A macrokinetic model‐based comparative meta‐analysis of recombinant protein production by Pichia pastoris under AOX1 promoter

Abstract

An unstructured macrokinetic model for heterologous production of Rhizopus oryzae lipase (ROL) by a Pichia pastoris PAOX1 based system was developed. Mean specific rates and state variables for various fed-batch cultures, under methanol limited and non-limited conditions were used for modeling. The most representative kinetic functions (viz., Monod, Haldane, Pirt and Luedeking-Piret) were used for the specific rates of cell growth (µ), substrate consumption (qS ) and product formation (qP ). The performance of two different process models was assessed via simulation tests, using an overall validation procedure. The best model describes the cellular growth by a non-monotonic substrate function, substrate uptake via Pirt's equation and product formation through a Luedeking-Piret equation. A comparative meta-analysis of heterologous protein production of various target proteins by Pichia pastoris under AOX1 promoter was conducted and a general strategy for improving protein production from process kinetics was developed as a key to bioprocess optimization.

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

“…By achieving stable specific secretion rates of around >0.4 mg g −1  h −1 , this P. pastoris strain reached high q p values. Specific secretion rates of recombinant proteins are often below 0.4 mg g −1  h −1 for P. pastoris using P AOX1 or P GAP (Barrigon et al 2015; Looser et al 2015). For instance, published specific secretion rates (recalculated in mg product built per g cell dry weight and per hour) are 0.17 mg g −1  h −1 with a glycoengineered P. pastoris strain producing recombinant IgG1 antibody (P AOX1 , methanol, μ =  0.014 h −1 , Potgieter et al 2010), 0.26 mg g −1  h −1 recombinant human serum albumin (P AOX1 , methanol, μ =  0.002 h −1 , Kobayashi et al 2000), 0.42 mg g −1  h −1 recombinant α-galactosidase (P AOX1 , methanol, μ =  0.03 h −1 , Zhang et al 2005) and 0.69 mg g −1  h −1 recombinant porcine trypsinogen (P AOX1 , substrate mixture methanol-glucose, μ =  0.07 h −1 , Paulova et al 2012).…”

“…maximum product formation) was determined at a specific glycerol uptake rate of 0.054 g g −1  h −1 , corresponding to a specific growth rate of 0.024 h −1 (Capone et al, 2015). However, for the widely applied P. pastoris system, in general, maximum recombinant product formation has been reported at various specific growth rates (Barrigon et al 2015; Looser et al 2015). The same promoter and substrate combination tended to have a greater influence on product formation kinetics than similar categories of recombinant products (Looser et al 2015).…”

Towards optimal substrate feeding for heterologous protein production in Pichia pastoris (Komagataella spp) fed‐batch processes under PAOX1 control: a modeling aided approach

“…A material balance based model of the bioprocess for ROL production by the P. pastoris Mut + phenotype was first developed to properly describe the behavior of substrate, biomass and ROL production …”

“…The model parameters used for the simulations performed concerning MIP are listed in Table . The ones corresponding to μ , q P and q S were adapted first from the data obtained considering the new experimental data . The intrinsic yields and maintenance coefficients of q O2 and q CO2 , which are now included in the extended model, are also indicated .…”

“…Hensing et al discussed bioprocess optimization for the yeasts Hansenula polymorpha and Kluyveromyces marxianus depending on several shapes of q P in respect to μ . Looser et al presented examples of production kinetics, comparing some of them from both P AOX1 and P GAP ‐based cultures, where q P generally increased with μ in P GAP cases, while P AOX1 strategies mostly described profiles with the highest q P at moderate μ . Accordingly, P GAP‐ based systems commonly have a linear dependence between q P and μ and are carried out using pre‐programmed feeding profiles to maintain constant values of μ close to the maximum to achieve high production levels.…”