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Endogenous 2μ Plasmid Editing for Pathway Engineering in Saccharomyces cerevisiae
Bo-Xuan Zeng1,2, Ming-Dong Yao1,2, Wen-Hai Xiao1,2
1Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.
Frontiers in Microbiology
|March 5, 2021
Summary
Researchers engineered a new multi-copy yeast plasmid system (pE2μ) using CRISPR/Cas9, improving plasmid stability and gene expression for enhanced production of valuable compounds like dihydroartemisinic acid.
Area of Science:
- Synthetic Biology
- Molecular Biology
- Biotechnology
Background:
- Conventional 2-micron plasmid (pC2μ) systems in *Saccharomyces cerevisiae* face challenges with plasmid stability and copy number control, particularly during long-term fermentation.
- These limitations hinder efficient multi-copy gene overexpression for pathway engineering.
Purpose of the Study:
- To develop an improved multi-copy yeast expression system based on the endogenous 2-micron plasmid (pE2μ).
- To enhance plasmid stability, copy number control, and reduce cell-to-cell variation in gene expression.
- To increase the production of valuable compounds through metabolic engineering.
Main Methods:
- Utilized CRISPR/Cas9 technology to edit the endogenous *Saccharomyces cerevisiae* 2-micron plasmid (pE2μ).
- Constructed a novel pE2μ multi-copy system by inserting target DNA and eliminating the original pE2μ plasmid.
- Integrated the essential *TPI1* gene onto the pE2μ plasmid for enhanced viability and copy number.
Main Results:
- The developed pE2μ-based plasmids (pE2μRAF1, pE2μREP2) exhibited higher copy numbers and slower loss rates compared to a pC2μ control (pRS425RK).
- Moving *TPI1* to pE2μRAF1 increased plasmid viability to nearly 100% and boosted plasmid copy number by 73.95%.
- The pE2μ system demonstrated significantly reduced cell-to-cell expression variation and achieved 90 generations of stable cultivation without loss when *TPI1* was complemented.
- Dihydroartemisinic acid (DHAA) production increased 4.73-fold (to 620.9 mg/L) using the pE2μ system compared to the pC2μ system.
Conclusions:
- The engineered pE2μ multi-copy system offers superior plasmid stability and expression control in *Saccharomyces cerevisiae* compared to conventional pC2μ systems.
- This improved system provides a robust platform for metabolic engineering and the high-titer biosynthesis of valuable products.
- The enhanced stability and expression efficiency make the pE2μ system highly promising for industrial applications and synthetic biology.
