Abstract
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.
Highlights
Proper protein glycosylation is important because glycosylation affects the stability, biological activity, and immunogenicity of a protein [1]
We propose first to delete the ALG3 (α-1,3- mannosyltransferase) gene because it is responsible for the first mannosylation step in the endoplasmic reticulum (ER) and its deletion will prevent the conversion of Man5GlcNAc2 to Man6GlcNAc2 (S1 Fig)
The proportions of Man7GlcNAc2 and Man8GlcNAc2 were higher in S. cerevisiae than in K. marxianus 4G5, the proportion of Man8GlcNAc2 was much lower in S. cerevisiae
Summary
Proper protein glycosylation is important because glycosylation affects the stability, biological activity, and immunogenicity of a protein [1]. Many clinically approved therapeutic proteins are glycosylated. Efforts to engineer glycosylation pathways have been made in a wide variety of cell types including bacterial, fungal, and mammalian cells [2, 3]. Mammalian cell lines are usually preferred because they produce complex glycans similar to those in humans. The requirements for complex nutrients in culture media and the special.
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