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Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies
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A Stable, Autonomously Replicating Plasmid Vector Containing Pichia pastoris Centromeric DNA.

Yasuyuki Nakamura1, Teruyuki Nishi1,2, Risa Noguchi3

  • 1Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.

Applied and Environmental Microbiology
|May 27, 2018
PubMed
Summary

We identified centromeric DNA sequences in Pichia pastoris, enabling the development of a stable, autonomously replicating plasmid. This new tool enhances genetic manipulation and protein production in Pichia pastoris.

Keywords:
Pichia pastorisautonomously replicating plasmidcentromereinverted repeatplasmid retention

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Yeast Genetics

Background:

  • Pichia pastoris is a key methylotrophic yeast for recombinant protein production due to high-density growth and secretion capabilities.
  • Current genetic engineering relies on integrative vectors; existing autonomously replicating plasmids (ARPs) using PARS1 are unstable.
  • The identification of centromeric DNA sequences in P. pastoris offers a new avenue for developing stable episomal vectors.

Purpose of the Study:

  • To identify centromeric DNA sequences in Pichia pastoris chromosomes.
  • To characterize the autonomous replication activity of these sequences.
  • To construct a novel, stable, autonomously replicating plasmid vector for P. pastoris.

Main Methods:

  • Transcriptome sequencing (RNA-seq) and genome sequence analysis to identify centromeric DNA.
  • Detailed examination of the chromosome 2 centromeric DNA (Cen2) sequence.
  • Construction and testing of a new episomal plasmid vector harboring the full-length Cen2 sequence.

Main Results:

  • Centromeric DNA sequences with long inverted repeats were identified on all four P. pastoris chromosomes.
  • An approximately 111-bp region of Cen2 exhibited autonomous replication activity.
  • The full-length Cen2 sequence is essential for accurate plasmid replication and distribution, leading to a stable ARP.

Conclusions:

  • A stable, autonomously replicating plasmid vector for Pichia pastoris was successfully constructed using the full-length Cen2 sequence.
  • This novel episomal vector provides high transformation efficiency and plasmid stability.
  • The developed vector facilitates genetic manipulation, improving recombinant protein production in P. pastoris.