烟酰胺腺嘌呤二核苷酸（NAD）生物活性和功能多样，是最常用的氧化还原辅酶。因此，扰动胞内NAD及其还原态水平的生物学效应难以预测。申请人持续致力于创建基于非天然辅酶的生物正交代谢体系，已创制出多种非天然辅酶的合成酶及关联的氧化还原酶，首次实现利用胞外无机化能，选择性驱动胞内物质代谢途径。本重点项目拟研究烟酰胺胞嘧啶二核苷酸（NCD）的代谢及其生物学效应，以支撑其有效行使非天然辅酶功能。利用代谢工程策略强化前体供给，构建高效NCD合成体统；利用同位素标记技术和代谢组学方法，研究NCD降解转化途径及其分子基础；分析不同NCD水平下宿主细胞转录组、蛋白质组和代谢物组的差异，结合生物信息学分析，系统认识NCD的生物学效应；最终改造模式微生物获得NCD自给表型稳定可控的工程菌株。项目成果将为理性调控NCD自给的表型奠定科学基础，为建立正交能量代谢系统乃至正交生命提供新材料、新工具和新思路。

The omnipresent metabolite nicotinamide adenosine dinucleotide (NAD) has multiple biological activities, and functions as the most important redox cofactor. Thus, it is well-known that changes of the levels of NAD and its reduced form result in largely unpredictable outcomes. Over the years, we devoted to devising non-natural cofactor-based bioorthogonal systems. We successfully engineered enzymes for synthesis and utilization of these non-natural cofactors and constructed strains capable of using extracellular inorganic chemical as the terminal electron source to drive a specific redox transformation. In this project, we plan to study the metabolism and biological effects of nicotinamide cytosine dinucleotide (NCD), a promising non-natural, biocompatible cofactor. We will use metabolic engineering tools to enhance the metabolic flux to CTP and NMN, two key precursors for NCD biosynthesis, identify products and pathways of NCD degradation by the endogenous enzymes. Next, we will use a systems approach to determine cellular responses at the transcriptomic, proteomic and metabolomic levels in the presence of NCD, and integrate those data to define the biological effects of NCD and related metabolic models. Finally, the information and genetic materials will be used to engineer Escherichia coli and Saccharomyces cerevisiae to obtain strains for stable and controllable biosynthesis of NCD. The results of this project will greatly enrich our understanding on the metabolism and function of pyridine nucleotides. These new biological materials, tools and strategies should be valuable for the chemical biology and synthetic biology community to engineer orthogonal energy metabolism and orthogonal organisms.