Open AccessConstructing a human complex type N-linked glycosylation pathway in Kluyveromyces marxianus
Author(s) -
Ming-Hsuan Lee,
Tsui-Ling Hsu,
Jinn-Jy Lin,
YuJu Lin,
Yi-Ying Kao,
Jui-Jen Chang,
Wen-Hsiung Li
Publication year - 2020
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0233492
Subject(s) - glycosylation , kluyveromyces marxianus , n linked glycosylation , glycan , biology , gene , mannose , biochemistry , protein subunit , glycoprotein , gene knockout , saccharomyces cerevisiae
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 Man 3 GlcNAc 2 and the human complex N-glycan Man 3 GlcNAc 4 , where Man is mannose and GlcNAc is N-acetylglucosamine . This study lays a cornerstone of glycosylation engineering in K . marxianus toward producing human glycoproteins.