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, nucleic acids, and critical metabolic pathways. An NADH synthesis pathway in artificial cells starting from D-ribose was constructed with 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 NADH synthesis pathway lays the foundation for fabricate artificial cells with complicated metabolic net works.
