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

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.