Efficient Biosynthesis of Polyhydroxybutyrate from Xylose via an In Vitro Synthetic Enzymatic Biosystem with Self-Sustained Cofactor Regeneration.

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