초록
<P><I>Yarrowia lipolytica</I> 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 <I>in vivo</I> and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in <I>Yarrowia lipolytica</I> so that it produces Man<SUB>3</SUB>GlcNAc<SUB>2</SUB> 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 <I>ALG3</I> gene resulted in modification of proteins mainly with Man<SUB>5</SUB>GlcNAc<SUB>2</SUB> and GlcMan<SUB>5</SUB>GlcNAc<SUB>2</SUB> glycans, and to a lesser extent with Glc<SUB>2</SUB>Man<SUB>5</SUB>GlcNAc<SUB>2</SUB> glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed <I>ALG6</I>. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric <I>Apergillus niger</I> glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man<SUB>3</SUB>GlcNAc<SUB>2</SUB> structures, which are substrates for the synthesis of complex-type glycans. The final <I>Yarrowia lipolytica</I> strain produces proteins glycosylated with the trimannosyl core N-glycan (Man<SUB>3</SUB>GlcNAc<SUB>2</SUB>), 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 <I>in vivo</I> or <I>in vitro</I> modification with the appropriate glycosyltransferases. The results demonstrate the high potential of <I>Yarrowia lipolytica</I> to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.</P>