Quo vadis? The challenges of recombinant protein folding and secretion in Pichia pastoris

Puxbaum, Verena; Mattanovich, Diethard; Gasser, Brigitte

doi:10.1007/s00253-015-6470-z

Cited by 137 publications

(110 citation statements)

References 116 publications

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“…Existing shortcomings of the P. pastoris production system are addressed either by genetic strain engineering (Idiris et al, 2010, Puxbaum et al, 2015 or at the level of the cultivation process in bioreactors. Generally, bioprocess design aims at identifying optimum conditions for biomass growth and product formation, including pH, temperature, oxygen and nutrient supply [e.g.…”

Section: Introductionmentioning

confidence: 99%

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Looser

Brühlmann

Bumbak

et al. 2015

Biotechnology Advances

291

268

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Pichia pastoris, a methylotrophic yeast, is an established system for the production of heterologous proteins, particularly biopharmaceuticals and industrial enzymes. To maximise and optimise the production of recombinant products, recent molecular research has focused on numerous issues including the design of expression vectors, optimisation of gene copy number, co-expression of secretory proteins such as chaperones, engineering of glycosylation and secretory pathways, etc. However, the physiological effects of different cultivation strategies are often difficult to separate from the molecular effects of the gene construct (e.g., cellular stress through over-expression or incorrect post-translational processing). Hence, overall system optimisation is difficult, even though it is urgently required in order to describe and understand the behaviour of new molecular constructs. This review focuses on particular aspects of recombinant protein production related to variations in biomass growth and their implications for strain design and screening, as well as on the concept of rational comparisons between cultivation systems for the development of specific production processes in bioreactors. The relationship between specific formation rates of secreted recombinant proteins, qp, and specific growth rates, μ, has been analysed in a conceptual attempt to compare different systems, particularly those based on AOX1/methanol and GAP/glucose, and this has now evolved into a pivotal concept for bioprocess engineering of P. pastoris.

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Section: Introductionmentioning

confidence: 99%

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Looser

Brühlmann

Bumbak

et al. 2015

Biotechnology Advances

291

268

View full text Add to dashboard Cite

show abstract

“…Furthermore, this yeast can be engineered to mimic the human N -glycosylation pathway and specific types of O -glycosylation, becoming a potential alternative for mammalian cell culture for the production of recombinant therapeutic glycoproteins for human use [4, 5]. Overexpression of heterologous proteins can lead to saturation or overloading of the secretory pathway [6, 7]. The most important bottlenecks in terms of recombinant protein production and secretion are membrane translocation, signal peptide processing and folding within the endoplasmic reticulum (ER) [8].…”

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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“…[92] Yeasts combine the simplicity, low cost, ease of manipulation and short generation time of a unicellular organism with the ability to carry complex post-translational modifications, [93] including human-like protein glycosylation in engineered strains. [94] Saccharomyces cerevisiae and Pichia pastoris are the best characterized species, with glycoengineered strains of the latter shown to be capable of producing recombinant anti-human epidermal growth factor receptor 2 (HER2) antibodies similar to the FDA-approved and mammalian cellproduced trastuzumab used to treat metastatic breast cancer. [95] Insect cells possess eucaryotic protein expression and posttranslational modification machineries typical of mammalian cells.…”

Section: Expression Systems For Recombinant Productsmentioning

confidence: 99%

Adding Functions to Biomaterial Surfaces through Protein Incorporation

et al. 2016

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The concept of biomaterials has evolved from one of inert mechanical supports with a long-term, biologically inactive role in the body into complex matrices that exhibit selective cell binding, promote proliferation and matrix production, and may ultimately become replaced by newly generated tissues in vivo. Functionalization of material surfaces with biomolecules is critical to their ability to evade immunorecognition, interact productively with surrounding tissues and extracellular matrix, and avoid bacterial colonization. Antibody molecules and their derived fragments are commonly immobilized on materials to mediate coating with specific cell types in fields such as stent endothelialization and drug delivery. The incorporation of growth factors into biomaterials has found application in promoting and accelerating bone formation in osteogenerative and related applications. Peptides and extracellular matrix proteins can impart biomolecule- and cell-specificities to materials while antimicrobial peptides have found roles in preventing biofilm formation on devices and implants. In this progress report, we detail developments in the use of diverse proteins and peptides to modify the surfaces of hard biomaterials in vivo and in vitro. Chemical approaches to immobilizing active biomolecules are presented, as well as platform technologies for isolation or generation of natural or synthetic molecules suitable for biomaterial functionalization.

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Quo vadis? The challenges of recombinant protein folding and secretion in Pichia pastoris

Cited by 137 publications

References 116 publications

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Cultivation strategies to enhance productivity of Pichia pastoris: A review

The effect of hypoxia on the lipidome of recombinant Pichia pastoris

Adding Functions to Biomaterial Surfaces through Protein Incorporation

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