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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.
Highlights
The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production, due to a highly efficient secretion mechanism and the possibility of reaching high product titres for simpler enzymes such as phytase and complex proteins containing multiple post-translational modifications, e.g. monoclonal antibodies[1,2,3,4,5]
Filamentous fungi and higher eukaryotes that are used in biotechnological applications display similar or more pronounced clonal variabilities due to a predominant non-homologous end joining (NHEJ) pathway[25,26,27], while in the model yeast Saccharomyces cerevisiae homologous recombination is dominant over the NHEJ pathway[28]
In our previous study[24], we concentrated on analysing the clonal variability found in 845 P. pastoris strains transformed with a GFP expression cassette targeted for AOX1 replacement
Summary
The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production, due to a highly efficient secretion mechanism and the possibility of reaching high product titres for simpler enzymes such as phytase and complex proteins containing multiple post-translational modifications, e.g. monoclonal antibodies[1,2,3,4,5]. The most frequently used approach for introducing the target gene in P. pastoris is still the integration of an expression cassette into the genome via homologous recombination. The most popular target for integration is the AOX1 (alcohol oxidase 1) locus that represents the stronger expressed of the two alcohol oxidases in P. pastoris This approach usually involves the utilization of the AOX1 promoter (pAOX1) as homologous sequence and as promoter of the target gene, because it offers very high expression levels and tight regulation[15]. The application of expression cassettes with two homologous sequences targeted for mediating the replacement of AOX1 is one possible technique. In our previous study[24], we concentrated on analysing the clonal variability found in 845 P. pastoris strains transformed with a GFP expression cassette targeted for AOX1 replacement. No off-target integrations were found in clones with a high productivity, suggesting that the impact of such events on productivity was low
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