改造铁氧还蛋白-NADP还原酶用于构建光驱动的非天然辅酶还原体系

Light driving cofactor reduction will improve economical efficiency of biomanufacture. Cyanobacterium derived ferredoxin-NADP reductase (FNR) efficiently generates NADPH by transferring electron, however, the follow-up energy transmission is not pathway selective. The bioorthogonal redox metabolic circuits was constructed based on non-natural cofactor nicotinamide cytosine dinucleotide (NCD), which can transfer energy pathway-selectively. But it uses phosphite as electron donor, which is expensive and toxic. The project intends to obtain NCD specific FNR mutant (FNR*) based on series of NCD specific modules, and analysis of sequence and structure related to cofactor specificity. The mutagenesis will be carried out based on rational design according to sequence and structure information of FNR, and then be screened from “hot-site screening” libraries or site saturation mutagenesis libraries. The light driving reducion system for non-natural redox cofactor will be constructed on the basis of FNR*. And the performance of the system will be evaluated by assembling bioorthogonal redox metabolic circuits, which supply NCDH selectively for NCD specific oxidoreductase mutant catalyzing production of building block chemicals. The light driving NCD specific reducing system will generate pathway specific [H] efficiently and economically, which will facilitate efficiency, economy and controllability of biomanufacture.

利用光能驱动产生还原力，可提高生物制造的经济性。蓝细菌光合系统的铁氧还蛋白-NADP还原酶(FNR)可高效接收电子，产生NADPH，但难以途径选择性传递还原力。申请人组装了基于非天然辅酶烟酰胺胞嘧啶二核苷酸（NCD）的生物正交氧化还原代谢电路，可选择性传递还原力，但其终端电子供体为亚磷酸，成本高、有弱毒性。本项目拟基于前期获得的一系列NCD特异的模块，及对FNR辅酶特异性相关序列和结构的分析，对FNR进行理性设计，利用“热点快速筛查”及饱和突变等蛋白改造策略，获得高活性及特异性还原NCD的突变型FNR（FNR*），构建基于FNR*的光驱动非天然辅酶还原体系，用于组装依赖于NCD的生物正交代谢电路，为NCD特异的突变型氧化还原酶提供还原力，推动平台化学品合成，测试代谢电路性能。构建光驱动的NCD还原体系，提供高效、廉价，且可选择性传递的还原力，将提高光驱动生物制造的效率、经济性及可控性。

降低蓝细菌光合系统铁氧还蛋白-NADP还原酶(FNR)的辅酶特异性，使特异性还原NADP的FNR可还原NAD或非天然辅酶烟酰胺胞嘧啶二核苷酸（NCD），可提高光能驱动产生还原力在生物制造中的应用效率，提高生物制造的经济性。本项目组通过对FNR辅酶特异性相关序列和结构的分析，对FNR进行理性设计，利用“热点快速筛查”及饱和突变等蛋白改造策略，获得了可还原NAD的FNR突变型。在此基础上，结合前期获得的一系列NCD特异性突变型的突变方式，对FNR进行理性突变，筛选到了高效还原NCD的突变型FNR。构建了高效辅酶转运系统和基于突变型FNR的咖啡酸生产工程菌，将还原力高效传递到咖啡酸生产途径，提高了咖啡酸生产效率。本项目的研究结果对于构建光驱动的高效还原体系，提高还原力依赖型生物制造的效率、经济性及可控性具有重要的意义。