title: Cell-surface expression of Aspergillus saitoi-derived functional α-1,2-mannosidase on Yarrowia lipolytica for glycan remodeling.
abstract: Expression of proteins on the surface of yeast has a wide range of applications, such as development of live vaccines, screening of antibody libraries, and use as whole-cell biocatalysts. The hemiascomycetes yeast Yarrowia lipolytica has been raised as a potential host for heterologous expression of recombinant proteins. In this study, we report the expression of Aspergillus saitoi α-1,2-mannosidase, encoded by the msdS gene, on the cell surface of Y. lipolytica. As the first step to achieve the secretory expression of msdS protein, four different signal sequences-derived from the endogenous Y. lipolytica Lip2 and Xpr2 prepro regions and the heterologous A. niger α-amylase and rice α-amylase signal sequences-were analyzed for their secretion efficiency. It was shown that the YlLip2 prepro sequence was most efficient in directing the secretory expression of msdS in fully N-glycosylated forms. The surface display of msdS was subsequently directed by fusing GPI anchoring motifs derived from Y. lipolytica cell wall proteins, YlCwp1p and YlYwp1p, respectively, to the C-terminus of the Lip2 prepro-msdS protein. The expression of actively functional msdS protein on the cell surface was confirmed by western blot, flow cytometry analysis, along with the α-1,2-mannosidase activity assay using intact Y. lipolytica cells as the enzyme source. Furthermore, the glycoengineered Y. lipolytica Δoch1Δmpo1 strains displaying α-1,2-mannosidase were able to convert Man8GlcNAc2 to Man5GlcNAc2 efficiently on their cell-wall mannoproteins, demonstrating its potential used for glycoengineering in vitro or in vivo.
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea.
authors.forename: Hye Yun
authors.initials: HY
authors.lastname: Moon
authors.forename: Trinh Luu
authors.initials: TL
authors.lastname: Van
authors.forename: Seon Ah
authors.initials: SA
authors.lastname: Cheon
authors.forename: Jinho
authors.initials: J
authors.lastname: Choo
authors.forename: Jeong-Yoon
authors.initials: JY
authors.lastname: Kim
authors.forename: Hyun Ah
authors.initials: HA
authors.lastname: Kang
mesh_terms: D001230:Aspergillus / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004789:Enzyme Activation; D005434:Flow Cytometry; D015966:Gene Expression Regulation, Fungal; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D009154:Mutation; D011134:Polysaccharides / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D025062:Yarrowia / Q000235:genetics / Q000378:metabolism*; D043323:alpha-Mannosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D043323:alpha-Mannosidase