Construction of an Alternative NAD⁺ De Novo Biosynthesis Pathway

Nicotinamide adenine dinucleotide (NAD⁺) is a life essential molecule involved in versatile biological processes. To date, only two de novo biosynthetic routes to NAD⁺ are described, both of which start from a proteinogenic amino acid and are tightly controlled. Here, a de novo quinolinic acid pathway starting from chorismate, which provides an alternative route (named as the C3N pathway) to NAD⁺ biosynthesis, is established. Significantly, the C3N pathway yields extremely high cellular concentrations of NAD(H) in E. coli. Its utility in cofactor engineering is demonstrated by introducing the four-gene C3N module to cell factories to achieve higher production of 2,5-dimethylpyrazine and develop an efficient C3N-based whole-cell bioconversion system for preparing chiral amines. The wide distribution and abundance of chorismate in most kingdoms of life implies a general utility of the C3N pathway for modulating cellular levels of NAD(H) in versatile organisms.