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Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production
Published on: September 20, 2016
Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis
Bo Liu1, Xin Gong, Shaohong Chang
1Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China.
Researchers engineered Kluyveromyces lactis yeast to produce therapeutic glycoproteins without non-human hypermannosylation. This modification improves protein properties for medical applications by reducing mannose residues on N-linked oligosaccharides.
Area of Science:
- Biotechnology
- Glycobiology
- Yeast Engineering
Background:
- Kluyveromyces lactis secretes glycoproteins with heterogeneous mannan residues.
- Hypermannosylation in K. lactis creates non-human glycosylation patterns, potentially impacting therapeutic protein efficacy (half-life, distribution, immunogenicity).
Purpose of the Study:
- To engineer a K. lactis strain for producing non-hypermannosylated glycoproteins.
- To reduce outer-chain mannose residues on N-linked oligosaccharides for improved therapeutic protein characteristics.
Main Methods:
- Utilized two-step homologous recombination to knockout the OCH1 and MNN1 genes in K. lactis.
- OCH1 encodes alpha1,6-mannosyltransferase; MNN1 homologue encodes a putative alpha1,3-mannosyltransferase.
Main Results:
- The Kloch1 mutant showed defective hyperglycosylation, unable to add mannose to the core oligosaccharide.
- N-linked oligosaccharides on HSA/GM-CSF in Kloch1 mutants were Man(13-14)GlcNAc(2); in Kloch1 mnn1 mutants, Man(9-11)GlcNAc(2).
- Wild-type K. lactis produced heterogeneous oligosaccharides, Man(>30)GlcNAc(2).
Conclusions:
- KlOch1p is crucial for outer-chain mannosylation of N-linked oligosaccharides in K. lactis.
- KlMnn1p contributes to outer hypermannosylation, likely as an alpha1,3-mannosyltransferase.
- Kloch1 and Kloch1 mnn1 mutants offer a foundational host for producing glycoproteins lacking outer-chain hypermannoses, suitable for therapeutic protein production.
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