Harnessing CO fixation and reducing power recycling for enhanced polyhydroxyalkanoates industrial bioproduction.

Palm oil is an attractive feedstock for bioproduction due to its high carbon content and low cost. However, its metabolism generates excess reducing power, leading to redox imbalances and reduced metabolic efficiency in industrial fermentations. Through a model-driven approach integrating flux balance analysis, we activated the Calvin-Benson-Bassham (CBB) cycle in Cupriavidus necator to recycle surplus reducing power and restore metabolic balance in polyhydroxyalkanoate (PHA) bioproduction. Computational simulations predicted that constitutive activation of the CBB cycle enhanced CO fixation and accelerated biomass generation when utilizing palm oil as the carbon source. Model-guided optimization revealed that precise tuning of CBB activation strength was critical, as both insufficient and excessive activation led to metabolic inefficiencies. At the 2-liter bench-scale, CBB activation tuning resulted in biomass changes ranging from -18 % to 21 % and PHA yield changes ranging from -36 % to 25 %. Mechanistic studies demonstrated that CBB activation improves metabolic efficiency through reducing power recycling and carbon redistribution. In the 15 m industrial-scale fermentations, the engineered strain achieved a 20 % higher PHA yield. These results demonstrate that recycling surplus reducing power is a scalable and robust strategy for enhanced bioproduction efficiency.