Recombinant multicopy plasmids in yeast - interactions with the endogenous 2 μm.

Area of Science:

• Molecular Biology
• Yeast Genetics
• Biotechnology

Background:

• Flp recombinase facilitates site-specific recombination in Saccharomyces cerevisiae, driving amplification of the endogenous 2 μm plasmid.
• High copy numbers of YEp-type vectors are desirable for research and industrial applications, potentially achieved through similar recombination mechanisms.

Purpose of the Study:

• To investigate the mechanisms of YEp-type plasmid amplification in yeast.
• To analyze the types and frequencies of plasmid recombination events under different conditions.

Main Methods:

• Cultivation of yeast strains with six isomeric YEp-type model expression plasmids.
• Back transformation of shuttle vectors into Escherichia coli for analysis.
• High-resolution analysis of 586 ampicillin-resistant clones to detect plasmid structural changes.

Main Results:

• Approximately 11% of analyzed plasmids showed alterations, indicating a lower limit of structural changes.
• Two categories of recombination were observed: Flp-based intermolecular recombination and Flp-independent homologous recombination.
• Flp-based recombination involved interactions with the 2 μm plasmid, potentially mediating YEp-type amplification.
• Flp-independent recombination led to plasmid oligomerization, even in 2μm-free strains.

Conclusions:

• Both Flp-mediated and Flp-independent recombination contribute to YEp-type plasmid amplification and high copy numbers in yeast.
• The gene of interest and its expression can influence host physiology beyond plasmid maintenance sequences.