Abstract
Degradation of cellulose is of major interest in the quest for alternative sources of renewable energy, for its positive effects on environment and ecology, and for use in advanced biotechnological applications. Due to its microcrystalline organization, celluose is extremely difficult to degrade, although numerous microbes have evolved that produce the appropriate enzymes. The most efficient known natural cellulolytic system is produced by anaerobic bacteria, such as C. thermocellum, that possess a multi-enzymatic complex termed the cellulosome. Our laboratory has devised and developed the designer cellulosome concept, which consists of chimaeric scaffoldins for controlled incorporation of recombinant polysaccharide-degrading enzymes. Recently, we reported the creation of a combinatorial library of four cellulosomal modules comprising a basic chimaeric scaffoldin, i.e., a CBM and 3 divergent cohesin modules. Here, we employed selected members of this library to determine whether the position of defined cellulolytic enzymes is important for optimized degradation of a microcrystalline cellulosic substrate. For this purpose, 10 chimaeric scaffoldins were used for incorporation of three recombinant Thermobifida fusca enzymes: the processive endoglucanase Cel9A, endoglucanase Cel5A and exoglucanase Cel48A. In addition, we examined whether the characteristic properties of the T. fusca enzymes as designer cellulosome components are unique to this bacterium by replacing them with parallel enzymes from Clostridium thermocellum. The results support the contention that for a given set of cellulosomal enzymes, their relative position within a scaffoldin can be critical for optimal degradation of microcrystaline cellulosic substrates.
Highlights
The cellulosome was first demonstrated in the anaerobic thermophilic cellulolytic bacterium, Clostridium thermocellum [1,2,3] and comprises an extracellular multi-enzymatic complex which efficiently degrades cellulose, the major component of plant cell wall
The recombinant form of Cel9A named a-9A was obtained by removing the CBM2 of the wild type at the C-terminus and by adding a dockerin module from A. cellulolyticus at the N-terminus
We tried to construct a 9A-a chimaera, where the A. cellulolyticus dockerin is positioned at the C-terminus of the enzyme to coincide with the original position of the wild-type enzyme's CBM2
Summary
The cellulosome was first demonstrated in the anaerobic thermophilic cellulolytic bacterium, Clostridium thermocellum [1,2,3] and comprises an extracellular multi-enzymatic complex which efficiently degrades cellulose, the major component of plant cell wall. Additional support was obtained from the establishment of an Israeli Center of Research Excellence (I-CORE Center No 152/11) managed by the ISF, Jerusalem, Israel, by the Weizmann Institute of Science Alternative Energy Research Initiative (AERI) and the Helmsley Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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