Is heterogeneity in large-scale bioreactors a real problem in recombinant protein synthesis by Pichia pastoris?

Culture medium heterogeneity is inherent in industrial bioreactors. The loss of mixing efficiency in a large-scale bioreactor yields to the formation of concentration gradients. Consequently, cells face oscillatory culture conditions that may deeply affect their metabolism. Herein, cell response to transient perturbations, namely high methanol concentration combined with hypoxia, has been investigated using a two stirred-tank reactor compartiments (STR-STR) scale-down system and a Pichia pastoris strain expressing the gene encoding enhanced green fluorescent protein (eGFP) under the control of the alcohol oxidase 1 (AOX1) promoter. Cell residence times under transient stressing conditions were calculated based on the typical hydraulic circulation times of bioreactors of tens and hundreds cubic metres. A significant increase in methanol and oxygen uptake rates was observed as the cell residence time was increased. Stressful culture conditions impaired biomass formation and triggered cell flocculation. More importantly, both expression levels of genes under the control of pAOX1 promoter and eGFP specific fluorescence were higher in those oscillatory culture conditions, suggesting that those a priori unfavourable culture conditions in fact benefit to recombinant protein productivity. Flocculent cells were also identified as the most productive as compared to ovoid cells. KEY POINTS: • Transient hypoxia and high methanol trigger high level of recombinant protein synthesis • In Pichia pastoris, pAOX1 induction is higher in flocculent cells • Medium heterogeneity leads to morphological diversification.