinding module (CBM) modules, thereby imitating the diversity that exists in nature - long 27 to 35 aminoacid intermodular linkers, short 5 amino-acid linkers or without such linkers (Figure 1b)
The results indicated a lack of preferred scaffoldin arrangement using the specific C. thermocellum cellulases and cellulosic substrates described in this study - including the non-native scaffoldins - which were fully functional in their contribution to degradation of cellulosic substrates

Summary

Introduction

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. One of the most potent cellulosedegrading microorganisms is the well-studied bacterium, Clostridium thermocellum This anaerobic thermophilic cellulolytic bacterium secretes a multi-enzymatic complex called the cellulosome, first discovered in 1983 [1,2]. In C. thermocellum the scaffoldin is composed of nine repeating cohesin modules, each of which can bind cohesively to a complementary dockerin-module, borne by a cellulosomal enzyme. Enhanced synergism among the catalytic units and close association between the cell-bound cellulosome and substrate serve to minimize diffusion of the enzymes and their hydrolytic products, providing the bacterium with a competitive advantage over other organisms [22]

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