The effect of hypoxia on the lipidome of recombinant Pichia pastoris

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0699-4) contains supplementary material, which is available to authorized users.

Rationale‐based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)

“…Interestingly, there were no significant differences in q S between clones, which suggests that this rate was unaffected by gene dosage. On the other hand, the overall biomass substrate yield ( Y X/S ) remained fairly constant with a value of about 0.5 g X g S –1 , which is consistent with most reported values (Garcia‐Ortega et al , ; Çalik et al, ; Adelantado et al , ). However, MCC exhibited a slight decrease in Y X/S at the lowest μ values (0.05 and 0.025 h ‐1 ) possibly due to requirement of more energy for maintenance ( m s ) than the single‐copy strain (0.029 vs. 0.0023 g S g X –1 h –1 ).…”

Whole-cell (+)-ambrein production in the yeast Pichia pastoris

“…Under standard cultivation conditions, squalene levels in wild type P. pastoris are usually below detection limit ( Adelantado et al, 2017 ). To our knowledge, the only study on triterpenoid production in P. pastoris in which proteins involved in sterol biosynthesis were targeted focused on accumulation of 2,3-oxidosqualene ( Liu et al, 2015 ).…”

Continuous Cultivation as a Tool Toward the Rational Bioprocess Development With Pichia Pastoris Cell Factory

“…On the other hand, endoplasmic reticulum associated protein degradation (ERAD) is one other limiting factor for RPP (Dragosits et al, 2010 ; Adelantado et al, 2017 ). UPR and ERAD have been found to jointly control protein folding to a considerable extent and also, generally, heterologous protein synthesis, in P. pastoris (Delic et al, 2013 ; Zahrl et al, 2018 ).…”