title: A dynamic fed batch strategy for a Pichia pastoris mixed feed system to increase process understanding.
abstract: 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 Muts strain producing the model enzyme horseradish peroxidase. Specific substrate uptake rates (qs) were controlled separately, and a stepwise increased qGly-control scheme was applied to investigate the effect of various substrate fluxes on the culture. The qs-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.
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria.
authors.forename: Dénes
authors.initials: D
authors.lastname: Zalai
authors.forename: Christian
authors.initials: C
authors.lastname: Dietzsch
authors.forename: Christoph
authors.initials: C
authors.lastname: Herwig
authors.forename: Oliver
authors.initials: O
authors.lastname: Spadiut
mesh_terms: D018533:Biomass; D003470:Culture Media; D005285:Fermentation; D006735:Horseradish Peroxidase / Q000096:biosynthesis / Q000235:genetics*; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D006735:Horseradish Peroxidase