title: Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris.
abstract: 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 homogeneous 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.
authors.affiliation: Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA.
authors.forename: Byung-Kwon
authors.initials: BK
authors.lastname: Choi
authors.forename: Piotr
authors.initials: P
authors.lastname: Bobrowicz
authors.forename: Robert C
authors.initials: RC
authors.lastname: Davidson
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hamilton
authors.forename: David H
authors.initials: DH
authors.lastname: Kung
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Robert G
authors.initials: RG
authors.lastname: Miele
authors.forename: Juergen H
authors.initials: JH
authors.lastname: Nett
authors.forename: Stefan
authors.initials: S
authors.lastname: Wildt
authors.forename: Tillman U
authors.initials: TU
authors.lastname: Gerngross
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D008364:Mannosyltransferases / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D008364:Mannosyltransferases