Constructing a human complex type N-linked glycosylation pathway in Kluyveromyces marxianus
| Autor: | Jinn-Jy Lin, Yu-Ju Lin, Ming-Hsuan Lee, Yi-Ying Kao, Wen-Hsiung Li, Tsui-Ling Hsu, Jui-Jen Chang |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Glycosylation
Glycobiology Mannose Yeast and Fungal Models Artificial Gene Amplification and Extension Mannosyltransferases Biochemistry Polymerase Chain Reaction chemistry.chemical_compound Gene Knockout Techniques Kluyveromyces Guide RNA N-linked glycosylation Gene Knock-In Techniques Post-Translational Modification chemistry.chemical_classification 0303 health sciences Multidisciplinary biology Chemistry Organic Compounds Eukaryota Proteases Recombinant Proteins Enzymes Nucleic acids Metabolic Engineering Experimental Organism Systems Mannosylation Physical Sciences Medicine Saccharomyces Cerevisiae Biotechnology Research Article Science Saccharomyces cerevisiae Genes Fungal Carbohydrates N-Acetylglucosaminyltransferases Research and Analysis Methods 03 medical and health sciences Saccharomyces Model Organisms Kluyveromyces marxianus Polysaccharides Mannosidases Genetics Humans Molecular Biology Techniques Molecular Biology 030304 developmental biology Glycoproteins 030306 microbiology Organic Chemistry Organisms Fungi Chemical Compounds Biology and Life Sciences Proteins DNA biology.organism_classification Yeast carbohydrates (lipids) Animal Studies Enzymology RNA CRISPR-Cas Systems Homologous recombination Glycoprotein |
| Zdroj: | PLoS ONE PLoS ONE, Vol 15, Iss 5, p e0233492 (2020) |
| ISSN: | 1932-6203 |
| Popis: | Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (β-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins. |
| Databáze: | OpenAIRE |
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