Yeast cells harboring human alpha-1,3-fucosyltransferase at the cell surface engineered using Pir, a cell wall-anchored protein.
Human alpha-1,3-fucosyltansferase (FucT) encoded by the FUT6 gene was displayed at the cell surface of yeast cells engineered using the yeast cell wall protein Pir1 or Pir2, and the FucT activity was detected at the surface of cells producing the Pir1-HA-FUT6 or Pir2-FLAG-FUT6 fusion proteins. To obtain higher activity, we engineered the host yeast cells in which endogenous PIR genes of the PIR1-4 gene family were disrupted. Among the disruptants, the pir1Delta pir2Delta pir3Delta strain with the PIR1-HA-FUT6 fusion gene showed the highest FucT activity, which was about three-fold higher than that of the wild-type strain. Furthermore, the co-expression of both the Pir1-HA-FUT6 and the Pir2-FLAG-FUT6 fusions showed an approximately 1.5-fold higher activity than that in the cell wall displaying Pir1-HA-FUT6 alone. The present method was thus effective for producing yeast cells that can easily synthesize various oligosaccharides, such as Le(x) and sLe(x), using Pir-glycosyltransferase fusions in combination with the deletion of endogenous PIR genes.
Abe
Hiroko
H
Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), AIST Central 6, Tsukuba, Ibaraki 305-8566, Japan.
Ohba
Mika
M
Shimma
Yoh-ichi
Y
Jigami
Yoshifumi
Y
D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D017931:DNA Primers; D005647:Fucosyltransferases / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase