Functional genomics of the Clostridium thermocellum cellulosome

嗜热梭状芽胞杆菌的纤维素体是一个大的胞外多酶复合体，由40多种不同的模块化酶组成，排列在一个非催化结构蛋白(支架蛋白)上。纤维素体的复杂结构是其对结晶纤维素的极高效率的基础，可能是由于局部协同作用。因此，纤维素体对生物质的转化最有价值。高温假单胞菌的基因组已经被测序，这使得功能基因组学成为可能。本项目计划1)用蛋白质组学方法确定和鉴定纤维素体的所有成分。2)用反向遗传学方法克隆新成分，测序和表达，用于生化鉴定。3)整个复合体的组成将在体内进行研究：在不同底物上生长后蛋白质模式的变化指向晶体纤维素水解所需的纤维素体成分。4)基本成分也将通过它们在诱导的非纤维分解突变体中的缺失来鉴定。5)结合细菌细胞壁的纤维体成分的分析为细菌的附着和组装机制提供了线索。

The cellulosome of the thermophilic bacterium Clostridium thermocellum is a large, extracellular multienzyme complex consisting of more than 40 different modular enzymes, arranged on a non-catalytic structural protein (scaffoldin). The complex structure of the cellulosome is the basis for its extreme efficiency in the hydrolysis of crystalline cellulose, probably due to local synergism. Therefore the cellulosome is of the highest interest for the conversion of biomass. The genome of C. thermocellum has been sequenced, making functional genomics feasable. This project plans 1) to determine and to identify all components of the cellulosome with proteomic methods. 2) New components will be cloned with reverse genetics, sequenced and expressed for biochemical characterisation. 3) The composition of the whole complex will be investigated in vivo: changes in protein pattern after growth on different substrates point to the cellulosomal components necessary for the hydrolysis of crystalline cellulose. 4) Essential components will also be identified by their absence in induced non-cellulolytic mutants. 5) Analysis of the cellulosome-components bound to the bacterial cell wall gives an indication for the mechanism of attachment and assembly.