Title |
Cell-Surface Expression of Aspergillus saitoi-Derived Functional α-1,2-Mannosidase on Yarrowia lipolytica for Glycan Remodeling |
Author |
Hye Yun Moon1, Trinh Luu Van1, Seon Ah Cheon1, Jinho Choo1, Jeong-Yoon Kim2, and Hyun Ah Kang1* |
Address |
1Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea, 2Department of Microbiology, School of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea |
Bibliography |
Journal of Microbiology, 51(4),506-514, 2013,
|
DOI |
10.1007/s12275-013-3344-x
|
Key Words |
Yarrowia lipolytica, surface display, a-1,2-mannosidase, GPI-anchor |
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. |