Rewiring <i>Cupriavidus necator</i> for Enhanced Polyhydroxybutyrate Production via Genetic Toolkits and Feeding Strategy

Cupriavidus necator is a promising bacterium for producing polyhydroxybutyrate (PHB), a biodegradable bioplastic. However, the cell growth is restricted due to a lack of glucose transporters and low glucokinase activity. To address this limitation, we first developed comprehensive genetic toolkits to express sfGFP as a proof-of-concept in C. necator. A plasmid-driven T7RNA polymerase (PDT7) system under the J23109 promoter achieved a 10-fold increase in fluorescence compared to strains without PDT7. However, PDT7 imposed a metabolic burden at higher expression levels and remained less effective than the constitutive Trc promoter, which consistently exhibited the highest transcriptional strength. Based on its robust and balanced performance, the Trc promoter was optimized to drive expression of galP (galactose permease) and glk (glucokinase) genes from Escherichia coli, (annotated as Tgg-H16), enabling enhanced glucose uptake, biomass and PHB biosynthesis. Further enhancement was achieved by supplementing a mixed carbon source, that is, 10 g/L glucose and 10 g/L fructose, which shortened the lag phase and supported higher PHB yields. Finally, a stepwise feeding strategy in fermentation boosted PHB production to 30.9 g/L. Rewiring carbon flux in C. necator through genetic circuit design demonstrates the feasibility of improving carbon uptake, while integration with tailored cultivation strategies enables upcycling sustainable PHB production.