Multilevel Metabolic Engineering of Cupriavidus necator for CO 2 ‐Derived Single‐Cell Protein Production

ABSTRACT 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 CO 2 . 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 Δ epr‐1 Δ glcR achieved a high crude protein content of 70.13%. However, when the GS13 module was introduced into this background, OD 600 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 .