# Development and Application of a Cumate‐Inducible Promoter, P gc , in Komagataella pastoris

## TL;DR

Researchers developed a new promoter system in yeast to control protein production more precisely and efficiently.

## Contribution

A novel cumate-inducible promoter, P_gc, was engineered for precise gene expression control in Komagataella pastoris.

## Key findings

- The P_gc promoter achieved an 11-fold increase in recombinant protein production upon induction.
- Promoter substitution assays confirmed the effectiveness of P_gc in temporal gene expression control.

## Abstract

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.

A novel cumate‐inducible promoter, P
gc
, was engineered in P. pastoris by optimising the CuO operator and CymR repressor (A). This system achieved an approximately 11‐fold increase in recombinant protein production upon induction (B).