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Glycobiology Advance Access published online on November 26, 2002
Glycobiology, doi:10.1093/glycob/cwg014
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Submitted on May 17, 2002
Revised on September 13, 2002
Accepted on September 14, 2002

© 2002 Oxford University Press

ORIGINAL ARTICLES

The in vitro oligosaccharide synthesis using yeast intact cells that display glycosyltransferases at the cell surface through cell wall-anchored protein Pir

Hiroko Abe 1, Yoh-ichi Shimma 1, Yoshifumi Jigami 1*

1 Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), AIST Central 6, Tsukuba, Ibaraki 305-8566, Japan

* To whom correspondence should be addressed. E-mail: jigami.yoshi{at}aist.go.jp.

Abstract

A glycosyltransferase was fused to the yeast cell wall protein Pir, which forms the Pir1-4 protein family and is incorporated into the cell wall by an unknown linkage, so as to be displayed at the yeast cell surface. We first expressed the PIR1-HA-gma12+ fusion, in which gma12+ encodes {alpha}-1, 2-galactosyltransferase from the fission yeast Schizosaccharomyces pombe, under the Saccharomyces cerevisiae GAPDH promoter. The {alpha}-1, 2-galactosyltransferase activity was detected at the surface of the intact cells that produce Pir1-HA-Gma12 fusion. To further demonstrate sequential oligosaccharide synthesis, two plasmids containing PIR1-HA-KRE2 and PIR2-FLAG-MNN1 fusion genes were constructed, where KRE2 and MNN1 encode {alpha}-1, 2-mannosyltransferase and {alpha}-1, 3-mannosyltransferasefrom S. cerevisiae, respectively. The yeast intact cells transformed with these two plasmids added mannoses initially with an {alpha}-1, 2-linkage and subsequently with an {alpha}-1, 3-linkage to the {alpha}-1, 2-mannobiose acceptor in the presence of a GDP-mannose donor, demonstrating that Pir1 and Pir2 can be used as anchors to simultaneously immobilize several glycosyltransferases at the yeast cell surface. Based on the high acceptor specificity of glycosyltransferases, we have proposed a simple in vitro method for oligosaccharide synthesis using the yeast intact cell as a biocatalyst.


Key words; glycosyltransferase / intact cell enzyme / oligosaccharide synthesis / Pir / S. cerevisiae
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