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Engineering Yarrowia lipolytica to produce glycoproteins homogeneously modified with the universal Man(3)GlcNAc(2) N-glycan core

Karen De Pourcq UGent, Petra Tiels UGent, Annelies Van Hecke UGent, Steven Geysens, Wouter Vervecken and Nico Callewaert UGent (2012) PLOS ONE. 7(6).
abstract
Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as "generally recognized as safe." Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man(3)GlcNAc(2) structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man(5)GlcNAc(2) and GlcMan(5)GlcNAc(2) glycans, and to a lesser extent with Glc(2)Man(5)GlcNAc(2) glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an alpha-1,2-mannosidase to obtain Man(3)GlcNAc(2) structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
ASPARAGINE-LINKED OLIGOSACCHARIDES, II BETA-SUBUNIT, SACCHAROMYCES-CEREVISIAE MUTANT, RETICULUM GLUCOSIDASE-II, MANNOSE-TYPE GLYCANS, ENDOPLASMIC-RETICULUM, PICHIA-PASTORIS, ALG3 GENE, HETERODIMERIC STRUCTURE, SUBSTRATE-SPECIFICITY
journal title
PLOS ONE
PLoS One
volume
7
issue
6
article_number
e39976
pages
12 pages
Web of Science type
Article
Web of Science id
000305892100160
JCR category
MULTIDISCIPLINARY SCIENCES
JCR impact factor
3.73 (2012)
JCR rank
7/56 (2012)
JCR quartile
1 (2012)
ISSN
1932-6203
DOI
10.1371/journal.pone.0039976
language
English
UGent publication?
yes
classification
A1
copyright statement
I have retained and own the full copyright for this publication
id
2968147
handle
http://hdl.handle.net/1854/LU-2968147
date created
2012-08-06 12:42:48
date last changed
2012-08-10 09:26:02
@article{2968147,
  abstract     = {Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as {\textacutedbl}generally recognized as safe.{\textacutedbl} Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man(3)GlcNAc(2) structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man(5)GlcNAc(2) and GlcMan(5)GlcNAc(2) glycans, and to a lesser extent with Glc(2)Man(5)GlcNAc(2) glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an alpha-1,2-mannosidase to obtain Man(3)GlcNAc(2) structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.},
  articleno    = {e39976},
  author       = {De Pourcq, Karen and Tiels, Petra and Van Hecke, Annelies and Geysens, Steven and Vervecken, Wouter and Callewaert, Nico},
  issn         = {1932-6203},
  journal      = {PLOS ONE},
  keyword      = {ASPARAGINE-LINKED OLIGOSACCHARIDES,II BETA-SUBUNIT,SACCHAROMYCES-CEREVISIAE MUTANT,RETICULUM GLUCOSIDASE-II,MANNOSE-TYPE GLYCANS,ENDOPLASMIC-RETICULUM,PICHIA-PASTORIS,ALG3 GENE,HETERODIMERIC STRUCTURE,SUBSTRATE-SPECIFICITY},
  language     = {eng},
  number       = {6},
  pages        = {12},
  title        = {Engineering Yarrowia lipolytica to produce glycoproteins homogeneously modified with the universal Man(3)GlcNAc(2) N-glycan core},
  url          = {http://dx.doi.org/10.1371/journal.pone.0039976},
  volume       = {7},
  year         = {2012},
}

Chicago
De Pourcq, Karen, Petra Tiels, Annelies Van Hecke, Steven Geysens, Wouter Vervecken, and Nico Callewaert. 2012. “Engineering Yarrowia Lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man(3)GlcNAc(2) N-glycan Core.” Plos One 7 (6).
APA
De Pourcq, Karen, Tiels, P., Van Hecke, A., Geysens, S., Vervecken, W., & Callewaert, N. (2012). Engineering Yarrowia lipolytica to produce glycoproteins homogeneously modified with the universal Man(3)GlcNAc(2) N-glycan core. PLOS ONE, 7(6).
Vancouver
1.
De Pourcq K, Tiels P, Van Hecke A, Geysens S, Vervecken W, Callewaert N. Engineering Yarrowia lipolytica to produce glycoproteins homogeneously modified with the universal Man(3)GlcNAc(2) N-glycan core. PLOS ONE. 2012;7(6).
MLA
De Pourcq, Karen, Petra Tiels, Annelies Van Hecke, et al. “Engineering Yarrowia Lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man(3)GlcNAc(2) N-glycan Core.” PLOS ONE 7.6 (2012): n. pag. Print.