A Combined System for Engineering Glycosylation Efficiency and Glycan Structure in Saccharomyces cerevisiae

Nasab, Farnoush Parsaie; Aebi, Markus; Bernhard, Gesche; Frey, Alexander D.

doi:10.1128/aem.02817-12

Cited by 60 publications

(39 citation statements)

References 44 publications

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“…However, prokaryotic glycans do not show similarities with human glycosylation patterns, resulting in immunogenic biopharmaceuticals [102,103]. Yeast glycans present an excess of mannose residues assembled together in "hyper-mannosidic" structures [104], which greatly differ from the human patterns [4,105]. Although CHO cells possess human-like glycosylation machinery, some discrepancies still persist [4].…”

Section: N-glycosylationmentioning

confidence: 99%

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

Barolo

Abbriano

Commault

et al. 2020

Cells

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Microalgae exhibit great potential for recombinant therapeutic protein production, due to lower production costs, immunity to human pathogens, and advanced genetic toolkits. However, a fundamental aspect to consider for recombinant biopharmaceutical production is the presence of correct post-translational modifications. Multiple recent studies focusing on glycosylation in microalgae have revealed unique species-specific patterns absent in humans. Glycosylation is particularly important for protein function and is directly responsible for recombinant biopharmaceutical immunogenicity. Therefore, it is necessary to fully characterise this key feature in microalgae before these organisms can be established as industrially relevant microbial biofactories. Here, we review the work done to date on production of recombinant biopharmaceuticals in microalgae, experimental and computational evidence for N- and O-glycosylation in diverse microalgal groups, established approaches for glyco-engineering, and perspectives for their application in microalgal systems. The insights from this review may be applied to future glyco-engineering attempts to humanize recombinant therapeutic proteins and to potentially obtain cheaper, fully functional biopharmaceuticals from microalgae.

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Section: N-glycosylationmentioning

confidence: 99%

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

Barolo

Abbriano

Commault

et al. 2020

Cells

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“…One of the main limitations of producing recombinant pharmaceuticals in yeast is the different N -glycan pattern compared to mammalian expression platforms [ 2 ]. However, producing correctly folded and glycosylated proteins for therapeutic applications in S. cerevisiae is about to become possible with the advancements of glycoengineered strains [ 3 , 4 ]. With further optimization of the host and the production process, the yeast has great potential to become a viable platform to produce competitive yields of complex, functional mammalian target proteins, such as antibodies.…”

Section: Introductionmentioning

confidence: 99%

Enhancing antibody folding and secretion by tailoring the Saccharomyces cerevisiae endoplasmic reticulum

2016

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BackgroundThe yeast Saccharomyces cerevisiae provides intriguing possibilities for synthetic biology and bioprocess applications, but its use is still constrained by cellular characteristics that limit the product yields. Considering the production of advanced biopharmaceuticals, a major hindrance lies in the yeast endoplasmic reticulum (ER), as it is not equipped for efficient and large scale folding of complex proteins, such as human antibodies.ResultsFollowing the example of professional secretory cells, we show that inducing an ER expansion in yeast by deleting the lipid-regulator gene OPI1 can improve the secretion capacity of full-length antibodies up to fourfold. Based on wild-type and ER-enlarged yeast strains, we conducted a screening of a folding factor overexpression library to identify proteins and their expression levels that enhance the secretion of antibodies. Out of six genes tested, addition of the peptidyl-prolyl isomerase CPR5 provided the most beneficial effect on specific product yield while PDI1, ERO1, KAR2, LHS1 and SIL1 had a mild or even negative effect to antibody secretion efficiency. Combining genes for ER enhancement did not induce any significant additional effect compared to addition of just one element. By combining the Δopi1 strain, with the enlarged ER, with CPR5 overexpression, we were able to boost the specific antibody product yield by a factor of 10 relative to the non-engineered strain.ConclusionsEngineering protein folding in vivo is a major task for biopharmaceuticals production in yeast and needs to be optimized at several levels. By rational strain design and high-throughput screening applications we were able to increase the specific secreted antibody yields of S. cerevisiae up to 10-fold, providing a promising strain for further process optimization and platform development for antibody production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0488-5) contains supplementary material, which is available to authorized users.

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“…Relative to mammalian cell culture, yeasts offer generally high yields of recombinant proteins, are well-characterized, give serum-free growth media, are readily adapted to large-scale fermentation processes and have greatly reduced costs [ 7 ]. Humanized glycoproteins have already been produced in a variety of yeasts, such as Saccharomyces cerevisiae [ 8 ], Pichia pastoris [ 9 , 10 ], Yarrowia lipolytica [ 11 ], Hansenula polymorpha [ 12 ], Schizosaccharomyces pombe [ 13 ] and Ogataea minuta [ 14 , 15 ].…”

Section: Introductionmentioning

confidence: 99%

Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae

Geyuan

Zhang

et al. 2016

Microb Cell Fact

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BackgroundTo humanize yeast N-glycosylation pathways, genes involved in yeast specific hyper-mannosylation must be disrupted followed by the introduction of genes catalyzing the synthesis, transport, and addition of human sugars. However, deletion of these genes, for instance, OCH1, which initiates hyper-mannosylation, could cause severe defects in cell growth, morphogenesis and response to environmental challenges.ResultsIn this study, overexpression of RHO1, which encodes the Rho1p small GTPase, is confirmed to partially recover the growth defect of Saccharomyces cerevisiae Δalg3Δoch1 double mutant strain. In addition, transmission electron micrographs indicated that the cell wall structure of RHO1-expressed cells have an enhanced glucan layer and also a recovered mannoprotein layer, revealing the effect of Rho1p GTPase on cell wall biosynthesis. Similar complementation phenotypes have been confirmed by overexpression of the gene that encodes Fks2 protein, a catalytic subunit of a 1,3-β-glucan synthase. Besides the recovery of cell wall structure, the RHO1-overexpressed Δalg3Δoch1 strain also showed improved abilities in temperature tolerance, osmotic potential and drug sensitivity, which were not observed in the Δalg3Δoch1-FKS2 cells. Moreover, RHO1 overexpression could also increase N-glycan site occupancy and the amount of secreted glycoproteins.ConclusionsOverexpression of RHO1 in ‘humanized’ glycoprotein producing yeasts could significantly facilitate its future industrial applications for the production of therapeutic glycoproteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0575-7) contains supplementary material, which is available to authorized users.

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A Combined System for Engineering Glycosylation Efficiency and Glycan Structure in Saccharomyces cerevisiae

Cited by 60 publications

References 44 publications

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

Enhancing antibody folding and secretion by tailoring the Saccharomyces cerevisiae endoplasmic reticulum

Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae

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