Optimization and application of a tetracycline-inducible gene expression system in Kluyveromyces marxianus

Abstract Kluyveromyces marxianus is a promising microbial cell factory owing to its rapid growth, thermotolerance, broad carbon-source utilization, and food-safe status. Tetracycline (Tet)-inducible systems provide powerful means for precise gene regulation in various hosts. However, Tet systems with strong induction and low leakiness remain poorly developed in K. marxianus. Here, we optimized a Tet-inducible system in K. marxianus by engineering promoter–Tet-operator architectures. The optimized rtTA-based system achieved up to 14-fold induction, exhibited a rapid response within 20 min, and functioned robustly across different temperatures and culture media. This system enabled efficient induction of genomic gene expression and supported higher heterologous protein production compared with constitutive promoters. Furthermore, we developed a TetR-based inducible guide RNA promoter derived from the native K. marxianus tRNAGly promoter, enabling low-leakage CRISPR-based gene disruption, activation, and interference. Together, these tools expand the genetic toolbox for precise gene regulation in both fundamental and industrial applications in K. marxianus. Key points A strong, rapid and robust Tet-inducible system was optimized in K. marxianus.The system efficiently induced genomic genes and heterologous protein expression.An inducible guide RNA promoter used for low-leakage CRISPR-based gene regulation. Graphical Abstract