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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]

Background: Haloacid dehalogenase (HAD)-like hydrolases represent the largest superfamily of phosphatases, which release inorganic phosphate from phosphate containing compounds, such as sugar phosphates. The HAD-like phosphatases with highly substrate specificity, which perform irreversible dephosphorylation, are always integrated into in vitro synthetic enzymatic biosystems as the last enzymatic step for the cost-efficient production of biochemicals. Therefore, identification and characterization of substrate specificity of HAD-like phosphatases are important for exploring their application. Results: In this study, a hyperthermophilic HAD-like phosphatase from Archaeoglobus fulgidus (AfPase) was cloned, expressed, and characterized. AfPase was identified as a type I Mg2+-dependent HAD-like phosphatase with high optimal temperature and thermostability. Among the tested phosphate containing compounds, AfPase exhibited the highest catalytic activity on p-nitrophenyl phosphate, followed by dihydroxyacetone phosphate (DHAP). On the basis of the high catalytic activity of AfPase to generate 1,3-dihydroxyacetone (DHA) from DHAP, an in vitro synthetic enzymatic biosystem containing this phosphatase and other five enzymes was constructed for the biosynthesis of DHA from inexpensive maltodextrin in one pot. About 14 mM (1.26 g/L) DHA was produced from 10 g/L maltodextrin. Conclusions: A hyperthermophilic HAD-like phosphatase from Archaeoglobus fulgidus was characterized carefully, and the success of an in vitro synthetic enzymatic biosystem containing this phosphatase provided a promising approach for DHA production from maltodextrin. [ABSTRACT FROM AUTHOR]