Multilevel Metabolic Engineering of Cupriavidus necator for CO₂-Derived Single-Cell Protein Production

Enhancing nitrogen assimilation and coordinating carbon-energy metabolism in the autotrophic chassis Cupriavidus necator remain key challenges for maximizing single-cell protein (SCP) production from CO₂. In this study, multilevel engineering was performed on C. necator. First, glnA was overexpressed to enhance nitrogen assimilation, which increased crude protein content to 72.75% in the best-performing strain, GS13. Preliminary physiological characterization suggested that this increase correlated with enhanced heme level. Second, glcR was disrupted to alter carbon allocation toward amino acid precursor metabolism, and epr-1 was deleted to inhibit extracellular protein degradation during the secretion process. The double-knockout strain Δepr-1ΔglcR achieved a high crude protein content of 70.13%. However, when the GS13 module was introduced into this background, OD₆₀₀ increased to 13.12 while crude protein content decreased to 68.05%, indicating a non-additive interaction between the engineered modules. Overall, our study provided improved strains and a modular framework for future SCP optimization and mechanistic studies of carbon-nitrogen coordination in C. necator.