脚架蛋白介导的多酶复合体的底物通道效应及其连接肽的功能解析

Mini-scaffoldin-mediated synthetic multi-enzyme complex can greatly improve the catalytic efficiency of cascade enzymes, which facilitated by substrate channeling. Additionally, synthetic multi-enzyme complex is easy to be purified and reconstructed, which has become an important biocatalytic module for synthetic biology projects. Up to now, what will be the effect of linker peptides (amino acid composition, length, and stiffness) on synthetic multi-enzyme complex (enzymatic properties and the overall structure) has not yet been determined. In this project, by using a one-step purification and co-immobilization method, thermostable triosephosphate isomerase (TIM) and bifunctional fructose-1,6-bisphosphate aldolase/phosphatase (FBPA/P) was constructed into a synthetic multi-enzyme complex TIM–FBPA/P. The rigidity and length of linker between two cohesin modules in mini-scaffoldin was reconstructed, and the substrate channeling of the multi-enzyme complex was characterized. We will use homology modeling to clarify the 3D structure information of the modules in the multi-enzyme complex TIM–FBPA/P. Meanwhile, small angle x-ray scattering, chemical cross-linking and mass spectrometry were used to analysis of the overall structure of the synthetic multi-enzyme complex. Then, we try to elucidate the function of linker in the TIM–FBPA/P. Based on the above research, further rational engineering of linker will be performed in order to obtain more efficient mini-scaffoldin-mediated synthetic multi-enzyme complex, which may give us clue about biocatalysis designing in accordance with human needs.

脚架蛋白介导的多酶复合体易于纯化和改造，具有底物通道效应，可以极大提高级联酶的催化效率，是合成生物学中生物催化的重要策略。到目前为止，连接肽的氨基酸组成、长度和刚柔性对多酶复合体的酶学性质和整体结构的影响，尚不清楚。本项目将以嗜热丙糖磷酸异构酶TIM和嗜热双功能醛缩酶/果糖-1,6-二磷酸酶FBPA/P作为研究材料，一步纯化、共固定获得脚架蛋白介导的TIM–FBPA/P多酶复合体。利用合成生物学手段改造脚架蛋白中两个cohesin之间连接肽的刚柔性和长度，并对重构多酶复合体的底物通道效应进行表征。通过蛋白同源建模，获得多酶复合体中各个模块的3D结构信息。同时，采用小角度x-射线散射、化学交联和质谱分析等方法，分析TIM–FBPA/P人工多酶复合体的整体结构，进而阐明连接肽在多酶复合体中的功能。为进一步设计改造脚架蛋白介导的人工多酶复合体，建立高效定制工业酶催化剂平台提供指导。

多酶复合体具有底物通道效应，可以极大提高级联酶的催化效率，是体外多酶体系进行生物催化的重要策略。脚架蛋白易于纯化和改造、成本低廉，是介导多酶复合体形成的理想材料。然而，连接肽的氨基酸组成、长度和刚柔性对多酶复合体的酶学性质和整体结构的影响，尚不清楚。本研究首先设计了一个体外多酶体系用于催化纤维素底物进行生物制造，实现了纤维素到肌醇的化学计量的转化，表明该无细胞体外多酶体系是新型、高效的生物制造平台。通过对纤维素利用途径的CDP和PGM构建多酶复合体，研究了功能模块在脚架蛋白上的排列顺序、数量等对复合体底物通道效应的影响。将底物通道效应最高的复合体应用到纤维素产电体外多酶体系中，能够极大提高初始电流密度和最大功率密度。对TIM–FBPA复合体中脚架蛋白的linker长度进行研究，结果表明含有25个氨基酸长度linker的复合体具有最高的初始反应速度。在相同linker长度下，复合体中含有两端是刚性单元、中间是柔性单元的linker（FRRRF）展现出最高的底物通道效应。经过同源建模、生物信息学分析，精确的linker长度和刚柔性能够影响复合体的整体构象，从而影响复合体的初始反应速率。基于上述分析，精确调控复合体的初始反应速度是一个复杂的系统工程，涉及到多酶结构、连接肽长度、刚柔性等，这为未来人工设计多酶复合体提供理性指导。