期刊
MICROBIAL CELL FACTORIES
卷 16, 期 -, 页码 -出版社
BMC
DOI: 10.1186/s12934-017-0699-4
关键词
Lipidomics; Pichia pastoris; Hypoxia; Recombinant protein production; Antibody fragment; Protein secretion; Unfolded protein response
资金
- Spanish Ministry of Economy and Competitiveness [CTQ2013-42391-R]
- Catalan Government [2014-SGR-452]
- Austrian Federal Ministry of Science, Research and Economy (BMWFW)
- Austrian Federal Ministry of Traffic, Innovation and Technology (bmvit)
- Styrian Business Promotion Agency SFG
- Standortagentur Tirol
- Government of Lower Austria
- ZIT-Technology Agency of the City of Vienna through the COMET-Funding Program
- German Research Foundation (DFG) [INST 186/822-1]
- German Federal Ministry of Education and Research (BMBF BioFung project) [0315595A]
Background: 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. 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. Results: To 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. Conclusions: Our 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.
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