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Constructing a human complex type N-linked glycosylation pathway in Kluyveromyces marxianus
Ming-Hsuan Lee1,2,3, Tsui-Ling Hsu4, Jinn-Jy Lin3
1Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan.
Plos One
|May 30, 2020
Summary
This study engineered Kluyveromyces marxianus yeast for producing human therapeutic glycoproteins. Researchers modified genes to reduce yeast mannosylation and introduce human N-glycan structures, achieving desired glycosylation patterns.
Area of Science:
- Biotechnology
- Molecular Biology
- Synthetic Biology
Background:
- Glycosylation significantly impacts protein stability, activity, and immunogenicity.
- Producing human therapeutic glycoproteins requires hosts with accurate glycan synthesis capabilities.
- Microbial systems offer cost-effective, rapid, and serum-free protein production with genetic tractability.
Purpose of the Study:
- To develop CRISPR/Cas9 gene editing for multiple knockouts and knockins in Kluyveromyces marxianus.
- To engineer K. marxianus for producing human N-glycan structures on therapeutic proteins.
- To overcome challenges like hyper-mannosylation in yeast expression systems.
Main Methods:
- Utilized CRISPR/Cas9 for simultaneous gene knockouts and knockins in K. marxianus.
- Knocked out genes ALG3 and OCH1 to reduce hyper-mannosylation.
- Knocked out KU70 to enhance homologous recombination efficiency.
- Knocked in MdsI, GnTI, and GnTII genes to modify N-glycan structures.
Main Results:
- Successfully generated two K. marxianus strains with engineered glycosylation pathways.
- Achieved reduced mannosylation by knocking out ALG3 and OCH1.
- Introduced human N-glycan structures by knocking in MdsI, GnTI, and GnTII.
- Produced core N-glycan Man3GlcNAc2 and human complex N-glycan Man3GlcNAc4.
Conclusions:
- Established a foundational protocol for glycosylation engineering in K. marxianus.
- Demonstrated the potential of K. marxianus as a host for producing human glycoproteins with specific glycan structures.
- Paved the way for advanced glycoengineering in yeast for biopharmaceutical applications.
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