Development and Application of a Cumate‐Inducible Promoter, <scp>P</scp> _<i>gc</i> , in <i>Komagataella pastoris</i>

Komagataella pastoris is extensively used as a microbial cell factory for the production of recombinant proteins and high-value compounds. However, tightly controlled promoter systems responsive to safe and economical inducers are required for precise metabolic and pathway engineering in this yeast species. Cumate-inducible promoters are an ideal choice due to the safety and low cost of cumate. In this study, we systematically optimised the insertion sites of the CuO operator sequence within the strong promoter P_GCW14 to isolate a high-activity variant that we designated as P_GCWCuO03. To fine-tune the expression of the repressor protein CymR, we developed a truncated promoter of P_GAP, designated as P_GAP200. Based on the optimal promoter P_GCWCuO03 and the CymR expression unit, we constructed a robust CymR/CuO-mediated cumate-inducible promoter, designated as P_gc, in K. pastoris. P_gc demonstrated outstanding induction properties, resulting in an approximately 11-fold increase in target protein production following induction. Promoter substitution assays validated the effectiveness of P_gc in temporal gene expression control, highlighting the significant potential of this promoter for both basic research and industrial bioprocessing applications in synthetic biology and biotechnology in K. pastoris.