Glycobiology Advance Access originally published online on November 26, 2002
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Glycobiology, 2003, Vol. 13, No. 2 87-95
© 2003 Oxford University Press
In vitro oligosaccharide synthesis using intact yeast cells that display glycosyltransferases at the cell surface through cell wall–anchored protein Pir
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (aist), Aist Central 6, Tsukuba, Ibaraki 305-8566, Japan
Received on May 17, 2002; revised on September 13, 2002; accepted on September 14, 2002
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 to be displayed at the yeast cell surface. We first expressed the PIR1-HA-gma12+ fusion, in which gma12+ encodes 
-1,2-galactosyltransferase from the fission yeast Schizosaccharomyces pombe under the Saccharomyces cerevisiae GAPDH promoter. The -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 in which KRE2 and MNN1 encode -1,2-mannosyltransferase and -1,3-mannosyltransferase from S. cerevisiae, respectively. The intact yeast cells transformed with these two plasmids added mannoses initially with an -1,2 linkage and subsequently with an -1,3 linkage to the -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 propose a simple in vitro method for oligosaccharide synthesis using the yeast intact cell as a biocatalyst.
1 To whom correspondence should be addressed; e-mail: jigami.yoshi{at}aist.go.jp
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