Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

Schwarzhans JP, Wibberg D, Winkler A, Luttermann T, Kalinowski J, Friehs K (2016)
Scientific Reports 6: 38952.

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Journal Article | Original Article | Published | English
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.
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Article Processing Charge funded by the Deutsche Forschungsgemeinschaft and the Open Access Publication Fund of Bielefeld University.
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Schwarzhans JP, Wibberg D, Winkler A, Luttermann T, Kalinowski J, Friehs K. Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing. Scientific Reports. 2016;6: 38952.
Schwarzhans, J. P., Wibberg, D., Winkler, A., Luttermann, T., Kalinowski, J., & Friehs, K. (2016). Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing. Scientific Reports, 6, 38952. doi:10.1038/srep38952
Schwarzhans, J. P., Wibberg, D., Winkler, A., Luttermann, T., Kalinowski, J., and Friehs, K. (2016). Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing. Scientific Reports 6:38952.
Schwarzhans, J.P., et al., 2016. Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing. Scientific Reports, 6: 38952.
J.P. Schwarzhans, et al., “Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing”, Scientific Reports, vol. 6, 2016, : 38952.
Schwarzhans, J.P., Wibberg, D., Winkler, A., Luttermann, T., Kalinowski, J., Friehs, K.: Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing. Scientific Reports. 6, : 38952 (2016).
Schwarzhans, Jan Philipp, Wibberg, Daniel, Winkler, Anika, Luttermann, Tobias, Kalinowski, Jörn, and Friehs, Karl. “Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing”. Scientific Reports 6 (2016): 38952.
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6 Citations in Europe PMC

Data provided by Europe PubMed Central.

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Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.
Vogl T, Gebbie L, Palfreyman RW, Speight R., Appl Environ Microbiol 84(6), 2018
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Kranz A, Vogel A, Degner U, Kiefler I, Bott M, Usadel B, Polen T., J Biotechnol 258(), 2017
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Schwarzhans JP, Luttermann T, Geier M, Kalinowski J, Friehs K., Biotechnol Adv 35(6), 2017
PMID: 28760369
Systems biotechnology for protein production in Pichia pastoris.
Zahrl RJ, Peña DA, Mattanovich D, Gasser B., FEMS Yeast Res 17(7), 2017
PMID: 28934418

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