Artificial ATP-Free in Vitro Synthetic Enzymatic Biosystems Facilitate Aldolase-Mediated C–C Bond Formation for Biomanufacturing

Asymmetric C–C bond formation mediated by aldolase provides one of the most efficient ways to produce valuable chemicals in biomanufacturing. Dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GA3P) are two important platform compounds for asymmetric C–C bond formation. In this study, several artificial ATP-free in vitro synthetic enzymatic biosystems were constructed to produce valuable chemicals via facile synthesis of GA3P and DHAP from starch and pyrophosphate. Six cascade enzymes were used for the biotransformation of starch and pyrophosphate to GA3P or DHAP: alpha-glucan phosphorylase (αGP), phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), pyrophosphate phosphofructokinase (PPi-PFK), d-fructose 1,6-bisphosphate aldolase (FruA), and triosephosphate isomerase (TIM). These two compounds were then used to produce various chemicals, including 2-deoxy-d-ribose (DR) and rare ketoses. After the optimization of reaction conditions, ∼23.2 mM DR with a product yield of 96.7% and 15.2 mM d-allulose with a product yield of 95.0% were produced, both achieving near-stoichiometric yields through downstream aldol additions and dephosphorylation reactions in one pot. In addition, more than 80% of the product yields of DR and many rare ketoses, such as d-allulose, l-tagatose, d-sorbose, l-fructose, and d-xylulose, from high concentrations of substrates were obtained, showing high industrial potential. This in vitro biomanufacturing platform may provide a promising and cost-effective approach for biomanufacturing value-added chemicals through asymmetric C–C bond formation in the near future.