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Applied and Environmental Microbiology, September 2002, p. 4517-4522, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4517-4522.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Construction of Yeast Strains with High Cell Surface Lipase Activity by Using Novel Display Systems Based on the Flo1p Flocculation Functional Domain

Takeshi Matsumoto,1 Hideki Fukuda,1 Mitsuyoshi Ueda,2 Atsuo Tanaka,2 and Akihiko Kondo3*

Division of Molecular Science, Graduate School of Science and Technology,1 Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, Nada-ku, Kobe 657-8501,3 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan2

Received 18 March 2002/ Accepted 11 June 2002

We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3' region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5' upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.

* Corresponding author. Mailing address: Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan. Phone: 81-78-803-6196. Fax: 81-78-803-6206. E-mail: kondo{at}cx.kobe-u.ac.jp.

Applied and Environmental Microbiology, September 2002, p. 4517-4522, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4517-4522.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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