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For the biosynthesis of nicotinamide mononucleotide (NMN), three artificial pathways including the nicotinamide ribose phosphorylation pathway, adenosine phosphate pyrophosphorylation pathway, and adenosine phosphate hydrolysis (APH) pathway were designed and successfully conducted to produce NMN in vitro. The APH pathway, using AMP nucleosidase, ribose-phosphate diphosphokinase, and nicotinamide phosphoribosyltransferase (NAMPT), exhibited the highest level of NMN synthesis. To further improve NMN production viathe APH pathway, various NAMPT orthologues were screened. The effects of temperature, pH, metal ions and enzyme ratios were further systematically investigated, and the accumulation of ADP was identified limiting pathway efficiency. Subsequently, an ATP recycling process was achieved by adding polyphosphate kinase 2 to convert ADP to ATP. With the optimized four multienzyme cascade catalysis systems, the NMN titer was increased to 9 mmol L−1(3.0 g L−1) from 600 μmol L−1(0.2 g L−1) in vitro. This is the first study to use a multienzyme cascade catalysis process for NMN biosynthesis.