Abstract
BackgroundCultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. In addition, transcriptomic analyses of hypoxic P. pastoris pointed out important regulation of lipid metabolism and unfolded protein response (UPR). Notably, UPR that plays a role in the regulation of lipid metabolism, amino acid metabolism and protein secretion, was found to be upregulated under hypoxia.ResultsTo improve our understanding of the interplay between lipid metabolism, UPR and protein secretion, the lipidome of a P. pastoris strain producing an antibody fragment was studied under hypoxic conditions. Furthermore, lipid composition analyses were combined with previously available transcriptomic datasets to further understand the impact of hypoxia on lipid metabolism. Chemostat cultures operated under glucose-limiting conditions under normoxic and hypoxic conditions were analyzed in terms of intra/extracellular product distribution and lipid composition. Integrated analysis of lipidome and transcriptome datasets allowed us to demonstrate an important remodeling of the lipid metabolism under limited oxygen availability. Additionally, cells with reduced amounts of ergosterol through fluconazole treatment were also included in the study to observe the impact on protein secretion and its lipid composition.ConclusionsOur results show that cells adjust their membrane composition in response to oxygen limitation mainly by changing their sterol and sphingolipid composition. Although fluconazole treatment results a different lipidome profile than hypoxia, both conditions result in higher recombinant protein secretion levels.
Highlights
Cultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions
Hypoxia and fluconazole treatment do not exert synergistic effects on protein secretion Previous studies cultivating P. pastoris under hypoxic chemostat conditions revealed that stringent hypoxia leads to bioreactor wash out [14]
To establish less severe working hypoxic conditions, which still result in respirofermentative metabolism but prevent culture instability, P. pastoris producing Fab 2F5 was grown in glucose-limited chemostat cultures using different concentrations of oxygen in the inlet gas
Summary
Cultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. P. pastoris is a eukaryote, and provides the potential for producing soluble, correctly folded recombinant proteins that have undergone all post-translational modifications required for functionality. This yeast can be Adelantado et al Microb Cell Fact (2017) 16:86 protein trafficking pathway, and minimizing post-secretory degradation [9]. There is increasing evidence that metabolic bottlenecks in the supply chain for building blocks and energy play an important role in recombinant yeast [10, 11] In this context, environmental conditions have a significant impact on the levels of recombinant proteins. Lower culture temperature [12, 13], low oxygen availability [14], as well as adequate substrate feeding strategies in high cell density cultures [15, 16], and type of carbon source(s) [17, 18] resulted in positive effects on protein secretion
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