• Developed a T7-independent CFPS system using the endogenous AOX1 promoter in Komagataella phaffii, eliminating the need for costly T7 RNA polymerase.
• Achieved a record GFP yield of 596.0 mg/L in K. phaffii CFPS through systematic optimization of key components.
• Identified a significant synergistic effect between potassium glutamate and magnesium glutamate, enhancing protein synthesis efficiency.
• Provides a foundation for scalable bioproduction and broadens the applicability of eukaryotic CFPS systems.