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 pyruvate-derived chemicals, and may promote the development of cell-free synthetic enzymatic biosystems for biomanufacturing.
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