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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 α-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.

Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core