Metabolic pathways are connected through their utilization of nucleotides as supplier of energy, allosteric effectors, and their role in activation of intermediates. Therefore, any attempt to exploit a given living organism in a biotechnological process will have an impact on nucleotide metabolism and vice versa. Here, we provide an overview of the pyrimidine and purine biosynthesis, the interconversion pathways, the formation of deoxyribonucleotides, and the salvage reactions used in the utilization of exogenous precursors. The enzymatic and genetic regulation of these pathways are addressed. The aim of this article is to provide knowledge of nucleotide metabolism and its regulation to facilitate interpretation of data arising from genetics, proteomics, and transcriptomics in connection with biotechnological processes and beyond.