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Development of precise genome editing and multi-copy integration tools in Hansenula polymorpha DL-1
Xiaoyi Zou1,2, Jiaqi Miao1,2, Hongbiao Li1,2
1Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China.
Synthetic and Systems Biotechnology
|July 30, 2025
Summary
This study developed advanced synthetic biology tools for Hansenula polymorpha yeast, improving genome editing and gene integration for sustainable manufacturing of valuable compounds.
Area of Science:
- Microbial biotechnology
- Synthetic biology
- Yeast genetics
Background:
- Hansenula polymorpha DL-1 is a thermotolerant yeast with potential for sustainable manufacturing.
- Its utility is limited by strong non-homologous end joining (NHEJ) and few genetic tools.
Purpose of the Study:
- To develop an optimized synthetic biology toolkit for H. polymorpha DL-1.
- To enhance genome editing efficiency and homologous recombination (HR) rates.
- To enable stable single-copy and multi-copy gene integration for increased production.
Main Methods:
- Established a high-efficiency CRISPR-Cas9 genome editing system.
- Suppressed NHEJ by knocking out KU80 and overexpressing S. cerevisiae HR genes.
- Identified 36 neutral sites for single-copy integration.
- Developed multi-copy integration tools targeting rDNA and Ty elements.
Main Results:
- Achieved 97.2% editing efficiency with CRISPR-Cas9.
- Increased HR rates to 88.9% by modifying NHEJ and HR pathways.
- Enabled stable single-copy integration without disrupting native genes.
- Achieved a ~60-fold increase in beta-carotene and enhanced squalene production to 187.2 mg/L via multi-copy integration.
Conclusions:
- The developed toolkit significantly enhances the genetic tractability of H. polymorpha DL-1.
- This facilitates its use as a versatile platform for efficient sustainable production of value-added compounds.
