Hypoxic fed‐batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins

Abstract

High cell density cultivation of Pichia pastoris has to cope with several technical limitations, most importantly the transfer of oxygen. By applying hypoxic conditions to chemostat cultivations of P. pastoris expressing an antibody Fab fragment under the GAP promoter, a 2.5-fold increase of the specific productivity q(P) at low oxygen supply was observed. At the same time the biomass decreased and ethanol was produced, indicating a shift from oxidative to oxidofermentative conditions. Based on these results we designed a feedback control for enhanced productivity in fed batch processes, where the concentration of ethanol in the culture was kept constant at approximately 1.0% (vv(-1)) by a regulated addition of feed medium. This strategy was tested successfully with three different protein producing strains, leading to a three- to sixfold increase of the q(P) and threefold reduced fed batch times. Taken together the volumetric productivity Q(P) increased 2.3-fold.

Genome‐scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production

“…5. This relationship between the optimal protein production rate and the oxygen uptake rate was observed in hypoxic fedbatch cultures where the productivities of three different proteins produced by P. pastoris increased when oxygen limitation was applied [38].…”

“…Thus, flux variability analysis was carried out to investigate the impact that oxygen uptake rate has on protein production within a range of possible oxygen uptake rates; the oxygen uptake rate was used as a constraint to simulate the hypoxic environment for PpaMBEL1254_HSA and PpaMBEL1254_hSOD.The growth rate was also examined for each state to determine an optimal physiological state that balances growth and heterologous protein production. Also, it was noted from fed-batch cultures during the heterologous protein production that ethanol is also produced [38]. In that experiment, the ethanol concentration increased from 2 to 10 g/L when the cell concentration increased from 65 to 71 g DCW/L.This gives …”

“…Because each protein sequence is unique, different versions of the metabolic model were constructed to reflect those differences (Table 3). Here, the production of the proteins HSA and hSOD is represented in the metabolic models PpaMBEL1254_HSA and PpaMBEL1254_hSOD, respectively [27,38]. Before examining the effect of oxygen limitation on the production of heterologous proteins, the in silico maximum production rates were calculated.…”

“…In P. pastoris, it has been reported that hypoxic culture condition leads to the increase in productivity of the heterologous proteins by two-to threefold over fully aerobic condition [38]. Thus, flux variability analysis was carried out to investigate the impact that oxygen uptake rate has on protein production within a range of possible oxygen uptake rates; the oxygen uptake rate was used as a constraint to simulate the hypoxic environment for PpaMBEL1254_HSA and PpaMBEL1254_hSOD.The growth rate was also examined for each state to determine an optimal physiological state that balances growth and heterologous protein production.…”

Anaerobic α-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae

“…Considering that industrial processes for production of pharmaceutical proteins (10) and consolidated bioprocesses are carried out under anaerobic conditions, there is a need for more fundamental knowledge on how protein production is affected by the oxygen supply. It has been reported previously that low oxygen levels could enhance production of certain proteins, such as glucoamylase (11), 3H6 Fab (12), and human trypsinogen (12). In order to investigate whether this is due to less oxidative stress under anaerobic conditions, we studied the impact of oxygen supply on the production of heterologous ␣-amylase and we combined macroscopic flux analysis with genome-wide transcription analysis under aerobic and anaerobic conditions.…”