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Stable multicopy integration of vector sequences in Hansenula polymorpha

R Gatzke1, U Weydemann, Z A Janowicz

  • 1Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany.

Applied Microbiology and Biotechnology
|October 1, 1995
PubMed
Summary

Plasmids containing the Saccharomyces cerevisiae URA3 gene replicate autonomously in Hansenula polymorpha, enabling genetic modification. Selective pressure drives plasmid integration into the host genome for stable, high-copy-number expression.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Yeast Genetics

Background:

  • Autonomous replication of plasmids in yeast is crucial for genetic engineering.
  • The URA3 gene from Saccharomyces cerevisiae is a common selectable marker.
  • Hansenula polymorpha is an important methylotrophic yeast with biotechnological applications.

Purpose of the Study:

  • To investigate the autonomous replication and stability of plasmids lacking a specific origin of replication in Hansenula polymorpha.
  • To determine the functional role of the Saccharomyces cerevisiae URA3 gene in H. polymorpha plasmid maintenance.
  • To analyze the integration mechanism and stability of plasmid sequences in the H. polymorpha genome.

Main Methods:

  • Transformation of Hansenula polymorpha with non-replicative plasmids bearing the S. cerevisiae URA3 gene.

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  • Assessment of autonomous replication and copy number using molecular techniques.
  • Evaluation of plasmid stability under selective and non-selective growth conditions.
  • Analysis of plasmid integration events into the H. polymorpha genome.

    • Main Results:

    • Plasmids with the S. cerevisiae URA3 gene demonstrated autonomous replication in H. polymorpha.
    • These plasmids exhibited low mitotic stability and were rescued in E. coli.
    • Selective propagation induced integration of plasmid sequences into the H. polymorpha genome at a single, non-homologous site.
    • Integration resulted in high-copy-number tandem arrays, conferring stability under non-selective conditions.
    • Conversely, using a homologous URA3 gene resulted in low-copy-number transformants.

    Conclusions:

    • The S. cerevisiae URA3 gene can confer autonomous replication and stabilization functions in H. polymorpha.
    • Non-homologous integration under selective pressure provides a stable, high-copy-number expression system in H. polymorpha.
    • This strategy offers a valuable tool for genetic manipulation and biotechnological applications in H. polymorpha.