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

Ruben Hohnholz1, Tilman Achstetter1

  • 1Department of Industrial Microbiology, City University of Applied Sciences Bremen, Neustadtswall 30, D-28199 Bremen, Germany.

FEMS Yeast Research
|April 3, 2019
PubMed

Insights

Yeast plasmids achieve high copy numbers through Flp-mediated recombination and Flp-independent homologous recombination. These mechanisms are crucial for plasmid amplification in research and industrial applications.

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.
Keywords:
Saccharomyces cerevisiae2 μmYEp-typeplasmid heterogeneityrecombination

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