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.

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

“…Thus, we could accurately assess clonal variation and pinpoint the expression spread. In the clones that we sequenced, we did not observe the cointegration of DNA from the E. coli plasmid host, leading to impaired growth as reported by Schwarzhans et al (27). We did, however, observe the loss of a large fragment of approximately 69 kbp in one of our strains with a growth defect (QTV84), suggesting deletions as an additional possible cause for clonal variation in P. pastoris.…”

“…This strain showed impaired growth, likely attributable to the 17 genes deleted, which included five plasma membrane transporters (including two sugar transporters and an iron transporter), one S. cerevisiae Gal4-like transcription factor, two glycoside hydrolases, and a putative cell wall flocculin, among others (Table S6). Such rare integration events have been reported in the past yet were mostly attributed to the integration of E. coli sequences (27), whereas the large deletion observed in our case suggests a different mechanism. Only one potential insertion site could be clearly observed with a SacI-cut plasmid (strain QTV86); this was in the AOX1 promoter, as expected.…”

“…A considerable decrease in the cost of next-generation sequencing and WGS over the last decade (54) has permitted comparisons of interclonal variation rather than sequencing only a single strain. Recently, Karl Friehs' group thoroughly investigated integration event-induced changes in recombinant protein productivity in P. pastoris, focusing on the nature of multicopy integration events and clones with abnormal colony morphology in two studies by Schwarzhans et al (26,27). While these studies provide insights into P. pastoris clonal variation, our alternative study design focused on different aspects and overcame some experimental limitations.…”

“…1) were based on phleomycin D1 (zeocin) antibiotic selection, as auxotrophic markers had resulted in mixed cultures in previous studies (26). We linearized the expression plasmids with the respective restriction enzymes and gel purified the bands (to avoid carryover of uncut vector or foreign DNA present [26,27]). P. pastoris cells were transformed in triplicate with each plasmid to test variability and potential influence of the transformation events.…”

“…With further decreases in the cost of these technologies, systematic studies of the underlying mechanisms for clonal variation in P. pastoris transformants have become feasible. Recently, milestone papers by the group of Karl Friehs investigated the influence of integration events on protein expression (26,27), providing insight into various mechanisms of multicopy integration (e.g., orientation of the cassettes [26]) and noncanonical integration events influencing growth phenotypes (27). While these integration events can have a profound influence on strain productivity and physiology, it remains partly unclear to what extent they occur, how they are influenced by expression cassette properties, and how they bias typical standard expression approaches in P. pastoris.…”