The ubiquitous nicotinamide adenine dinucleotide (NAD) engages in diverse biological processes, leading to non-selective energy transfer toward target synthetic reactions. To achieve selective energy transfer in complex biological systems, we previously constructed artificial systems mediated by the non-natural cofactor nicotinamide cytosine dinucleotide (NCD), which can be specifically recognized by engineered enzymes with minimal cross-talk with natural cofactors. For enhanced energy transfer and higher product yields, efficient conversion of NCD to NCDH is required to deliver reducing power in NCD-mediated biosynthetic pathways. Here, we established a comprehensive strategy for selective reduction in intracellular NCD. First, coupled enzymatic colorimetric assays with high specificity were validated for quantifying NAD, NADP, and NCD. With phosphite as the energy source, we selectively elevated the intracellular NCDH/NCD ratio with minimal perturbation to NADH/NAD and NADPH/NADP ratios in E. coli. To overcome the limitation of phosphite transmembrane transport, cell-free systems were constructed to confirm that phosphite could drive near-complete NCD reduction. Finally, cells were treated with polymyxin B, which promoted phosphite uptake and thereby enabled maximal reduction in intracellular NCD. An NCDH/NCD ratio of 47 was achieved, demonstrating that 98% of the intracellular NCD pool existed in the reduced form. This work demonstrates that NCD can function as an independent redox cofactor for selective regulation, providing viable strategies for artificial cofactor-driven biosynthesis.

The ubiquitous cofactors nicotinamide adenine dinucleotide (NAD) and its reduced form play pivotal roles in energy metabolism and redox homeostasis. Recently, an NAD analog nicotinamide cytosine dinucleotide (NCD) has emerged for selective energy transfer in vivo . However, the biological effects of NCD remain elusive. Here we document cellular responses to NCD biosynthesis by multi-omics analysis of NCD self-sufficient Escherichia coli strains. Proteomics analysis revealed that NCD biosynthesis led to disturbance in up to 4% of the proteome, most of which were nonessential proteins or isozymes. By combining omics data and flux analysis, we identified fructose 6-phosphate, cytidine triphosphate, aspartate and glutamate as key branch nodes in the perturbation network. Importantly, we constructed several lines of E. coli cells capable of synthesizing NCD at different levels and confirmed that these cells grew similarly to their parental strains in either synthetic minimal or nutrient-rich media. Furthermore, we demonstrated that upregulation of the NCDH/NCD ratio caused little disturbance to those of natural nicotinamide cofactors. Our results assure that biosynthesis of NCD has minimal effects on cell growth and that the redox states of NCD(H) are largely segregated from those of natural redox cofactors, thereby facilitating a broader application of NCD as a non-natural cofactor in chemical biology and synthetic biology.