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Pichia pastoris as a host system for transformations.

J M Cregg, K J Barringer, A Y Hessler

    Molecular and Cellular Biology
    |December 1, 1985
    PubMed
    Summary
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    We engineered the yeast Pichia pastoris for DNA transformations using a HIS4 gene marker. This system, enhanced by autonomous replication sequences, significantly boosted transformation efficiency in P. pastoris.

    Area of Science:

    • Microbiology
    • Molecular Biology
    • Biotechnology

    Background:

    • Developing efficient DNA transformation systems is crucial for genetic engineering.
    • Pichia pastoris is a methylotrophic yeast with significant biotechnological potential.
    • Existing transformation methods for P. pastoris can be inefficient.

    Purpose of the Study:

    • To develop Pichia pastoris as a robust host for DNA transformations.
    • To identify and characterize selectable markers and replication sequences for P. pastoris.
    • To improve the efficiency of DNA delivery into P. pastoris cells.

    Main Methods:

    • Utilized an auxotrophic P. pastoris mutant defective in histidinol dehydrogenase.
    • Isolated and characterized the P. pastoris HIS4 gene as a selectable marker.

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  • Employed a modified spheroplast (CaCl2-polyethylene glycol)-fusion procedure for DNA transfer.
  • Identified P. pastoris DNA fragments (PARS1, PARS2) with autonomous replication sequence activity.

    • Main Results:

    • Successfully transformed the P. pastoris mutant host using plasmids with either P. pastoris or Saccharomyces cerevisiae HIS4 genes.
    • Achieved transformation frequencies up to 10(5)/micrograms with plasmids containing PARS1 and PARS2.
    • Demonstrated that PARS1 and PARS2 enable plasmids to be maintained as autonomous elements in P. pastoris.

    Conclusions:

    • Pichia pastoris is a viable and efficient host for DNA transformations.
    • The P. pastoris HIS4 gene serves as an effective selectable marker.
    • The identified PARS elements significantly enhance transformation efficiency and plasmid stability in P. pastoris.