The PARS sequence increase the efficiency of stable Pichia pastoris transformation

• Linear plasmids with the PARS sequence transform P. pastoris with high efficiency.
• The linear plasmids integrate in the P. pastoris genome. They are not episomal.
• A novel mechanism for genomic integration is suggested.
• Constructs which are inherently more efficient for transformation can be designed.

The methylotrophic yeast Pichia pastoris is a popular host for recombinant expression of proteins.Plasmids containing the Pichia autonomously replicating sequence (PARS) transform P. pastoris with higher efficiency than linear DNA equipped with termini designed for homologous recombination. Moreover, PARS containing constructs provide higher protein yields. Unfortunately, these autonomous plasmids are inherently unstable and the preferred method of P. pastoris transformation is therefore stable integration in the genome by homologous recombination. In the present study we report that a novel combination of PARS and linearization of plasmids for P. pastoris transformation serves to significantly increase the transformation efficiency. Moreover, it is demonstrated that the constructs do not re-circularize but integrate stably into the P. pastoris genome.