N-Linked Glycoengineering Service in Yeast

N-Glycosylation in Yeast and Mammals

Yeast and other mammalian cells share similar initial biosynthetic pathways for crafting N-glycans. Briefly, Glc₃Man₉GlcNAc₂, a lipid-linked oligosaccharide (LLO), is synthesized within the endoplasmic reticulum (ER) through the concerted action of various glycosyltransferases. This LLO is subsequently attached to asparagine residues on the emerging polypeptide chain and trimmed down to Man₈GlcNAc₂. The newly formed glycoconjugate is then transported to the Golgi apparatus. Further glycan processing in the Golgi differs between yeast and mammalian cells. Mammalian cells employ α-mannosidases to further trim the Man₈GlcNAc₂ N-glycan, yielding a substrate that allows for the creation of hybrid- and complex-type N-glycans via glycosyltransferases. In contrast, yeast cells elongate the existing high mannose structure by adding extra mannose sugars, ultimately resulting in the production of hyper-mannosylated glycans.

Fig.1 N-glycan processing in yeast and mammalian cells.¹

N-Linked Glycoengineering Services in Yeast at Creative Biolabs

With a wealth of experience in Cell Line Glycoengineering Services, Creative Biolabs has implemented multiple glycoengineering strategies and genetic techniques, such as Knockdown, Knockout, and Transient Overexpression, to enhance the yeast N-glycosylation pathway for the synthesis of recombinant glycoproteins and therapeutic proteins with uniform and humanized glycans. After eliminating undesirable yeast-specific glycan structures, we focus on humanizing the N-linked glycosylation pathway in yeast. This includes introducing the necessary glycosidases and glycosyltransferases to enable the production of hybrid- and complex-type glycans, closely resembling human-type glycosylation patterns.

Removal of yeast-specific N-glycans

Elimination of yeast glycosyltransferase such as Och1p, MNN1.
Knockout of Bmt2p for β-Mannose depletion.
Intervention in the LLO assembly.
Overexpression of Endo-β-N-Acetylglucosaminidases (ENGases).

Humanization of the N-glycosylation pathway

Overexpression of human N-acetylglucosaminyltransferases, such as GnT-I, GnT-II.
Overexpression of β-1,4-galactosyltransferase (GalT) in Golgi.
Introduction of sialyltransferase (ST) for terminal sialylation.
Improvement of N-glycan site occupancy.

Features of Our Services

Multi-targeted and optional glycoengineering strategies
Highly efficient and precise knockin or knockout of genes
High-quality glycoprotein production with fully humanized N-glycans
Customized glycoengineering tailored to specific needs

Published Data

Technology: N-linked glycoengineering in yeast

Journal: Nature Protocols

IF: 17.021

Published: 2009

Results: A protocol for modifying the N-glycosylation pathway in Pichia pastoris has been developed, which involves the disruption of the endogenous glycosyltransferase gene and the introduction of heterologous glycosylation.

Fig.2 N-linked glycoengineering strategy in Pichia pastoris.²

Scientists at Creative Biolabs have amassed a wealth of expertise in the field of glycoengineering in yeast. We are committed to offering a full spectrum of customized glycoengineering services, tailored to meet your unique needs. If you have any special requests related to glycoengineering, we encourage you to contact us without hesitation.

References

Laukens, Bram, et al. "Engineering yeast for producing human glycoproteins: where are we now?" Future Microbiology 10.1 (2015): 21-34.
Jacobs, Pieter P., et al. "Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology." Nature Protocols 4.1 (2009): 58-70.

For Research Use Only.

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