- Cell-Surface Expression of Aspergillus saitoi-Derived Functional α-1,2-Mannosidase on Yarrowia lipolytica for Glycan Remodeling
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Hye Yun Moon , Trinh Luu Van , Seon Ah Cheon , Jinho Choo , Jeong-Yoon Kim , Hyun Ah Kang
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J. Microbiol. 2013;51(4):506-514. Published online August 30, 2013
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DOI: https://doi.org/10.1007/s12275-013-3344-x
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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 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.
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Citations
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Maria Orfanidou, Eleftheria Panagiotidou, Antonios M. Makris, Eleni Theodosiou
Biotechnology for the Environment.2025;[Epub] CrossRef - Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications
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Biogerontology.2022; 23(3): 341. CrossRef - Hydrolytic secretome engineering in Yarrowia lipolytica for consolidated bioprocessing on polysaccharide resources: review on starch, cellulose, xylan, and inulin
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Van-Trinh Luu, Hye Yun Moon, Jee Youn Hwang, Bo-Kyu Kang, Hyun Ah Kang
Journal of Microbiology.2017; 55(8): 655. CrossRef - Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica
Rémi Dulermo, François Brunel, Thierry Dulermo, Rodrigo Ledesma-Amaro, Jérémy Vion, Marion Trassaert, Stéphane Thomas, Jean-Marc Nicaud, Christophe Leplat
Microbial Cell Factories.2017;[Epub] CrossRef -
Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering
Catherine Madzak
Applied Microbiology and Biotechnology.2015; 99(11): 4559. CrossRef - Biotechnological applications of Yarrowia lipolytica: Past, present and future
Hu-Hu Liu, Xiao-Jun Ji, He Huang
Biotechnology Advances.2015; 33(8): 1522. CrossRef - Functional characterization of extracellular chitinase encoded by the YlCTS1 gene in a dimorphic yeast Yarrowia lipolytica
Jeong-Nam Park, Chang Pyo Han, Dong-Jik Lee, Seon Ah Cheon, Hyun Ah Kang
Journal of Microbiology.2014; 52(4): 284. CrossRef - Characterization of putative glycosylphosphatidylinositol-anchoring motifs for surface display in the methylotrophic yeast Hansenula polymorpha
Seon Ah Cheon, Jinhee Jung, Jin Ho Choo, Doo-Byoung Oh, Hyun Ah Kang
Biotechnology Letters.2014; 36(10): 2085. CrossRef
- Functional Analysis of a Hansenula polymorpha MNN2-2 Homologue Encoding a Putative UDP-N-acetylglucosamine Transporter Localized in the Endoplasmic Reticulum
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Jeong-Nam Park , Jinho Choo , Hyun Ah Kang
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J. Microbiol. 2011;49(6):1012-1017. Published online December 28, 2011
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DOI: https://doi.org/10.1007/s12275-011-1520-4
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The Kluyveromyces lactis UDP-GlcNAc transporter (KlMnn2-2p) is responsible for the biosynthesis of N-glycans containing N-acetylglucosamine. A putative gene of Hansenula polymorpha encoding a KlMnn2-2p homologue, HpMNN2-2, was identified and investigated for its function. The deletion mutant strain of HpMNN2-2 (Hpmnn2-2Δ) showed increased sensitivity to geneticin, hygromycin B, and tunicamycin. However, the Hpmnn2-2Δ strain exhibited increased resistance to Calcofluor white, an inhibitor of chitin biosynthesis, along with a reduced chitin content. The localization of HpMnn2-2p at the endoplasmic reticulum-enriched membrane, different from the Golgi localization of a K. lactis homologue, further supports the involvement of HpMnn2-2p in cell wall chitin biosynthesis.
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Environmental Microbiology.2022; 24(3): 1200. CrossRef - Evaluation of Ogataea (Hansenula) polymorpha for Hyaluronic Acid Production
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The Journal of Microbiology.2012; 50(2): 341. CrossRef
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