Multiple-copy integration in the yeast Yarrowia lipolytica

Area of Science:

• Molecular Biology
• Yeast Genetics
• Biotechnology

Background:

• Gene amplification is crucial for overproducing proteins.
• Understanding rDNA integration and stability is key for industrial applications.
• Yarrowia lipolytica is a valuable host for recombinant protein production.

Purpose of the Study:

• To construct and evaluate plasmids for gene amplification in the rDNA of Yarrowia lipolytica.
• To investigate the impact of URA3 promoter deletions on rDNA integration and copy number.
• To assess the stability of integrated plasmids and their effect on reporter gene expression.

Main Methods:

• Construction of plasmids with rDNA integration fragments, defective URA3, and XPR2 reporter gene.
• Transformation of Y. lipolytica and selection for transformants.
• Analysis of plasmid copy number using PCR and DNA hybridization.
• Evaluation of plasmid stability and chromosomal localization.

Main Results:

• Plasmids pINA773, pINA767, and pINA772 facilitated multiple integrations into the rDNA.
• Copy numbers ranged from 5 to 60, with higher copy numbers integrating into multiple chromosomes.
• Plasmid stability was maintained at lower copy numbers but affected by induction of alkaline extracellular protease (AEP) synthesis at higher copy numbers.

Conclusions:

• Specific plasmid constructs enable efficient gene amplification in Y. lipolytica rDNA.
• High plasmid copy numbers can lead to chromosomal instability, particularly under inducing conditions.
• Optimized gene amplification strategies are essential for stable, high-level protein production in Y. lipolytica.