Journal
JOURNAL OF MICROBIOLOGY
Volume 51, Issue 4, Pages 506-514Publisher
MICROBIOLOGICAL SOCIETY KOREA
DOI: 10.1007/s12275-013-3344-x
Keywords
Yarrowia lipolytica; surface display; a-1,2-mannosidase; GPI-anchor
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Funding
- Korean Ministry of Rural Development Administration [ADFC2013-PJ009586]
- CASSY fellowship from Chung-Ang University
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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 bio-catalysts. The hemiascomycetes yeast Yarrowia hpolytica has been raised as a potential host for heterologous expression of recombinant proteins. In this study, we report the expression of Aspergillus saitoi alpha-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 alpha-amylase and rice a-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 alpha-1,2-mannosidase activity assay using intact Y. lipolytica cells as the enzyme source. Furthermore, the glycoengineered Y. lipolytica Delta och1 Delta mpo1 strains displaying alpha-1,2-mannosidase were able to convert Man(8)GlcNAc(2) to Man(5)GlcNAc(2) efficiently on their cell-wall mannoproteins, demonstrating its potential used for glycoengineering in vitro or in vivo.
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