Engineering xylose utilization in Cupriavidus necator for enhanced poly(3-hydroxybutyrate) production from mixed sugars

[Display omitted] •Engineered strains utilize xylose for efficient poly(3-hydroxybutyrate) production.•RXI22 strain achieves 76 wt% poly(3-hydroxybutyrate) production from mixed sugars.•RXI22 strain produced 1.8 times more poly(3-hydroxybutyrate) than RXW62 strain.•Strains are promising host for producing polyhydroxyalkanaote from lignocellulose.•Study expands microbial carbon source use for polyhydroxyalkanaote synthesis. Lignocellulosic biomass is a promising renewable feedstock for biodegradable plastics like polyhydroxyalkanoates (PHAs). Cupriavidus necator, a versatile microbial host that synthesizes poly(3-hydroxybutyrate) (PHB), the most abundant type of PHA, has been studied to expand its carbon source utilization. Since C. necator NCIMB11599 cannot metabolize xylose, we developed xylose-utilizing strains by introducing synthetic xylose metabolic pathways, including the xylose isomerase, Weimberg, and Dahms pathways. Through rational and evolutionary engineering, the RXI22 and RXW62 strains were able to efficiently utilize xylose as the sole carbon source, producing 64.2 wt% (wt%) and 61.4 wt% PHB, respectively. Among the engineered strains, the xylose isomerase-based RXI22 strain demonstrated the most efficient co-fermentation performance, with a PHB content of 75.7 wt% and a yield of 0.32 (g PHB/g glucose and xylose) from mixed sugars. The strains developed in this study represent an enhanced PHA producer, offering a sustainable route for converting lignocellulosic biomass into bioplastics.