热纤梭菌纤维小体的产物抑制机理及工程改造

项目名称： 热纤梭菌纤维小体的产物抑制机理及工程改造

项目编号： No.31470210

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 崔球

作者单位： 中国科学院青岛生物能源与过程研究所

项目金额： 82万元

中文摘要： 纤维小体是自然界中最高效的纤维素降解分子机器，强产物抑制效应是其产业化应用的主要障碍之一。本项目围绕纤维小体应用于木质纤维素的整合生物加工糖化的核心瓶颈，分别从酶组分的蛋白质工程改良、纤维小体的体外设计/优化、热纤梭菌的纤维小体系统的遗传改造三个层次开展研究：首先以外切酶Cel48S及β-葡萄糖苷酶Bgl21为对象，通过结构比较及分子模拟计算，解析产物抑制的分子机理及构效关系，利用蛋白质理性设计缓解纤维二糖或葡萄糖的产物抑制效应；其次，设计及构建人工纤维小体，重点优化Cel48S及Bgl21的催化结构域的距离及剂量配比，获得酶组分间催化动力学平衡、底物载量高，葡萄糖终浓度大的纤维小体改良方案，最后，对热纤梭菌DSM1313菌株进行遗传改造，将其打造成改良纤维小体的全菌催化剂平台，解决纤维小体的规模化制备瓶颈，为纤维小体在纤维素基生物能源及生物基化学品方面的工业化应用奠定必要的理论基础。

中文关键词： 热纤梭菌；纤维小体；遗传改造；木质纤维素；产物抑制

英文摘要： Cellulosome, nature's most effective cellulose degrading molecular machine, suffers serious product inhibition, which hampers its industrial application. The central theme of this proposal focuses on the core bottle-necks of employing cellulosome for enzymatic lignocellulose hydrolysis via Consolidated Bio-Processing route. The research plan consists of three parts: the protein engineering of individual key enzymes, the construction and optimization of designer cellulosome, and the genetic manipulation of cellulosome system of Clostridium thermocellum. Firstly, the structure detail and the product inhibition mechanism of a thermostable beta-glucosidase Bgl21 and a cellobiohydrolase Cel48S of Clostridium thermocellum will be analyzed by structure comparison and molecular simulation, which will then guide the protein engineering to alleviate the product inhibition effect of glucose and cellubiose. Secondly, we will construct and analyze a series of designer cellulosomes in order to obtain information for constructing an optimal cellulosome with enzymatic kinetic balance, and tolerant to high substrate loading and high product concentration. Finally, Clostridium thermocellum strain DSM1313 will be genetically converted into a designer cellulosome producer. This work will pave the road for the industrial application of cellulosome in lignocellulose-based bioenergy and bio-based chemicals.

英文关键词： Clostridium thermocellum;Cellulosome;Genetic engineering;Lignocellulose;Product inhibition