Cell Surface Expression of Bacterial Esterase A by Saccharomyces cerevisiae and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response

Applied and Environmental Microbiology - Tập 72 Số 11 - Trang 7140-7147 - 2006
Frank Breinig1, Björn Diehl1, Sabrina Rau1, Christina Zimmer1, Helmut Schwab2, Manfred Schmitt1
1Angewandte Molekularbiologie, Universität des Saarlandes, D-66041 Saarbrücken, Germany
2Institut für Biotechnologie, Technische Universität Graz, A-8010 Graz, Austria

Tóm tắt

ABSTRACT Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p -nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg −1 protein for Kre1/EstA/Cwp2p and 72 mU mg −1 protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg −1 protein for Kre1/EstA/Cwp2p and 1.27 U mg −1 protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli , can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

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Applied and Environmental Microbiology

Tập 72 Số 11

7140-7147

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