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

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.Changes to the product quantity and quality as well as the robustness of bioprocesses can be triggered by a number of factors which affect the physiological state of the microorganisms. The expression of a foreign gene, the processing of the recombinant protein, and the exposure of the cell to metabolites, inductors, substrates, unfavorable environmental conditions, high cell density (HCD), and/or "aging" can all result in markedly different physiological responses (17).The interpretation of bioprocess data often fails to reflect the actual state of individual cells within a population. It is based typically on performance characterization using concentration measurements and the simplifying assumption of uniform ("averaged") performance of each cell. This practice (39) provides an example of the failure to distinguish between a homogenous population of equally compromised microorganisms and a heterogeneous population of cells each performing differently. An understanding of the state of the individual cells is critical in achieving high efficiency in recombinant protein production. Only by knowing the number of (vital) cells that express the target molecule at the highest possible rate can the number of such cells within the population be maximized and the propo...

Cultivation strategies to enhance productivity of Pichia pastoris: A review

“…In addition, values of the growth characteristics (i.e. µ max , Y x/s , m s , see nomenclature given in Table 1) are crucial since they give a direct indication of whether the strain is physiologically impaired due to the introduction and expression of a foreign gene (Cos et al, 2006, Heyland et al, 2011, Hyka et al, 2010. Heavily impaired strains tend to stop growing, die and lyse, which causes problems in bioreactor operation because of heavy foaming as well as impaired product quality due to the release of host proteins or intracellular proteases (Jahic et al, 2003, Jahic et al, 2006, Zhou and Zhang, 2002.…”

“…Optimum conditions for the production of a recombinant protein in P. pastoris differ according to the target molecule and promoter (Hyka et al, 2010). To develop a production process for a newly constructed strain of unknown behaviour, it is therefore essential to start with its physiological characterisation.…”

Effect of dilution rate and methanol‐glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastorisMut+ phenotype in continuous culture

“…34 This experimental approach also minimizes the effect of process conditions such as high agitation rate and oxygen enrichment, 35 that can lead to uncontrolled variables into the experimental design. Quadratic model was selected to analyze the ROL response surfaces.…”

Synergistic modular promoter and gene optimization to push cellulase secretion by Pichia pastoris beyond existing benchmarks

“…Nevertheless, contrary to P GAP , P DeS can be still quite tightly regulated by the available carbon source type and level. Moreover, studies performed with another synthetic derepressed promoter P(De) revealed that there is a positive effect on cell physiology compared to expression under the control of P AOX1 (Hyka et al, 2010) which might also apply for P DeS .…”