title: Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing.
abstract: The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production in scientific research and industry. Typically, the expression cassette is integrated into the genome via homologous recombination. Due to unknown integration events, a large clonal variability is often encountered consisting of clones with different productivities as well as aberrant morphological or growth characteristics. In this study, we analysed several clones with abnormal colony morphology and discovered unpredicted integration events via whole genome sequencing. These include (i) the relocation of the locus targeted for replacement to another chromosome (ii) co-integration of DNA from the E. coli plasmid host and (iii) the disruption of untargeted genes affecting colony morphology. Most of these events have not been reported so far in literature and present challenges for genetic engineering approaches in this yeast. Especially, the presence and independent activity of E. coli DNA elements in P. pastoris is of concern. In our study, we provide a deeper insight into these events and their potential origins. Steps preventing or reducing the risk for these phenomena are proposed and will help scientists working on genetic engineering of P. pastoris or similar non-conventional yeast to better understand and control clonal variability.
authors.affiliation: Fermentation Engineering, Bielefeld University, Universitätsstr. 25, Bielefeld, 33615, Germany.|Microbial Genomics and Biotechnology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstr. 27, Bielefeld, 33615, Germany.
authors.forename: Jan-Philipp
authors.initials: JP
authors.lastname: Schwarzhans
authors.affiliation: Genome Research of Industrial Microorganisms, CeBiTec, Bielefeld University, Universitätsstr. 27, Bielefeld, 33615, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wibberg
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstr. 27, Bielefeld, 33615, Germany.
authors.forename: Anika
authors.initials: A
authors.lastname: Winkler
authors.affiliation: Fermentation Engineering, Bielefeld University, Universitätsstr. 25, Bielefeld, 33615, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Luttermann
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstr. 27, Bielefeld, 33615, Germany.
authors.forename: Jörn
authors.initials: J
authors.lastname: Kalinowski
authors.affiliation: Fermentation Engineering, Bielefeld University, Universitätsstr. 25, Bielefeld, 33615, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Friehs
mesh_terms: D015825:Chromosomes, Fungal / Q000235:genetics*; D016681:Genome, Fungal; D059014:High-Throughput Nucleotide Sequencing; D010843:Pichia / Q000235:genetics*; D014170:Transformation, Genetic