Breakthrough in <italic>Komagataella phaffii</italic> cell-free protein synthesis: AOX1 promoter drives T7-independent expression efficiently

This study develops a cell-free protein synthesis (CFPS) system based on the endogenous alcohol oxidase 1 promoter in <i>K</i>. <i>phaffii</i>. The system avoids the dependence of the T7 promoter, thus eliminating the cost issues associated with the T7 RNA polymerase-dependent system in traditional CFPS systems. By integrating an alcohol oxidase 1 promoter-driven GFP expression cassette with optimized <i>K</i>. <i>phaffii</i> cell extract, key components are optimized via a one-factor experiment and a deterministic screening design. This study demonstrates that potassium glutamate and magnesium glutamate have a significant synergistic effect on this system. After optimization, the system achieves a GFP yield of 596.0 mg/L, providing a new record for GFP expression in <i>K</i>. <i>phaffii</i> CFPS systems. This work provides an important theoretical foundation for the further development of T7-independent <i>K</i>. <i>phaffii</i> CFPS systems and their potential applications in scalable bioproduction.