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β-Nicotinamide mononucleotide (NMN) is an important precursor in the synthesis of nicotinamide adenine dinucleotide (NAD+) and confers multiple health benefits, resulting in the rapid growth of NMN market capacity in the fields of food and health care. To overcome the drawbacks of NMN production by the existing chemical or microbial fermentation method, there is an urgent need to develop a prospective NMN production strategy with low cost, low pollution, and high yield. In this study, we demonstrated an artificial in vitro multi-enzyme cascade biocatalysis using starch and nicotinamide (Nam) as substrates for the synthesis of NMN in one-pot. This multi-enzyme cascade reaction was optimized in terms of pH value, buffer concentration, inorganic phosphate concentration, enzyme composition, and phosphoenolpyruvate concentration. Under optimized conditions, a high molar yield of 87.8% for NMN was achieved using 3.2 mM Nam as substrate, and a molar yield of 55.37% for NMN was also achieved under the initial Nam concentration of 9.21 mM. This in vitro enzymatic platform provides an environmental friendliness biomanufacturing technology for the production of NMN, showing a highly promising alternative approach for NMN production. • We created an innovative platform for the biomanufacturing of NMN from starch and Nam via in vitro multi-enzyme cascade biosystem. • This NMN production method is environmentally friendly. • The system can efficiently convert maltodextrin and Nam to NMN. [ABSTRACT FROM AUTHOR]