Pichia pastoris (syn. Komagataella phaffii) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae, where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (<10%) harboring ectopically integrated cassettes showed a greater variation spanning 25-fold range, surpassing specifically reference strains up to 6-fold. high-expression correlation between increased gene copy numbers and high reporter protein fluorescence levels. our results suggest that for comparing expression levels strains, the integration locus can be neglected as long sufficient of transformed are compared. optimization highly expressible proteins, increasing number appears dominant positive influence rather than locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (snps).importance yeasts commonly used biotechnological production hosts proteins metabolites. in yeast saccharomyces cerevisiae, carrying foreign genes integrate at targeted sites genome. contrast, often random positions nonconventional yeasts, such pichia pastoris (syn. komagataella phaffii). hence, cells from same transformation event behave differently, with significant clonal necessitating screening large strains. importance this study is we systematically investigated events more 700 findings provide novel insight into p. and, thus, how avoid pitfalls obtain reliable results. underlying mechanisms may also play role other hence could generally relevant recombinant copyright © 2018 american society microbiology.
Journal: Applied and environmental microbiology
DOI: 10.1128/AEM.02712-17
Year: 2018