Affordable Access

Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris

  • Byung-Kwon Choi
  • Piotr Bobrowicz
  • Robert C. Davidson
  • Stephen R. Hamilton
  • David H. Kung
  • Huijuan Li
  • Robert G. Miele
  • Juergen H. Nett
  • Stefan Wildt
  • Tillman U. Gerngross
The National Academy of Sciences
Publication Date
Apr 17, 2003
  • Biology
  • Engineering
  • Mathematics


The secretory pathway of Pichia pastoris was genetically re-engineered to perform sequential glycosylation reactions that mimic early processing of N-glycans in humans and other higher mammals. After eliminating nonhuman glycosylation by deleting the initiating α-1,6-mannosyltransferase gene from P. pastoris, several combinatorial genetic libraries were constructed to localize active α-1,2-mannosidase and human β-1,2-N-acetylglucosaminyltransferase I (GnTI) in the secretory pathway. First, >32 N-terminal leader sequences of fungal type II membrane proteins were cloned to generate a leader library. Two additional libraries encoding catalytic domains of α-1,2-mannosidases and GnTI from mammals, insects, amphibians, worms, and fungi were cloned to generate catalytic domain libraries. In-frame fusions of the respective leader and catalytic domain libraries resulted in several hundred chimeric fusions of fungal targeting domains and catalytic domains. Although the majority of strains transformed with the mannosidase/leader library displayed only modest in vivo [i.e., low levels of mannose (Man)5-(GlcNAc)2] activity, we were able to isolate several yeast strains that produce almost homogenous N-glycans of the (Man)5-(GlcNAc)2 type. Transformation of these strains with a UDP-GlcNAc transporter and screening of a GnTI leader fusion library allowed for the isolation of strains that produce GlcNAc-(Man)5-(GlcNAc)2 in high yield. Recombinant expression of a human reporter protein in these engineered strains led to the formation of a glycoprotein with GlcNAc-(Man)5-(GlcNAc)2 as the primary N-glycan. Here we report a yeast able to synthesize hybrid glycans in high yield and open the door for engineering yeast to perform complex human-like glycosylation.

Report this publication


Seen <100 times