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

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.

Proximity Effect among Cellulose-Degrading Enzymes Displayed on the Saccharomyces cerevisiae Cell Surface

“…Studies on cellulosomes have yielded unexpected results showing that decreasing the distance between cellulases in the cellulosome does not necessarily lead to increased cellulose-degrading activity (25,26); this was unexpected because of the lack of information on the natural fine structure of the cellulosome. In the previous study in which a designed minicellulosome was used, the proximity effects of cellulases at various distances were evaluated (25). The lengths of the linkers between cohesins (domains in the scaffoldin structure of the cellulosome that cellulases can bind (a) The distance between the different kinds of cellulases displayed on ALL-yeast (X) was calculated.…”

Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome

“…The chimeric scaffoldin enables control of the location of each cellulolytic enzyme in the cellulosomal complex, as well as its molar ratio. This Lego-like complex has been used to test the effects of enzymatic compositions, the relative positioning of the enzymes within the complex, and their spatial proximity, which, together, generate the synergistic action of the cellulosomal components (33)(34)(35). The majority of the T. fusca biomass-degrading enzymes have been converted previously to the cellulosomal mode (19,20,(36)(37)(38)(39)(40)(41), including a pair of lytic polysaccharide monooxygenases (LPMOs) involved in the oxidative cleavage of crystalline cellulose fibers (22).…”

A seawater-based open and continuous process for polyhydroxyalkanoates production by recombinant Halomonas campaniensis LS21 grown in mixed substrates

“…Industrial biotechnology aims to produce chemicals, materials and biofuels on a large scale using sustainable resources from agriculture [1][2][3]. By partially replacing depleting petrochemicals, industrial biotechnology should help sustainable development of human society [4][5][6][7][8][9].…”