Abolishment of N-glycan mannosylphosphorylation in glyco-engineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes

作者：Kim Yeong Hun; Kang Ji Yeon; Gil Jin Young; Kim Sang Yoon; Shin Keun Koo; Kang Hyun Ah; Kim Jeong Yoon; Kwon Ohsuk; Oh Doo Byoung

来源：Applied Microbiology and Biotechnology, 2017, 101(7): 2979-2989.

DOI：10.1007/s00253-017-8101-3

• Mannosylphosphate
• MNN4
• MNN6
• MNN14
• N-glycan

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 och1 Delta mnn1 Delta mnn4 Delta mnn6 Delta strain, which lacks yeast-specific hyper-mannosylation and the immunogenic alpha(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 och1 Delta mnn1 Delta mnn4 Delta mnn14 Delta 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.

• 出版日期2017-4