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  2. Life Sciences und Facility Management
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  4. Publikationen Life Sciences und Facility Management
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dc.contributor.authorHyka, Petr-
dc.contributor.authorZüllig, Thomas-
dc.contributor.authorRuth, Claudia-
dc.contributor.authorLooser, Verena-
dc.contributor.authorMeier, Christian-
dc.contributor.authorKlein, Joachim-
dc.contributor.authorMelzoch, Karel-
dc.contributor.authorMeyer, Hans-Peter-
dc.contributor.authorGlieder, Anton-
dc.contributor.authorKovar, Karin-
dc.date.accessioned2018-09-20T12:33:12Z-
dc.date.available2018-09-20T12:33:12Z-
dc.date.issued2010-05-04-
dc.identifier.issn0099-2240de_CH
dc.identifier.issn1098-5336de_CH
dc.identifier.urihttps://digitalcollection.zhaw.ch/handle/11475/10882-
dc.description.abstractMatching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut+ (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.de_CH
dc.language.isoende_CH
dc.publisherAmerican Society for Microbiologyde_CH
dc.relation.ispartofApplied and Environmental Microbiologyde_CH
dc.rightsLicence according to publishing contractde_CH
dc.subjectPichia pastorisde_CH
dc.subject.ddc570: Biologiede_CH
dc.titleCombined use of fluorescent dyes and flow cytometry to quantify the physiological state of Pichia pastoris during the production of heterologous proteins in high-cell-density fed-batch culturesde_CH
dc.typeBeitrag in wissenschaftlicher Zeitschriftde_CH
dcterms.typeTextde_CH
zhaw.departementLife Sciences und Facility Managementde_CH
zhaw.organisationalunitInstitut für Chemie und Biotechnologie (ICBT)de_CH
dc.identifier.doi10.1128/AEM.02475-09de_CH
zhaw.funding.euNode_CH
zhaw.issue13de_CH
zhaw.originated.zhawYesde_CH
zhaw.pages.end4496de_CH
zhaw.pages.start4486de_CH
zhaw.publication.statuspublishedVersionde_CH
zhaw.volume76de_CH
zhaw.publication.reviewPeer review (Publikation)de_CH
zhaw.webfeedBiokatalysede_CH
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Hyka, P., Züllig, T., Ruth, C., Looser, V., Meier, C., Klein, J., Melzoch, K., Meyer, H.-P., Glieder, A., & Kovar, K. (2010). Combined use of fluorescent dyes and flow cytometry to quantify the physiological state of Pichia pastoris during the production of heterologous proteins in high-cell-density fed-batch cultures. Applied and Environmental Microbiology, 76(13), 4486–4496. https://doi.org/10.1128/AEM.02475-09
Hyka, P. et al. (2010) ‘Combined use of fluorescent dyes and flow cytometry to quantify the physiological state of Pichia pastoris during the production of heterologous proteins in high-cell-density fed-batch cultures’, Applied and Environmental Microbiology, 76(13), pp. 4486–4496. Available at: https://doi.org/10.1128/AEM.02475-09.
P. Hyka et al., “Combined use of fluorescent dyes and flow cytometry to quantify the physiological state of Pichia pastoris during the production of heterologous proteins in high-cell-density fed-batch cultures,” Applied and Environmental Microbiology, vol. 76, no. 13, pp. 4486–4496, May 2010, doi: 10.1128/AEM.02475-09.
HYKA, Petr, Thomas ZÜLLIG, Claudia RUTH, Verena LOOSER, Christian MEIER, Joachim KLEIN, Karel MELZOCH, Hans-Peter MEYER, Anton GLIEDER und Karin KOVAR, 2010. Combined use of fluorescent dyes and flow cytometry to quantify the physiological state of Pichia pastoris during the production of heterologous proteins in high-cell-density fed-batch cultures. Applied and Environmental Microbiology. 4 Mai 2010. Bd. 76, Nr. 13, S. 4486–4496. DOI 10.1128/AEM.02475-09
Hyka, Petr, Thomas Züllig, Claudia Ruth, Verena Looser, Christian Meier, Joachim Klein, Karel Melzoch, Hans-Peter Meyer, Anton Glieder, and Karin Kovar. 2010. “Combined Use of Fluorescent Dyes and Flow Cytometry to Quantify the Physiological State of Pichia Pastoris during the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures.” Applied and Environmental Microbiology 76 (13): 4486–96. https://doi.org/10.1128/AEM.02475-09.
Hyka, Petr, et al. “Combined Use of Fluorescent Dyes and Flow Cytometry to Quantify the Physiological State of Pichia Pastoris during the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures.” Applied and Environmental Microbiology, vol. 76, no. 13, May 2010, pp. 4486–96, https://doi.org/10.1128/AEM.02475-09.


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