Conversion of D-glucose to L-lactate via pyruvate by an optimized cell-free enzymatic biosystem containing minimized reactions

Cell-free synthetic enzymatic biosystem is emerging to expand the traditional biotechnological mode by utilizing a number of purified/partially purified enzymes and coenzymes in a single reaction vessel for the production of desired products from low-cost substrates.Here,a cell-free synthetic biosystem containing minimized number of reactions was designed for the conversion of D-glucose to L-lactate via pyruvate.This NADH-balanced biosystem was comprised of only 5 thermophilic enzymes without ATP supplementation.After optimization of enzyme loading amounts,buffer concentration and cofactor concentration,D-glucose was converted to L-lactate with a product yield of∼90%.Our study has provided an emerging platform with potentials in producing pyruvatederived chemicals,and may promote the development of cell-free synthetic enzymatic biosystems for biomanufacturing.