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A Dual-Dimensional CRISPR Toolkit Enables One-Step High-Efficiency Multiplex Genome Editing in Komagataella phaffii
Zhixian Yu1, Kaidi Chen2, Gulikezi Maimaitirexiati2
1State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
Metabolic Engineering
|August 13, 2026
Summary
This study introduces Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) for engineering Komagataella phaffii, enabling efficient multiplex gene knockout and large-fragment integration for C1 bioeconomy applications.
Area of Science:
- Synthetic Biology
- Metabolic Engineering
- Industrial Biotechnology
Background:
- Engineering methanol-utilizing Komagataella phaffii (K. phaffii) is crucial for expanding the one-carbon (C1) product profile and advancing the C1-based bioeconomy.
- Existing methods for multiplex gene editing and large-fragment integration in K. phaffii face challenges with low efficiency and complex procedures.
- Development of robust tools is needed to overcome these limitations for complex metabolic pathway reconstruction.
Purpose of the Study:
- To establish a CRISPR toolkit, Efficient Multi-Gene Editing System 3.0 (EMGES 3.0), for streamlined multiplex gene editing and large-fragment integration in K. phaffii.
- To enhance the efficiency of gene knockout and DNA integration for complex metabolic engineering.
- To demonstrate the application of EMGES 3.0 in producing valuable compounds from methanol.
Main Methods:
- Engineered a repair-deficient chassis (Y09) by integrating five DNA repair modules (Δlig4, ppMRE11 overexpression, Δrad9, Δmph1, PapRecT-PaSSB co-expression) to enhance recombination.
- Optimized an episomal CRISPR vector (Nov_pGAP_panARS_pLAT1_Cas9) by replacing cenARS with panARS and using the PGAP promoter for sgRNA expression.
- Combined the engineered chassis and vector to create the EMGES 3.0 system for one-step multiplex gene knockout and large-fragment integration.
Main Results:
- EMGES 3.0 achieved dual-functional gene knockout efficiencies ranging from 76.6% to 100%.
- Demonstrated high efficiency for medium-long fragment insertion (> 4.5kb) at 93.3% and achieved 14.8% for one-step ultra-long fragment integration (> 16kb), a novel report.
- Successfully applied EMGES 3.0 for one-step production of free fatty acids (FFAs) and resveratrol using methanol as the sole carbon source.
Conclusions:
- EMGES 3.0 significantly improves transformation rate and editing efficiency in K. phaffii, enabling complex metabolic pathway reconstruction.
- The system provides a robust foundation for developing high-yield industrial strains and advancing K. phaffii as a synthetic biology chassis for C1 utilization.
- This tool facilitates the biosynthesis of complex compounds and accelerates the industrialization of C1-based bioeconomy.
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