Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production

2019 ◽  
Vol 39 (2) ◽  
pp. 258-271 ◽  
Author(s):  
Wan-Cang Liu ◽  
Sarah Inwood ◽  
Ting Gong ◽  
Ashish Sharma ◽  
Li-Yan Yu ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Cell Density ◽  
High Cell Density ◽  
High Cell ◽  
Fed Batch ◽  
High Cell Density Fermentation

Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris

2011 ◽  
Vol 79 (2) ◽  
pp. 217-222 ◽  
Author(s):  
Johanne Mørch Jensen ◽  
Malene Bech Vester-Christensen ◽  
Marie Sofie Møller ◽  
Birgit C. Bønsager ◽  
Hans Erik Mølager Christensen ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Cell Density ◽  
High Cell Density ◽  
High Cell ◽  
Secretory Expression ◽  
High Cell Density Fermentation ◽  
Limit Dextrinase

scholarly journals 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

2010 ◽  
Vol 76 (13) ◽  
pp. 4486-4496 ◽  
Author(s):  
Petr Hyka ◽  
Thomas Züllig ◽  
Claudia Ruth ◽  
Verena Looser ◽  
Christian Meier ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Cell Density ◽  
Fluorescent Dyes ◽  
High Cell Density ◽  
Physiological State ◽  
Stress Factors ◽  
High Cell ◽  
Fed Batch ◽  
Batch Cultures ◽  
The Individual

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.

A new methanol-feeding strategy for the improved production of β-galactosidase in high cell-density fed-batch cultures of Pichia pastoris Mut+ strains

2012 ◽  
Vol 17 (1) ◽  
pp. 76-83 ◽  
Author(s):  
Amir Maghsoudi ◽  
Safoura Hosseini ◽  
Seyed Abbas Shojaosadati ◽  
Ebrahim Vasheghani-Farahani ◽  
Mohsen Nosrati ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Cell Density ◽  
High Cell Density ◽  
Feeding Strategy ◽  
High Cell ◽  
Fed Batch ◽  
Batch Cultures ◽  
Methanol Feeding
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