Standardized Markerless Gene Integration for Pathway Engineering in Yarrowia lipolytica

Area of Science:

• Biotechnology
• Synthetic Biology
• Microbial Engineering

Background:

• Yarrowia lipolytica is a valuable yeast for producing lipid-based chemicals.
• Stable production strains require genomic integration, but current methods can be inefficient.
• Episomal expression necessitates specialized media and selective pressures.

Purpose of the Study:

• To develop a CRISPR-Cas9 tool for targeted, markerless gene integration in Yarrowia lipolytica.
• To identify genomic loci suitable for stable gene insertion without affecting cell growth.
• To facilitate rapid and reliable metabolic engineering in Y. lipolytica.

Main Methods:

• Developed a CRISPR-Cas9 system for targeted gene insertion.
• Screened multiple genomic loci in Y. lipolytica for compatibility with gene integration.
• Validated expression levels of integrated genes at different loci using a GFP cassette.
• Engineered a semisynthetic lycopene biosynthesis pathway by integrating four genes.

Main Results:

• Identified five genomic loci (AXP, XPR2, A08, D17, MFE1) suitable for markerless gene integration.
• Demonstrated consistent gene expression from AXP, XPR2, A08, and D17 loci.
• Successfully engineered a four-gene pathway for lycopene production.
• The tool allows for multiple gene integrations without marker recovery.

Conclusions:

• The developed CRISPR-Cas9 tool enables efficient and precise genomic engineering in Y. lipolytica.
• The identified integration sites offer predictable expression levels for pathway construction.
• This technology accelerates the development of engineered yeast strains for industrial applications.