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Peer-reviewedCC BY-NC-ND 4.0Applied Microbiology and Biotechnology

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

  • Jingqi Wang1,2
  • Baixiang Zhao1,2
  • Junyuan Zeng1,2
  • Jungang Zhou1,2
  • Hong Lu1,2
  • Yao Yu1,2
  1. 1State Key Laboratory of Genetics and Development of Complex Phenotypes, School of Life Sciences, Fudan University, Shanghai, 200438 China
  2. 2Shanghai Engineering Research Center of Industrial Microorganisms, Shanghai, 200438 China
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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:

Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s00253-026-13995-8.

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Cite

BibTeXRIS
BibTeX
@article{wang2026optimization,
  title = {{Optimization and application of a tetracycline-inducible gene expression system in Kluyveromyces marxianus}},
  author = {Jingqi Wang and Baixiang Zhao and Junyuan Zeng and Jungang Zhou and Hong Lu and Yao Yu},
  journal = {Applied Microbiology and Biotechnology},
  year = {2026},
  month = aug,
  doi = {10.1007/s00253-026-13995-8},
  url = {https://doi.org/10.1007/s00253-026-13995-8},
  note = {CC BY-NC-ND 4.0},
}
RIS
TY  - JOUR
TI  - Optimization and application of a tetracycline-inducible gene expression system in Kluyveromyces marxianus
AU  - Jingqi Wang
AU  - Baixiang Zhao
AU  - Junyuan Zeng
AU  - Jungang Zhou
AU  - Hong Lu
AU  - Yao Yu
PY  - 2026
DA  - 2026/08/08
JO  - Applied Microbiology and Biotechnology
DO  - 10.1007/s00253-026-13995-8
UR  - https://doi.org/10.1007/s00253-026-13995-8
AB  - 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:  Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s00253-026-13995-8.
SN  - 0175-7598
SN  - 1432-0614
ER  -