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D-Mannitol (hereinafter as mannitol) is a six-carbon sugar alcohol with diverse applications in food and pharmaceutical industries. To overcome the drawbacks of the chemical hydrogenation method commonly used for mannitol production at present, there is a need to search for novel prospective mannitol production strategies that are of high yield and low cost. In this study, we present a novel approach for the stoichiometric synthesis of mannitol via an in vitro synthetic enzymatic biosystem using the low-cost starch as substrate. By dividing the overall reaction pathway into three modules which could be executed sequentially in one pot, our design aimed at the stoichiometric conversion of starch-based materials into mannitol in an ATP-independent and cofactor-balanced manner. At optimized conditions, high product yields of around 95-98% were achieved using both 10 g/L and 50 g/L maltodextrin as substrate, indicating the potential of our designed system for industrial applications. This study not only provides a high-efficient strategy for the synthesis of mannitol but also expands the product scope of sugar alcohols by the in vitro synthetic enzymatic biosystems using low-cost starch-based materials as the input. Key points: • We described a design-build-test-learn pipeline to construct in vitro biosystems. • The designed system comprised six key enzymes and another three enzymes. • The system converted maltodextrin stoichiometrically to mannitol in one pot. [ABSTRACT FROM AUTHOR]