High-efficiency CO₂fixation and single-cell protein production in Cupriavidus necator via ARTP-driven metabolic rewiring

Autotrophic conversion of CO₂ to single-cell protein (SCP) represents a promising carbon-negative strategy to address global food security. By combining atmospheric and room temperature plasma (ARTP) mutagenesis with an innovative balloon-based high-throughput screening strategy, we isolated mutant R3, which grew 2.3-fold faster than wild-type H16 and accumulated >60% crude protein in 100 mL anaerobic bottles. Multi-omic analyses unexpectedly revealed that a single synonymous mutation, lysine-tRNA ligase (lysS), exerted a decisive influence on growth performance. Subsequent validation demonstrated that both enhanced sulfur metabolism and elevated gapdh expression were instrumental in accelerating strain proliferation. Under the optimized gas fermentation conditions (H₂ : O₂ : CO₂ = 7 : 1 : 1, 40 mL min⁻¹ flow rate, 600 rpm stirring speed), the dry cell weight (DCW) of the strain R3 reached 23.0 ± 0.8 g L⁻¹ after 5 days, with a crude protein content exceeding 60% in a 5 L bioreactor. The CO₂ fixation rate was about 17.1 g d⁻¹. The yield of carbon was approximately 21.7 g DCW per kg CO₂. The final protein concentration reached 13.8 g L⁻¹, with an average productivity of 2.76 g L⁻¹ d⁻¹. This work establishes ARTP-driven metabolic rewiring as a powerful tool and paves the way for carbon-negative SCP biomanufacturing.