基于理性设计的纤维小体结构优化及体外组装

Artificial cellulosome is a multienzyme complex with highly catalytic activity, which could adjust the kind and expression level of enzyme. So it can completely hydrolyze non-starch polysaccharides (NSP) in the dietaries. To solve the problem in the process of cellulosomic assembly, some strategies will be taken to optimize its structure. Based on the lignocellulose degradation enzyme (AExynM, Auman5A, AuFaeA and EG1) with good qualities and the cohesin-dockerin genes in rumen microbial metagenomics in our laboratory, cohesin-dockerin complex with strong affinity will be screened by rational design. Then the specificity between cohesin and dockerin will be analyzed by Native-PAGE and Isothermal Titration Calorimetry (ITC) experiments. Finally, by adjusting the proportion of cohesin modules and the enzyme components, yeast transformants displaying different cellulosomes on the cell surface will be analyzed to determine their abilities to hydrolyze different lignocelluloses. This project is expected to improve the feeding value of agricultural by-products by breaking the bottleneck in the production of NSP enzymes, such as the difficulty of cooperative fermentation and the inflexibility in adjusting the kind and expression level of enzyme. Moreover, this project could explore novel cohesin-dockerin complex, and provide new strategy and technical support for assembling of other cellulosomes and and multienzyme complexes.

人工纤维小体是一种具高催化活性的多酶复合体，可灵活调控酶的种类和表达量，从而达到充分降解饲粮中非淀粉多糖（NSP）的目的。针对纤维小体中组分蛋白的缺陷，本项目拟通过以下策略来优化纤维小体的组装。基于前期获得的优质木质纤维素降解酶基因（AExynM、Auman5A、AuFaeA及EG1）及瘤胃微生物宏基因组内的粘连蛋白和对接蛋白基因，首先借助理性设计的方法，获得理论上与种粘连蛋白亲和力强的多种对接蛋白；然后结合Native-PAGE及ITC实验验证粘连蛋白-对接蛋白之间的专一性；最后通过调节粘连蛋白模块的比例及酶组分的种类，在酿酒酵母细胞表面组装具不同催化功能及不同表达量的多酶组分纤维小体。本研究有望突破NSP复合酶生产中协同发酵困难或无法灵活协调、量化酶组分的瓶颈，对提高农业副产物的饲用价值具有重要意义，同时又可发掘新型粘连蛋白-对接蛋白，为其他类型纤维小体或多酶复合体的组装提供技术平台。

人工纤维小体是--种具高催化活性的多酶复合体，可灵活调控酶的种类和表达量，从而达到充分降解饲粮中非淀粉多糖(NSP)的目的。针对纤维小体中组分蛋白的缺陷，本项目通过海子水牛瘤胃微生物宏基因组测序，结合生物信息学分析，发现了4个粘连蛋白Coh基因和64个对接蛋白Doc基因；然后借助理性设计的方法，获得理论上结合力强的Coh1-Doc1、Coh2-Doc2及Coh3-Doc3。 提取瘤胃微生物宏基因组，将Doc1、Doc2和Doc3基因分别克隆并融入前期获得的具高催化活性木聚糖酶AExynM、葡聚糖酶EG1和β-甘露聚糖酶Anman5A基因的C末端，然后在大肠杆菌BL21中进行了表达，发现融合对接蛋白会降低酶的催化活性。将Coh1、Coh2 及Coh3分别克隆并在大肠杆菌中进行表达，利用组氨酸标签对六种重组蛋白进行纯化,借助非变性凝胶电泳实验验证Coh-Doc结合的特异性,发现Coh1-Doc1 及Coh3-Doc3可特异性结合。借助pPIC9K及pPICZa A表达质粒，实现.了AExynM-Doc1 和Anman5A-Doc3在毕赤酵母GS115中的共表达，二者酶活分别为132.20U/mL和33.84U/mL。本研究工作首次解析了海子水牛瘤胃微生物的宏基因组数据，为发掘新型具有工业应用潜力的木质纤维素降解酶奠定了理论基础，而且本研究发现的特异性结合Coh1-Doc1 及Coh3-Doc3，可为其他类型纤维小体或多酶复合体的组装提供技术平台，同时该研究实现了木聚糖酶和葡聚糖酶在酵母中的共表达，可突破NSP复合酶生产中协同发酵困难的瓶颈，对提高农业副产物的饲用价值具有重要意义。