Multi-omics analyses reveal cellular responses to biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide.

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.