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, alpha-glucan phosphorylase (αGP), phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), pyrophosphate phosphofructokinase (PPi-PFK), D-fructose 1,6-bisphosphate aldolase (FruA) and triosephosphate isomerase (TIM), were used for the biotransformation of starch and pyrophosphate to GA3P or DHAP. 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, about 23.2 mM DR with a product yield of 96.7% and ...