Abstract
A Pichia pastoris cell-surface display system was constructed using the Sed1 anchor system that has been developed in Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was used as the model protein and was fused to an anchor that consisted of 338 amino acids of Sed1. The resulting fusion protein CALBSed1 was expressed under the control of the alcohol oxidase 1 promoter (pAOX1). Immunofluorescence microscopy of immunolabeled Pichia pastoris revealed that CALB was displayed on the cell surface. Western blot analysis showed that the fusion protein CALBSed1 was attached covalently to the cell wall and was highly glycosylated. The hydrolytic activity of the displayed CALB was more than 220 U/g dry cells after 120 h of culture. The displayed protein also exhibited a higher degree of thermostability than free CALB.
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Bony M, Thines-Sempoux D, Barre P, Blondin B (1997) Localization and cell surface anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p. J Bacteriol 179:4929–4936
Fransson AB, Xu Y, Leijondahl K, Backvall JE (2006) Enzymatic resolution, desymmetrization, and dynamic kinetic asymmetric transformation of 1,3-cycloalkanediols. J Org Chem 71:6309–6316
Han S-Y, Pan Z-Y, Huang D-F, Ueda M, Wang X-N, Lin Y (2009) Highly efficient synthesis of ethyl hexanoate catalyzed by CALB-displaying Saccharomyces cerevisiae whole-cells in non-aqueous phase. J Mol Catal B 59:168–172
Jaeger KE, Reetz TM (1998) Microbial lipases form versatile tools for biotechnology. Trends Biotechnol 16:396–403
Kobori H, Sato M, Osumi M (1992) Relationship of actin organization to growth in the two forms of the dimorphic yeast Candida tropicalis. Protoplasma 167:193–204
Kondo A, Ueda M (2004) Yeast cell-surface display-applications of molecular display. Appl Microbiol Biotechnol 64:28–40
Larios A, Garcia HS, Oliart RM, Valerio-Alfaro G (2004) Synthesis of flavor and fragrance esters using Candida antarctica lipase. Appl Microbiol Biotechnol 65:373–376
Liu W, Zhao H, Jia B, Xu L, Yan Y (2009) Surface display of active lipase in Saccharomyces cerevisiae using Cwp2 as an anchor protein. Biotechnol Lett. doi:10.1007/s10529-009-0138-7
Lou WY, Zong MH (2006) Efficient kinetic resolution of (R,S)-1-trimethylsilylethanol via lipase-mediated enantioselective acylation in ionic liquids. Chirality 18:814–821
Matsumoto T, Fukuda H, Ueda M, Tanaka A, Kondo A (2002) Construction of yeast strains with high cell surface lipase activity by using novel display systems based on the Flo1p flocculation functional domain. Appl Environ Microbiol 68:4517–4522
Modi MK, Reddy JR, Rao BV, Prasad RB (2006) Lipase-mediated transformation of vegetable oils into biodiesel using propan-2-ol as acyl acceptor. Biotechnol Lett 28:637–640
Tanino T, Fukuda H, Kondo A (2004) Construction of a Pichia pastoris cell-surface display system using Flo1p anchor system. Biotechnol Prog 22:989–993
Tanino T, Ohno T, Aoki T, Fukuda H, Kondo A (2007) Development of yeast cells displaying Candida antarctica lipase B and their application to ester synthesis reaction. Appl Microbiol Biotechnol 75:1319–1325
Torres CF, Hill CG Jr, Otero C (2004) Lipase-catalyzed ethanolysis of borage oil: a kinetic study. Biotechnol Prog 20:756–763
Van der Vaart JM, te Biesebeke R, Chapman JW, Toschka FM, Verrips CT (1997) Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins. Appl Environ Microbiol 63:615–620
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This work was supported by grants from the National Natural Science Foundation of China (No: 30670053) and the Ministry of Science and Technology of the People’s Republic of China (National “863” Project No. 2006AA020203).
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Su, Gd., Zhang, X. & Lin, Y. Surface display of active lipase in Pichia pastoris using Sed1 as an anchor protein. Biotechnol Lett 32, 1131–1136 (2010). https://doi.org/10.1007/s10529-010-0270-4
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DOI: https://doi.org/10.1007/s10529-010-0270-4