High-titer nicotinamide adenine dinucleotide production via artificially designed pseudo-de novo biosynthesis pathway

Nicotinamide adenine dinucleotide (NAD⁺) is a pivotal cofactor essential for cellular metabolism and physiological homeostasis. Herein, a latent and evolutionarily conserved pseudo-de novo NAD⁺ synthesis pathway, termed the chimeric NAD⁺ synthesis from 2-nitrobenzoate mineralisation (CNNM) pathway, was identified and functionally characterised in microbes involved in the bioremediation of 2-nitrobenzoate (2-NBA). Using the key enzymes 2-NBA nitroreductase, 2-hydroxylaminobenzoate mutase, and 3-hydroxyanthranilate 3,4-dioxygenase, which catalyse the conversion of 2-NBA to quinolinate, as probes, a total of 136 microbial species harbouring the putative CNNM pathway were identified in the NCBI database. The corresponding genes were then reconstructed into a portable plug-and-play module for the bioconversion of 2-NBA to NAD⁺. Upon introducing this module into an NAD⁺‑auxotrophic Escherichia coli strain, its NAD⁺ biosynthetic capability was validated with plate-based growth assays. During bioremediation of 1 mM 2-NBA, the engineered strain achieved a theoretical NAD(H) yield of 97.89%. Remarkably, through a combination of adaptive laboratory evolution and systematic metabolic engineering, intracellular NAD(H) concentration reached 15.12 mM, representing the highest reported level to date. By harnessing the CNNM pathway, consolidated 2-NBA detoxification and NAD⁺ biosynthesis were accomplished in a single bioprocess. These findings support an eco-friendly approach to coupling 2-NBA valorisation with high-value NAD⁺ production, promoting the economic feasibility of 2-NBA bioremediation.