Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man8GlcNAc2 and Man5GlcNAc2

Microbial Cell Factories

Research

Nico Callewaert¹  Annelies Van Hecke²  Isabelle Dewerte²  Karen De Pourcq²  Wouter Vervecken³  Albena Valevska³ 

[1] Department for Molecular Biomedical Research, Unit for Medical Biotechnology,VIB, Technologiepark 927, B-9052, Ghent, Belgium;Department of Biochemistry and Microbiology, Ghent University, K.L.-Ledeganckstraat 35, B-9000, Ghent, Belgium;Department for Molecular Biomedical Research, Unit for Medical Biotechnology,VIB, Technologiepark 927, B-9052, Ghent, Belgium;Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, B-9052, Ghent, Belgium;Department for Molecular Biomedical Research, Unit for Medical Biotechnology,VIB, Technologiepark 927, B-9052, Ghent, Belgium;Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, B-9052, Ghent, Belgium;Oxyrane Belgium, Technologiepark 3, B-9052, Ghent, Belgium;

关键词: Yarrowia Lipolytica;    Mannose Residue;    Lipolytica Strain;    Swiss Prot Entry;    Cerezyme;   

DOI  :  10.1186/1475-2859-11-53

 received in 2011-04-26, accepted in 2012-03-21,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundProtein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation.ResultsWe engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases Yl Och1p and Yl Mnn9p; the former inactivation yielded a strain producing homogeneous Man8GlcNAc2 glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that Yl Och1p and Yl Mnn9p, despite some initial uncertainty about their function, are most likely the α-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained α-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man5GlcNAc2. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme.ConclusionsWe generated a Y. lipolytica expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man8GlcNAc2 or Man5GlcNAc2 N-glycans. This platform expands the utility of Y. lipolytica as a heterologous expression host and makes it possible to produce glycoproteins with homogeneously glycosylated N-glycans of the human high-mannose-type, which greatly broadens the application scope of these glycoproteins.

【 授权许可】

Unknown   
© De Pourcq et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.