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Published online on April 17, 2003, 10.1073/pnas.0931263100
PNAS | April 29, 2003 | vol. 100 | no. 9 | 5022-5027


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Applied Biological Sciences
 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*, and Tillman U. Gerngrossdagger

Thayer School of Engineering, Dartmouth College, Hanover, NH 03755

Communicated by Phillips W. RobbinsDagger , Boston Medical Center, Boston, MA, March 4, 2003 (received for review December 5, 2002)

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 alpha -1,6-mannosyltransferase gene from P. pastoris, several combinatorial genetic libraries were constructed to localize active alpha -1,2-mannosidase and human beta -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 alpha -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.

* Present address: GlycoFi, Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766.

dagger To whom correspondence should be addressed at: Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, NH 03755. E-mail: tillman.gerngross{at}dartmouth.edu.

Dagger The member who communicated this paper is a Science Advisor for GlycoFi, Inc.

www.pnas.org/cgi/doi/10.1073/pnas.0931263100
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