A modular single- and dual-gene expression toolkit for Kluyveromyces marxianus

Area of Science:

• Microbiology
• Biotechnology
• Synthetic Biology

Background:

• Kluyveromyces marxianus is a robust yeast with potential for industrial applications.
• Limited gene expression tools in K. marxianus hinder its use in synthetic biology.
• Development of efficient expression systems is crucial for expanding its applications.

Purpose of the Study:

• To develop novel single- and dual-gene expression systems for Kluyveromyces marxianus.
• To optimize gene expression based on screened genetic elements, carbon source, and culture conditions.
• To enhance protein production and enable subcellular targeting in K. marxianus.

Main Methods:

• Screening and development of promoters (pENO, pHTX) and intergenic sequences.
• Construction and testing of single- and dual-gene expression systems in multiple K. marxianus strains.
• Optimization of expression by varying carbon sources (galactose, glucose) and culture time.
• Utilizing Simian Virus 40 (SV40) nuclear localization signals (NLS) for subcellular targeting.

Main Results:

• Stable gene expression achieved across different K. marxianus strains.
• Single-gene system with bicistronic expression yielded 30.4 mg/L of leghemoglobin (VaHB) on galactose.
• Dual-gene system co-expressing HEM1 achieved 48.4 mg/L of VaHB on glucose.
• SV40 NLS successfully directed fluorescent proteins to the nucleus.

Conclusions:

• The developed toolkit provides efficient and context-responsive gene expression in K. marxianus.
• These systems enhance K. marxianus as a microbial chassis for industrial protein production.
• The study facilitates broader synthetic biology applications for K. marxianus.