Engineering the supply chain for protein production/secretion in yeasts and mammalian cells

Klein, Tobias; Niklas, Jens; Heinzle, Elmar

doi:10.1007/s10295-014-1569-2

Cited by 28 publications

(17 citation statements)

References 146 publications

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“…Strain engineering strategies for protein secretion are mainly focused on engineering the protein folding and quality control systems in the ER, the intracellular protein trafficking pathway, and minimizing post-secretory degradation [9]. In addition, 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.…”

Section: Introductionmentioning

confidence: 99%

The effect of hypoxia on the lipidome of recombinant Pichia pastoris

et al. 2017

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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.

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Section: Introductionmentioning

confidence: 99%

The effect of hypoxia on the lipidome of recombinant Pichia pastoris

et al. 2017

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“…Although a linear correlation between gene copy number and cytoplasmic protein production has been demonstrated in yeast, this was not true for protein secretion (41). Recombinant protein secretion is often hampered along the secretory pathway or because of limitations in the central carbon metabolism (5). This was also true for high-level maltase secretion, where we could overcome these limitations by influencing membrane composition and targeted amino acid feeding.…”

Section: Discussionmentioning

confidence: 94%

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion

Hung

Klein

Cassidy

et al. 2016

Molecular & Cellular Proteomics

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Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the ␣-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative pro-

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“…Indeed, they have enabled the overexpression or down-regulation of specific gene candidates to increase yield during culture and control product quality [3, 4, 8–10]. Several prominent strategies have targeted cell metabolism, cell cycle regulatory machinery, the protein secretion pathway, apoptosis, and protein glycosylation [9, 11]. …”

Section: Introductionmentioning

confidence: 99%

Improvements in protein production in mammalian cells from targeted metabolic engineering

Richelle

Lewis

2017

Current Opinion in Systems Biology

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Bioprocess optimization has yielded powerful clones for biotherapeutic production. However, new genomic technologies allow more targeted approaches to cell line development. Here we review efforts to enhance protein production in mammalian cells through metabolic engineering. Most efforts aimed to reduce toxic byproducts accumulation to enhance protein productivity. However, recent work highlights the possibility of regulating other desirable traits (e.g., apoptosis and glycosylation) by targeting central metabolism since these processes are interconnected. Therefore, as we further detail the pathways underlying cell growth and protein production and deploy diverse algorithms for their analysis, opportunities will arise to move beyond simple cell line designs and facilitate cell engineering strategies with complex combinations of genes that together underlie a phenotype of interest.

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Engineering the supply chain for protein production/secretion in yeasts and mammalian cells

Cited by 28 publications

References 146 publications

The effect of hypoxia on the lipidome of recombinant Pichia pastoris

The effect of hypoxia on the lipidome of recombinant Pichia pastoris

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion

Improvements in protein production in mammalian cells from targeted metabolic engineering

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