Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis.
Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding alpha1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative alpha1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The N-linked oligosaccharides assembled on a secretory glycoprotein, HSA/GM-CSF in Kloch1 mutant, contained oligosaccharide Man(13-14)GlcNAc(2), and in Kloch1 mnn1 mutant, contained oligosaccharide Man(9-11)GlcNAc(2), whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man(>30)GlcNAc(2). Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in K. lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation, most possibly encodes alpha1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production.
Liu
Bo
B
Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China.
Gong
Xin
X
Chang
Shaohong
S
Yang
Yili
Y
Song
Miao
M
Duan
Demin
D
Wang
Lina
L
Ma
Qingjun
Q
Wu
Jun
J
D004586:Electrophoresis; D005656:Fungal Proteins / Q000235:genetics*; D017353:Gene Deletion; D055786:Gene Knockout Techniques / Q000379:methods*; D017930:Genes, Reporter; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D008364:Mannosyltransferases / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D008364:Mannosyltransferases