title: Abolishment of N-glycan mannosylphosphorylation in glyco-engineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes.
abstract: Mannosylphosphorylated glycans are found only in fungi, including yeast, and the elimination of mannosylphosphates from glycans is a prerequisite for yeast glyco-engineering to produce human-compatible glycoproteins. In Saccharomyces cerevisiae, MNN4 and MNN6 genes are known to play roles in mannosylphosphorylation, but disruption of these genes does not completely remove the mannosylphosphates in N-glycans. This study was performed to find unknown key gene(s) involved in N-glycan mannosylphosphorylation in S. cerevisiae. For this purpose, each of one MNN4 and five MNN6 homologous genes were deleted from the och1Δmnn1Δmnn4Δmnn6Δ strain, which lacks yeast-specific hyper-mannosylation and the immunogenic α(1,3)-mannose structure. N-glycan profile analysis of cell wall mannoproteins and a secretory recombinant protein produced in mutants showed that the MNN14 gene, an MNN4 paralog with unknown function, is essential for N-glycan mannosylphosphorylation. Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan mannosylphosphorylation. Our results suggest that the S. cerevisiae och1Δmnn1Δmnn4Δmnn14Δ strain, in which all yeast-specific N-glycan structures including mannosylphosphorylation are abolished, may have promise as a useful platform for glyco-engineering to produce therapeutic glycoproteins with human-compatible N-glycans.
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea.|Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon, South Korea.
authors.forename: Yeong Hun
authors.initials: YH
authors.lastname: Kim
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea.
authors.forename: Ji-Yeon
authors.initials: JY
authors.lastname: Kang
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea.
authors.forename: Jin Young
authors.initials: JY
authors.lastname: Gil
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea.
authors.forename: Sang-Yoon
authors.initials: SY
authors.lastname: Kim
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea.
authors.forename: Keun Koo
authors.initials: KK
authors.lastname: Shin
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, South Korea.
authors.forename: Hyun Ah
authors.initials: HA
authors.lastname: Kang
authors.affiliation: Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon, South Korea.
authors.forename: Jeong-Yoon
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea. oskwon@kribb.re.kr.|Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, South Korea. oskwon@kribb.re.kr.
authors.forename: Ohsuk
authors.initials: O
authors.lastname: Kwon
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea. dboh@kribb.re.kr.|Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, South Korea. dboh@kribb.re.kr.
authors.forename: Doo-Byoung
authors.initials: DB
authors.lastname: Oh
mesh_terms: D002473:Cell Wall / Q000378:metabolism; D006801:Humans; D008358:Mannose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism; D008364:Mannosyltransferases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D060847:Metabolic Engineering; D010766:Phosphorylation; D011134:Polysaccharides / Q000378:metabolism*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C105147:MNN4 protein, S cerevisiae; D008360:Mannosephosphates; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C525358:MNN6 protein, S cerevisiae; D008364:Mannosyltransferases; D008358:Mannose
keywords: MNN14; MNN4; MNN6; Mannosylphosphate; N-glycan