A Study of the Physiological Response of Pichia pastoris Growing in a Continuous Culture to an Induced Stress Situation

Abstract

During our work, flow cytometry was used for a study of the influence of cultivation conditions on a physiological state, mainly on the viability and vitality of the recombinant Pichia pastoris strain producing ?-galactosidase. The fluorescent dyes propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol were used for the determination of the structural integrity and the membrane potential of Pichia pastoris cells, respectively. First the staining procedures were optimised and adapted for the Pichia pastoris yeast cells and later applied in a lab-scale continuous cultivation. Stained samples were analysed with the flow cytometer PAS III Partec or the epifluorescence microscope Nikon Eclipse E400. The following conditions were found to be optimum for the staining of Pichia pastoris cells: 57 ?g/ml propidium iodide in the sample, 5 min incubation time; 2 ?g/ml bis-(1,3-dibutylbarburic acid) trimethine oxonol in the sample, 20 min incubation time. The optimised staining methods were employed in a study of stress-induced physiological response to change of substrate from glycerol to methanol in a glycerol steady state growing culture of Pichia pastoris yeast. During the first 5 h of the transitional state an accumulation of methanol in the culture broth was accompanied by a decreasing concentration of biomass and an increasing amount of cells stained with propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol. After the adaptation phase the amount of cells stained with propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol reached steady levels of 2% and 5%, respectively.

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

“…Therefore, even the routine use of well-established dyes is ineffective without an appropriate adjustment to the method, particularly with regard to the dye concentration, the incubation conditions, and the incubation time. As the physiological characteristics of cells can also change during any sample handling procedure, the reliability and reproducibility of the results should be confirmed with appropriate control experiments and at least triplicate analyses (1,30,51).…”

“…The power of the technique lies in the use of specific, fluorescent dyes to discriminate between vital (i.e., active and viable) and compromised (or dead) cells. For instance, propidium iodide (PI), bis-(1,3-dibutylbarbituric acid)trimethine oxonol (BOX) (30), and ethidium bromide (EB) (34,46) can be used to stain defective cells with permeabilized membranes and damaged membrane integrity, while 5(6)-carboxyfluorescein diacetate (cFDA) (26) is used to stain intact cells with nonspecific esterase activity.…”