title: 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.
abstract: Matching 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.
authors.affiliation: Zurich University of Applied Sciences (ZHAW), Institute of Biotechnology, Campus Grüental, CH-8820 Wädenswil, Switzerland.
authors.forename: Petr
authors.initials: P
authors.lastname: Hyka
authors.forename: Thomas
authors.initials: T
authors.lastname: Züllig
authors.forename: Claudia
authors.initials: C
authors.lastname: Ruth
authors.forename: Verena
authors.initials: V
authors.lastname: Looser
authors.forename: Christian
authors.initials: C
authors.lastname: Meier
authors.forename: Joachim
authors.initials: J
authors.lastname: Klein
authors.forename: Karel
authors.initials: K
authors.lastname: Melzoch
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Meyer
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
authors.forename: Karin
authors.initials: K
authors.lastname: Kovar
mesh_terms: D001709:Biotechnology / Q000379:methods; D049109:Cell Proliferation; D015169:Colony Count, Microbial; D003470:Culture Media; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D000432:Methanol / Q000378:metabolism; D010641:Phenotype; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003470:Culture Media; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D000432:Methanol
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