Stoichiometric Conversion of Maltose for Biomanufacturing by In Vitro Synthetic Enzymatic Biosystems

Maltose is a natural α-(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However,maltose has scarcely been used for in vitro biosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP)yields β-glucose 1-phosphate (β-G1P) that cannot be utilized by α-phosphoglucomutase (α-PGM) commonly found in in vitrosynthetic enzymatic biosystems previously constructed by our group. Herein, we designed an in vitro synthetic enzymaticreaction module comprised of MP, β-phosphoglucomutase (β-PGM), and polyphosphate glucokinase (PPGK) for thestoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this syntheticmodule, we further constructed two in vitro synthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP),respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6mW/cm^(2),whereas the 5-enzyme in vitro FDP-producing biosystem yielded 187.0mM FDP from 50 g/L (139mM) maltose by adopting afed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novelstrategies for the high-efficient production of bioelectricity and value-added biochemicals.