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(1): 38952.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
OA 1.15 MB
Abstract / Bemerkung
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.
Scientific Reports
Open-Access-Publikationskosten wurden durch die Deutsche Forschungsgemeinschaft und die Universität Bielefeld gefördert.
Page URI


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(1): 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(1), 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(1): 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 6.1 (2016): 38952.
Alle Dateien verfügbar unter der/den folgenden Lizenz(en):
Copyright Statement:
Dieses Objekt ist durch das Urheberrecht und/oder verwandte Schutzrechte geschützt. [...]
Access Level
OA Open Access
Zuletzt Hochgeladen
MD5 Prüfsumme

13 Zitationen in Europe PMC

Daten bereitgestellt von Europe PubMed Central.

Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors.
Vogl T, Sturmberger L, Fauland PC, Hyden P, Fischer JE, Schmid C, Thallinger GG, Geier M, Glieder A., Biotechnol Bioeng 115(4), 2018
PMID: 29280481
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
PMID: 29330186
Presence of protein production enhancers results in significantly higher methanol-induced protein production in Pichia pastoris.
Gidijala L, Uthoff S, van Kampen SJ, Steinbüchel A, Verhaert RMD., Microb Cell Fact 17(1), 2018
PMID: 30005638
A Stable, Autonomously Replicating Plasmid Vector Containing Pichia pastoris Centromeric DNA.
Nakamura Y, Nishi T, Noguchi R, Ito Y, Watanabe T, Nishiyama T, Aikawa S, Hasunuma T, Ishii J, Okubo Y, Kondo A., Appl Environ Microbiol 84(15), 2018
PMID: 29802190
Engineered bidirectional promoters enable rapid multi-gene co-expression optimization.
Vogl T, Kickenweiz T, Pitzer J, Sturmberger L, Weninger A, Biggs BW, Köhler EM, Baumschlager A, Fischer JE, Hyden P, Wagner M, Baumann M, Borth N, Geier M, Ajikumar PK, Glieder A., Nat Commun 9(1), 2018
PMID: 30181586
Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii.
Cankorur-Cetinkaya A, Narraidoo N, Kasavi C, Slater NKH, Archer DB, Oliver SG., Biotechnol Bioeng 115(12), 2018
PMID: 30267565
Deletion of DNA ligase IV homolog confers higher gene targeting efficiency on homologous recombination in Komagataella phaffii.
Ito Y, Watanabe T, Aikawa S, Nishi T, Nishiyama T, Nakamura Y, Hasunuma T, Okubo Y, Ishii J, Kondo A., FEMS Yeast Res 18(7), 2018
PMID: 30010892
High precision genome sequencing of engineered Gluconobacter oxydans 621H by combining long nanopore and short accurate Illumina reads.
Kranz A, Vogel A, Degner U, Kiefler I, Bott M, Usadel B, Polen T., J Biotechnol 258(), 2017
PMID: 28433722
A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production.
Schwarzhans JP, Luttermann T, Wibberg D, Winkler A, Hübner W, Huser T, Kalinowski J, Friehs K., Front Microbiol 8(), 2017
PMID: 28512458
Towards systems metabolic engineering in Pichia pastoris.
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

66 References

Daten bereitgestellt von Europe PubMed Central.

Expression of the Aspergillus fumigatus phytase gene in Pichia pastoris and characterization of the recombinant enzyme.
Rodriguez E, Mullaney EJ, Lei XG., Biochem. Biophys. Res. Commun. 268(2), 2000
PMID: 10679211
Real-time monitoring of glycerol and methanol to enhance antibody production in industrial Pichia pastoris bioprocesses
Pichia pastoris: protein production host and model organism for biomedical research.
Gasser B, Prielhofer R, Marx H, Maurer M, Nocon J, Steiger M, Puxbaum V, Sauer M, Mattanovich D., Future Microbiol 8(2), 2013
PMID: 23374125
Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production.
Ahmad M, Hirz M, Pichler H, Schwab H., Appl. Microbiol. Biotechnol. 98(12), 2014
PMID: 24743983
Expression in the yeast Pichia pastoris
GAP promoter library for fine-tuning of gene expression in Pichia pastoris.
Qin X, Qian J, Yao G, Zhuang Y, Zhang S, Chu J., Appl. Environ. Microbiol. 77(11), 2011
PMID: 21498769
Promoter library designed for fine-tuned gene expression in Pichia pastoris.
Hartner FS, Ruth C, Langenegger D, Johnson SN, Hyka P, Lin-Cereghino GP, Lin-Cereghino J, Kovar K, Cregg JM, Glieder A., Nucleic Acids Res. 36(12), 2008
PMID: 18539608
Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris.
Prielhofer R, Maurer M, Klein J, Wenger J, Kiziak C, Gasser B, Mattanovich D., Microb. Cell Fact. 12(), 2013
PMID: 23347568
A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris.
Vogl T, Sturmberger L, Kickenweiz T, Wasmayer R, Schmid C, Hatzl AM, Gerstmann MA, Pitzer J, Wagner M, Thallinger GG, Geier M, Glieder A., ACS Synth Biol 5(2), 2015
PMID: 26592304
Compact multi-enzyme pathways in P. pastoris
Use of Combinatorial Genetic Libraries to Humanize N-Linked Glycosylation in the Yeast Pichia pastoris
New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris.
Lin Cereghino GP, Lin Cereghino J, Sunga AJ, Johnson MA, Lim M, Gleeson MA, Cregg JM., Gene 263(1-2), 2001
PMID: 11223254
Foreign gene expression in yeast: a review.
Romanos MA, Scorer CA, Clare JJ., Yeast 8(6), 1992
PMID: 1502852
Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway.
Krainer FW, Dietzsch C, Hajek T, Herwig C, Spadiut O, Glieder A., Microb. Cell Fact. 11(), 2012
PMID: 22330134
Deletion of the Pichia pastoris KU70 homologue facilitates platform strain generation for gene expression and synthetic biology.
Naatsaari L, Mistlberger B, Ruth C, Hajek T, Hartner FS, Glieder A., PLoS ONE 7(6), 2012
PMID: 22768112
High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene
Cultivation strategies to enhance productivity of Pichia pastoris: A review.
Looser V, Bruhlmann B, Bumbak F, Stenger C, Costa M, Camattari A, Fotiadis D, Kovar K., Biotechnol. Adv. 33(6 Pt 2), 2015
PMID: 26027890
Can too many copies spoil the broth?
Aw R, Polizzi KM., Microb. Cell Fact. 12(), 2013
PMID: 24354594
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., Microb. Cell Fact. 15(), 2016
PMID: 27206580
Highly efficient gene targeting in the Aspergillus niger kusA mutant.
Meyer V, Arentshorst M, El-Ghezal A, Drews AC, Kooistra R, van den Hondel CA, Ram AF., J. Biotechnol. 128(4), 2007
PMID: 17275117
Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells.
Guirouilh-Barbat J, Huck S, Bertrand P, Pirzio L, Desmaze C, Sabatier L, Lopez BS., Mol. Cell 14(5), 2004
PMID: 15175156
Nonhomologous end joining in yeast.
Daley JM, Palmbos PL, Wu D, Wilson TE., Annu. Rev. Genet. 39(), 2005
PMID: 16285867
Identification and deletion of the major secreted protein of Pichia pastoris.
Heiss S, Maurer M, Hahn R, Mattanovich D, Gasser B., Appl. Microbiol. Biotechnol. 97(3), 2012
PMID: 22801711
Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris.
Marx H, Mattanovich D, Sauer M., Microb. Cell Fact. 7(), 2008
PMID: 18664246
Construction of new Pichia pastoris X-33 strains for production of lycopene and β-carotene.
Araya-Garay JM, Feijoo-Siota L, Rosa-dos-Santos F, Veiga-Crespo P, Villa TG., Appl. Microbiol. Biotechnol. 93(6), 2011
PMID: 22159890
Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris.
Wriessnegger T, Augustin P, Engleder M, Leitner E, Muller M, Kaluzna I, Schurmann M, Mink D, Zellnig G, Schwab H, Pichler H., Metab. Eng. 24(), 2014
PMID: 24747046
Metabolic engineering of Pichia pastoris for the production of dammarenediol-II.
Liu XB, Liu M, Tao XY, Zhang ZX, Wang FQ, Wei DZ., J. Biotechnol. 216(), 2015
PMID: 26467715
Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production.
Nocon J, Steiger MG, Pfeffer M, Sohn SB, Kim TY, Maurer M, Rußmayer H, Pflugl S, Ask M, Haberhauer-Troyer C, Ortmayr K, Hann S, Koellensperger G, Gasser B, Lee SY, Mattanovich D., Metab. Eng. 24(), 2014
PMID: 24853352
Humanization of Yeast to Produce Complex Terminally Sialylated Glycoproteins
Genome wide distribution of illegitimate recombination events in Kluyveromyces lactis.
Kegel A, Martinez P, Carter SD, Astrom SU., Nucleic Acids Res. 34(5), 2006
PMID: 16549875
Novel genetic tools for Hansenula polymorpha.
Saraya R, Krikken AM, Kiel JA, Baerends RJ, Veenhuis M, van der Klei IJ., FEMS Yeast Res. 12(3), 2011
PMID: 22129301
Frontiers of yeast metabolic engineering: diversifying beyond ethanol and Saccharomyces.
Liu L, Redden H, Alper HS., Curr. Opin. Biotechnol. 24(6), 2013
PMID: 23541504
OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides.
Nakayama K, Nagasu T, Shimma Y, Kuromitsu J, Jigami Y., EMBO J. 11(7), 1992
PMID: 1628616
Knockout of an endogenous mannosyltransferase increases the homogeneity of glycoproteins produced in Pichia pastoris.
Krainer FW, Gmeiner C, Neutsch L, Windwarder M, Pletzenauer R, Herwig C, Altmann F, Glieder A, Spadiut O., Sci Rep 3(), 2013
PMID: 24252857
Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris.
Lin-Cereghino GP, Godfrey L, de la Cruz BJ, Johnson S, Khuongsathiene S, Tolstorukov I, Yan M, Lin-Cereghino J, Veenhuis M, Subramani S, Cregg JM., Mol. Cell. Biol. 26(3), 2006
PMID: 16428444
High-quality genome sequence of Pichia pastoris CBS7435.
Kuberl A, Schneider J, Thallinger GG, Anderl I, Wibberg D, Hajek T, Jaenicke S, Brinkrolf K, Goesmann A, Szczepanowski R, Puhler A, Schwab H, Glieder A, Pichler H., J. Biotechnol. 154(4), 2011
PMID: 21575661
Genome sequence of the recombinant protein production host Pichia pastoris.
De Schutter K, Lin YC, Tiels P, Van Hecke A, Glinka S, Weber-Lehmann J, Rouze P, Van de Peer Y, Callewaert N., Nat. Biotechnol. 27(6), 2009
PMID: 19465926
Interaction of Pik1p and Sjl proteins in membrane trafficking.
Nguyen PH, Hasek J, Kohlwein SD, Romero C, Choi JH, Vancura A., FEMS Yeast Res. 5(4-5), 2005
PMID: 15691741
Palindromic sequences in heteroduplex DNA inhibit mismatch repair in yeast.
Nag DK, White MA, Petes TD., Nature 340(6231), 1989
PMID: 2546083
Long palindromic sequences induce double-strand breaks during meiosis in yeast.
Nasar F, Jankowski C, Nag DK., Mol. Cell. Biol. 20(10), 2000
PMID: 10779335
Allotopic expression of mitochondrial-encoded genes in mammals: achieved goal, undemonstrated mechanism or impossible task?
Perales-Clemente E, Fernandez-Silva P, Acin-Perez R, Perez-Martos A, Enriquez JA., Nucleic Acids Res. 39(1), 2010
PMID: 20823090
In vivo ligation of linear DNA molecules to circular forms in the yeast Saccharomyces cerevisiae.
Suzuki K, Imai Y, Yamashita I, Fukui S., J. Bacteriol. 155(2), 1983
PMID: 6307979
A visual method for direct selection of high-producing Pichia pastoris clones.
Hu F, Li X, Lu J, Mao PH, Jin X, Rao B, Zheng P, Zhou YL, Liu SY, Ke T, Ma XD, Ma LX., BMC Biotechnol. 11(), 2011
PMID: 21418613
Effect of nitrogen source and inorganic phosphate concentration on methanol utilization and PEX genes expression in Pichia pastoris
Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris.
Vogl T, Ahmad M, Krainer FW, Schwab H, Glieder A., Microb. Cell Fact. 14(), 2015
PMID: 26169367
Contribution of horizontal gene transfer to the evolution of Saccharomyces cerevisiae.
Hall C, Brachat S, Dietrich FS., Eukaryotic Cell 4(6), 2005
PMID: 15947202
Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase.
Gmeiner C, Saadati A, Maresch D, Krasteva S, Frank M, Altmann F, Herwig C, Spadiut O., Microb. Cell Fact. 14(), 2015
PMID: 25567661

Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena.
Weis R, Luiten R, Skranc W, Schwab H, Wubbolts M, Glieder A., FEMS Yeast Res. 5(2), 2004
PMID: 15489201
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ., Nucleic Acids Res. 25(17), 1997
PMID: 9254694
Consed: a graphical tool for sequence finishing.
Gordon D, Abajian C, Green P., Genome Res. 8(3), 1998
PMID: 9521923
Complete genome sequencing of Agrobacterium sp. H13-3, the former Rhizobium lupini H13-3, reveals a tripartite genome consisting of a circular and a linear chromosome and an accessory plasmid but lacking a tumor-inducing Ti-plasmid.
Wibberg D, Blom J, Jaenicke S, Kollin F, Rupp O, Scharf B, Schneiker-Bekel S, Sczcepanowski R, Goesmann A, Setubal JC, Schmitt R, Puhler A, Schluter A., J. Biotechnol. 155(1), 2011
PMID: 21329740
Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes.
Zhu T, Guo M, Zhuang Y, Chu J, Zhang S., Appl. Microbiol. Biotechnol. 89(4), 2010
PMID: 20981418


Markieren/ Markierung löschen
Markierte Publikationen

Open Data PUB

Web of Science

Dieser Datensatz im Web of Science®


PMID: 27958335
PubMed | Europe PMC

Suchen in

Google Scholar