In vitro oligosaccharide synthesis using intact yeast cells that display glycosyltransferases at the cell surface through cell wall-anchored protein Pir.
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 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 in which KRE2 and MNN1 encode alpha-1,2-mannosyltransferase and alpha-1,3-mannosyltransferase from S. cerevisiae, respectively. The intact yeast 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 propose a simple in vitro method for oligosaccharide synthesis using the yeast intact cell as a biocatalyst.
Abe
Hiroko
H
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (aist), Aist Central 6, Tsukuba, Ibaraki 305-8566, Japan.
Shimma
Yoh-ichi
Y
Jigami
Yoshifumi
Y
D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology; D013997:Time Factors
D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005700:Galactosyltransferases