Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization
Schwarzhans JP, Wibberg D, Winkler A, Luttermann T, Kalinowski J, Friehs K (2016)
Microbial Cell Factories 15(1): 84.
Zeitschriftenaufsatz
| Veröffentlicht | Englisch
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Autor*in
Schwarzhans, Jan PhilippUniBi;
Wibberg, DanielUniBi;
Winkler, AnikaUniBi;
Luttermann, Tobias;
Kalinowski, JörnUniBi;
Friehs, KarlUniBi
Einrichtung
Abstract / Bemerkung
Background
The classic AOX1 replacement approach is still one of the most often used techniques for expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. Although this approach is largely successful, it frequently delivers clones with unpredicted production characteristics and a work-intense screening process is required to find the strain with desired productivity.
Results
In this project 845 P. pastoris clones, transformed with a GFP expression cassette, were analyzed for their methanol-utilization (Mut)-phenotypes, GFP gene expression levels and gene copy numbers. Several groups of strains with irregular features were identified. Such features include GFP expression that is markedly higher or lower than expected based on gene copy number as well as strains that grew under selective conditions but where the GFP gene cassette and its expression could not be detected. From these classes of strains 31 characteristic clones were selected and their genomes sequenced. By correlating the assembled genome data with the experimental phenotypes novel insights were obtained. These comprise a clear connection between productivity and cassette-to-cassette orientation in the genome, the occurrence of false-positive clones due to a secondary recombination event, and lower total productivity due to the presence of untransformed cells within the isolates were discovered. To cope with some of these problems, the original vector was optimized by replacing the AOX1 terminator, preventing the occurrence of false-positive clones due to the secondary recombination event.
Conclusions
Standard methods for transformation of P. pastoris led to a multitude of unintended and sometimes detrimental integration events, lowering total productivity. By documenting the connections between productivity and integration event we obtained a deeper understanding of the genetics of mutation in P. pastoris. These findings and the derived improved mutagenesis and transformation procedures and tools will help other scientists working on recombinant protein production in P. pastoris and similar non-conventional yeasts.
Stichworte
Pichia pastoris Recombinant protein production AOX1 promoter Genome sequencing Insertion locus Non-conventional yeast Expression cassette orientation False-positive
Erscheinungsjahr
2016
Zeitschriftentitel
Microbial Cell Factories
Band
15
Ausgabe
1
Art.-Nr.
84
ISSN
1475-2859
Finanzierungs-Informationen
Open-Access-Publikationskosten wurden durch die Deutsche Forschungsgemeinschaft und die Universität Bielefeld gefördert.
Page URI
https://pub.uni-bielefeld.de/record/2903655
Zitieren
Schwarzhans JP, Wibberg D, Winkler A, Luttermann T, Kalinowski J, Friehs K. Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization. Microbial Cell Factories. 2016;15(1): 84.
Schwarzhans, J. P., Wibberg, D., Winkler, A., Luttermann, T., Kalinowski, J., & Friehs, K. (2016). Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization. Microbial Cell Factories, 15(1), 84. doi:10.1186/s12934-016-0486-7
Schwarzhans, Jan Philipp, Wibberg, Daniel, Winkler, Anika, Luttermann, Tobias, Kalinowski, Jörn, and Friehs, Karl. 2016. “Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization”. Microbial Cell Factories 15 (1): 84.
Schwarzhans, J. P., Wibberg, D., Winkler, A., Luttermann, T., Kalinowski, J., and Friehs, K. (2016). Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization. Microbial Cell Factories 15:84.
Schwarzhans, J.P., et al., 2016. Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization. Microbial Cell Factories, 15(1): 84.
J.P. Schwarzhans, et al., “Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization”, Microbial Cell Factories, vol. 15, 2016, : 84.
Schwarzhans, J.P., Wibberg, D., Winkler, A., Luttermann, T., Kalinowski, J., Friehs, K.: Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization. Microbial Cell Factories. 15, : 84 (2016).
Schwarzhans, Jan Philipp, Wibberg, Daniel, Winkler, Anika, Luttermann, Tobias, Kalinowski, Jörn, and Friehs, Karl. “Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization”. Microbial Cell Factories 15.1 (2016): 84.
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