吡啶核苷酸辅酶是介导氧化还原代谢的重要辅酶，并广泛参与其它生物学过程；因此，改变吡啶核苷酸水平的生物学效应靶向性低，难以预测。申请人前期合成了多种吡啶核苷酸类似物，改造并获得了适配的氧化还原酶，初步形成了基于非天然辅酶的代谢调控研究思路。本项目拟改造烟酰胺单核苷酸（NMN）腺苷转移酶，赋予其腺苷三磷酸结合域识别嘧啶苷三磷酸的功能，获得烟酰胺嘧啶二核苷酸（NPyD）合酶，并建立相关生物合成方法。以NPyD的代表烟酰胺胞嘧啶二核苷酸（NCD）为例，研究利用NCD合酶、NCD专一性亚磷酸脱氢酶和亚磷酸敏感性宿主，构建高通量筛选平台，获得新的NCD偏好型氧化还原酶，丰富NCD关联的生物元件和作用模式。利用上述材料，设计构建具有NCD自给表型的工程菌，研究NCD介导代谢子系统的运行规律及其与全局代谢网络的相互作用。为化学生物学、合成生物学研究和先进细胞工厂设计提供新材料、新工具和新思路。

Pyridine nucleotide cofactors link major redox metabolic reactions, as well as many non-redox biochemical transformations. Therefore, it is intrinsically difficult to control one particular reaction precisely at the cofactor level. We have synthesized a number of pyridine nucleotide cofactor analogs, devised redox enzymes matching those analogs, and advocated a non-natural cofactor-based chemical biology. In this project, we plan to engineer the ATP binding site of nicotinamide mononucleotide (NMN) adenylyltransferase to accept pyrimidine triphosphate instead of ATP for direct biosynthesis of nicotinamide pyrimidine dinucleotides (NPyD) from NMN and pyrimidine triphosphate. We will then use the gene encoding the synthase for typical NPyD molecule nicotinamide cytosine dinucleotide (NCD) to engineer NCD self-sufficient strains. Phosphite-sensitive hosts will be engineered to overexpress NCD synthase and NCD-specific phosphite dehydrogenase for high-throughput screening genes encoding novel NCD-preferred oxidoreductase. Next, we will introduce these genetic elements into microbial hosts to set up various NCD-linked metabolic sub-systems, and analyze the mechanisms how these sub-systems work and interact with the global metabolic network. The results of this project should provide new biological materials, tools and strategies for the chemical biology and synthetic biology community to engineer advanced microbial strains for diverse applications and to address challenging problems related to pyridine nucleotide cofactors.