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.

Cultivation strategies to enhance productivity of Pichia pastoris: A review

“…The use of dynamic process conditions for fast physiological strain characterisation are summarised by reviews of Spadiut and Herwig, (2014) and Spadiut et al (2013). Various dynamic fedbatch approaches currently under development would allow the entire production range of µ to be covered in a single experiment (Lüthy et al, 2011, Spadiut et al, 2014b, Zalai et al, 2012. In these kinds of experiments, how product formation may adapt with time has also been investigated (Spadiut et al, 2014b).…”

“…In recent years, protocol design has shifted from classical "recipes" (Invitrogen, 2002, Stratton et al, 1998, Tolner et al, 2006 to more conceptual attempts (Maurer et al, 2006, Zalai et al, 2012, Zhang et al, 2007. Considering the maximum specific growth rate (µ max ) as the upper limit and the production kinetics q p (µ) as an indicator of the optimum production range of µ, design of a customised fedbatch feed profile to maximise product titre is possible (Fig.…”

“…These theoretical time courses of specific growth rates were calculated from the time courses of biomass concentrations (second row). x V x V µt tt (Dietzsch et al, 2011b) exponential fedbatch feedprofiles (Zhang et al, 2007) dynamic fedbatch approach (Zalai et al, 2012) standardised constant feed profiles (Invitrogen, 2002, Stratton et al, 1998, Tolner et al, 2006 standard protocols customised feed profile à maximum titer / productivity (high biomass for extended time) (Kobayashi et al, 2000, Zhang et al, 2005, Maurer et al, 2006 technical limits ( …”

Glyco-variant library of the versatile enzyme horseradish peroxidase

“…Since HRP is a versatile enzyme used in numerous, quite diverse industrial and medical applications, such as waste water treatment, fine chemical synthesis, immunoassays, biosensors and coupled enzyme assays (e.g., Krieg and Halbhuber 2003), the controllable recombinant production and subsequent efficient purification of single HRP isoenzymes is highly desired. Thus, we have not only investigated and improved the recombinant production of the isoenzyme HRP C1A with Pichia pastoris in the past few years (Dietzsch et al 2011a,b; Krainer et al 2012; Zalai et al 2012; Spadiut et al 2013), but also developed an efficient downstream process for the hypermannosylated enzyme recombinantly derived from this yeast (Spadiut et al 2012; Krainer, Pletzenauer, et al 2013). However, for medical applications where HRP is conjugated to antibodies, like antibody-directed enzyme-prodrug therapy (Folkes and Wardman 2001; Wardman 2002) and medical diagnostics (Romero et al 1999; Huang 2001; Palmgren et al 2011; Dotsikas and Loukas 2012), the degree of glycosylation of HRP is of utmost importance, since not only the stability of the enzyme but also the conjugation with antibodies is expected to change with varying glycosylation.…”

Effects of different media supplements on the production of an active recombinant plant peroxidase in aPichia pastorisΔoch1strain

“…[1][2][3] ). However, a significant disadvantage of this microorganism is its tendency to perform hyperglycosylation of proteins.…”