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A dynamic fed batch strategy for a Pichia pastoris mixed feed system to increase process understanding
Author(s) -
Zalai Dénes,
Dietzsch Christian,
Herwig Christoph,
Spadiut Oliver
Publication year - 2012
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.1551
Subject(s) - pichia pastoris , substrate (aquarium) , horseradish peroxidase , fed batch culture , glycerol , recombinant dna , chemistry , mixing (physics) , biochemistry , biology , enzyme , fermentation , gene , physics , quantum mechanics , ecology
Mixed substrate feeding strategies are frequently investigated to enhance the productivity of recombinant Pichia pastoris processes. For this purpose, numerous fed batch experiments or time‐consuming continuous cultivations are required to optimize control parameters such as the substrate mixing ratio and the applied methanol concentration. In this study, we decoupled the feeding of methanol and glycerol in a mixed substrate fed batch environment to gain process understanding for a recombinant P. pastoris Mut s strain producing the model enzyme horseradish peroxidase. Specific substrate uptake rates ( q s ) were controlled separately, and a stepwise increased q Gly ‐control scheme was applied to investigate the effect of various substrate fluxes on the culture. The q s ‐controlled strategy allowed a parallel characterization of the metabolism and the recombinant protein expression in a fed batch environment. A critical‐specific glycerol uptake rate was determined, where a decline of the specific productivity occurred, and a time‐dependent acceleration of protein expression was characterized with the dynamic fed batch approach. Based on the observations on recombinant protein expression, propositions for an optimal feeding design to target maximal productivities were stated. Thus, the dynamic fed batch strategy was found to be a valuable tool for both process understanding and optimization of product formation for P. pastoris in a mixed substrate environment. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012