体外无细胞多酶催化途径中电子传递模块的人工设计与组装

Synthetic enzymatic pathways containing electron transfer modules have many applications in the biosynthesis and bioconversion of various chemicals and energies. However, there is no systematic study on in vitro electron transfer so far, leading to the unclearness in the interaction and compatibility of its parts, and the lack of the design principle and the assembly method of its modules. In this work, we will target on the electron transfer module consisted of enzymes from the glucose 6-phosphate dehydrogenase to the hydrogenase in an in vitro enzymatic biohydrogen production pathway. Through introducing exogenous chemical or biological electron mediators, crosslinking enzyme-coenzyme conjugates, and assembling enzyme complexes, the in vitro electron transfer chain will be constructed with the breakthrough of energy barriers and configurational limitations, as well as the fast electron transfer rate and the increase in the overall reaction rate. With the help of enzyme kinetics, electrochemistry, and structural simulation, we can analyze the compatibility of various parts and optimize their ratio, configuration, position, and distance. The electron transfer mechanism will be elucidated and the new theory and method for artificially assembling electron transfer modules will be investigated. This study can not only promote the development of the in vitro biohydrogen production technology, but also pave the way for the design and optimization of other synthetic enzymatic pathways containing electron transfer modules.

含有电子传递模块的体外无细胞多酶催化途径在物质和能量的生物合成与转化中有许多重要应用。然而，目前还没有针对体外电子传递的系统研究，各元件相互作用和适配机制尚不清楚，缺乏设计与组装体外电子传递模块的原理和方法。本项目拟以体外多酶产氢途径中的由葡萄糖6磷酸脱氢酶到氢酶的电子传递模块为研究对象，通过引入包含化学或生物类的外源电子载体、交联酶-辅酶结合物、组装多酶复合体，构建体外人工电子传递链，突破能量壁垒和空间构型局限，实现电子快速传递和总反应速度的提升。通过酶动力学、电化学、和结构模拟等方法，分析各元件的适配性，优化各元件的配比、构象、空间位置、和距离等，解析体外电子快速传递的机制，探索人工设计和组装电子传递模块的新理论和新方法。本项目的完成不光将推动体外生物制氢技术的发展，还将为其它含有电子传递模块的体外无细胞多酶催化途径的设计和优化提供理论依据。

含有电子传递模块的体外无细胞多酶催化途径在物质和能量的生物合成与转化中有许多重要应用。然而，目前还没有针对体外电子传递的系统研究，各元件相互作用和适配机制尚不清楚，缺乏设计与组装体外电子传递模块的原理和方法。本研究以体外多酶产氢和产电途径中的电子传递模块为对象，通过引入外源电子载体、交联酶-辅酶、酶-电子载体、组装多酶复合体等策略，构建体外人工电子传递链。建立了新的酶-辅酶、酶-电子载体交联物的构建方法，降低局部电子传递距离；尝试了基于四亚基氢酶的复合体构建方法，并对其内部电子传递机理做了解析；对相关脱氢酶和氢酶做了表征，解析了多亚基氢酶在电极界面的定向吸附规律和电子传递机制，提升了脱氢酶-氢酶组成的电子传递链性能；将此电子传递链体系应用于生物传感器中，实现了真实样品木糖和二羟基戊二酸的精准测定，具有重要意义。本项目的完成不光将推动体外生物制氢技术的发展，还将为其它含有电子传递模块的体外无细胞多酶催化途径的设计和优化提供理论依据。