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

• Main Authors: De Pourcq Karen, Vervecken Wouter, Dewerte Isabelle, Valevska Albena, Van Hecke Annelies, Callewaert Nico
• Format: Article
• Language: English
• Published: BMC 2012-05-01
• Series: Microbial Cell Factories
• Online Access: http://www.microbialcellfactories.com/content/11/1/53

<p>Abstract</p> <p>Background</p> <p>Protein-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. <it>Yarrowia lipolytica</it> 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 <it>Y. lipolytica</it> to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation.</p> <p>Results</p> <p>We engineered <it>Y. lipolytica</it> to produce homogeneous human-type terminal-mannose glycosylated proteins, <it>i.e.</it> glycosylated with Man₈GlcNAc₂ or Man₅GlcNAc₂. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases <it>Yl</it>Och1p and <it>Yl</it>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 <it>Yl</it>Och1p and <it>Yl</it>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.</p> <p>Conclusions</p> <p>We generated a <it>Y. lipolytica</it> 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 <it>Y. lipolytica</it> 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.</p>