[en] Pichia pastoris is a widely used recombinant protein expression system, so much that to date, more than 500 biomolecules have been synthesized using this yeast, many of which are approved for production and commercialization by the biotechnological industry.
The pAOX1-based expression system (MUT+) is one of the most widely used to produce rProt in both research and industry. Under productive conditions, MUT+ shows a high consumption rate of methanol and oxygen, and the accumulation of methanol can lead to the accumulation of cytotoxic intermediates such as formaldehyde and formate. This converts methanol and oxygen critical variables in the design of an rProt production strategy.
Culture medium heterogeneity is inherent in large-scale bioreactors. The loss of mixing quality 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 compartments (STR-STR) scale-down (SD) 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 meters. 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 unfavorable culture conditions in fact benefit to recombinant protein productivity. Flocculent cells were also identified as the most productive as compared to ovoid cells.
It is known that overexpression of the pAOX1 promoter does not always leads to an improvement in rProt productivity, as many bottlenecks can impair the production ability of secreted rProt. For this reason, the production of extracellular Candida antarctica lipase was analyzed using the same configurations of SD systems as with the eGFP-producer strain. Extended cell residence time in microenvironments of high methanol concentration and low oxygen availability that are typically found at the upper part of a large-scale bioreactor near the methanol feeding point, triggers the unfolded protein response (UPR) and thus impairs protein secretion. Methanol/sorbitol co-feeding was shown herein as a means to reduce the UPR response and to restore productivity of the secreted protein.
