This study demonstrates an in vitro synthetic enzymatic biosystem for the high-yield enzymatic conversion of xylose to polyhydroxybutyrate (PHB). Through systematic optimization of reaction conditions, we achieved high product yield of PHB from xylose via a self-sustained cofactor regeneration system, providing sustainable biomanufacturing and advanced biorefining strategies.

In vitro biotransformation mediated by in vitro synthetic enzymatic biosystems (ivSEBs) represents a highly promising platform for sustainable biomanufacturing, offering enhanced reaction efficiency by circumventing cellular constraints. This study develops an ivSEB comprising 17 enzymes for the cell-free biosynthesis of polyhydroxybutyrate (PHB) from D-xylose via acetyl-coenzyme A (CoA). This ivSEB integrates partial glycolysis and the pentose phosphate pathway, enabling self-sustained balance of several cofactors including CoA, NADP⁺/NADPH, and ATP/ADP. Stoichiometric analysis demonstrates a theoretical molar yield of PHB from xylose of 111.1%. Through optimizing concentrations of cofactors and enzymes, the one-pot reaction produces 44.0 mM (3.8 g L^{− 1}) PHB from 44.8 mM (6.7 g L⁻¹) xylose, corresponding to a molar yield of 98.2%. Even at a higher substrate concentration (13.5 g L⁻¹), this yield (84.5%) remains robust. This study demonstrates the potential of the ivSEB as a scalable and efficient approach for the large-scale production of PHB and other xylose-based or acetyl-CoA-derived chemicals.