天然纤维小体是自然界中最高效的纤维素降解超分子机器，产物纤维糊精的严重抑制效应是阻碍其工业化应用的主要瓶颈之一。本项目围绕纤维糊精代谢酶的体外体内功能、胞内纤维糊精代谢机理及纤维糊精代谢增强三个方面依次开展研究。首先以热纤梭菌DSM1313菌株的3种ß-葡萄糖苷酶和3种纤维二糖/纤维糊精磷酸化酶这两类酶为对象，通过大肠杆菌进行异源表达，并进行纯化和酶学性质分析，初步解析纤维糊精代谢酶的体外功能及对纤维糊精降解的优先性；其次，对这两类酶进行体内单基因敲除和多基因敲除，通过对突变菌株的表型分析及代谢物水平、转录水平和蛋白质水平的多尺度研究，解析这两类酶的体内功能，并在此基础上阐明胞内纤维糊精代谢的分子机理；最后，通过对代谢关键酶进行启动子强化或敲入新的同工酶，增强胞内纤维糊精的代谢性能。本项目将为纤维小体酶的产物抑制作用的缓解以及热纤梭菌在木质纤维素CBP工艺中的应用奠定必要的理论基础。

The cellulosome is the most efficient extracellular supramolecular machine in the nature that can efficiently degrade cellulose. However, serious degraded production inhibition hampered its industrial application. The emphasis of this proposal focuses on the key bottleneck of employing cellulosome for emzymatic hydrolysis of cellulose by Consolidated Bioprocessing route. The research plan consists of three parts: the function of cellodextrins degradation enzymes, metabolic mechanism of intracellular cellodextrins and its metabolic boost. Firstly, 6 cellodextrins degradation enzymes from Clostridium thermocellum DSM 1313 will be heterologous expressed and purified, and then its properties will be analyzed, which will give clues to enzymes function and enzymes priority for cellodextrins degradation. Secondly, construct a serious of mutant strains with disruption of hydrolytic or phosphorolytic cleavage of cellodextrins, and then analyze properties of these mutant strains on metabolic, transcriptional and proteinic levels, which will shed light on intracellular functions of cellodextrins degradation enzymes and metabolic mechanism of cellodextrins. Finally, Clostridium thermocellum DSM 1313 will be genetically manipulated to boost its intracellular cellodextrins metabolism. This work will pave the way for reduction of production inhibition of cellulosome and industrial application of Clostridium thermocellum in CBP.