Construction of the reduced nicotinamide adenine dinucleotide salvage pathway in artificial cells and its application in amino acid synthesis

Reduced nicotinamide adenine dinucleotide (NADH) salvage pathway reconstitution is a crucial step toward autonomous artificial cells. In living systems, d-ribose is a fundamental precursor intricately involved in the synthesis of nucleotides, and other critical metabolic pathways. An NADH synthesis pathway in artificial cells starting from d-ribose was constructed with a five-enzyme cascade containing ribokinase, ribose-phosphate pyrophosphokinase, nicotinamide phosphoribosyltransferase, nicotinamide mononucleotide adenylyltransferase, and formate dehydrogenase (RK, RPPK, NAMPT, NMNAT, and FDH), which efficiently converted 10 mM d-ribose into 415 μM NADH within 80 minutes under optimized conditions. The produced NADH was further used to drive the amino acid metabolism, i.e., to convert NH₄⁺ and α-ketoglutarate to glutamate by introducing additional glutamate dehydrogenase (GDH) inside artificial cells. The successful reconstitution of the NADH synthesis pathway lays the foundation for fabricating artificial cells with complicated metabolic networks.