Engineering of the Yeast Yarrowia lipolytica for the Production of Glycoproteins Lacking the Outer-Chain Mannose Residues of N-Glycans

Abstract

ABSTRACT In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative α-1,6-mannosyltransferase in Y. lipolytica . The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of Yl OCH1 and the lack of in vitro α-1,6-mannosyltransferase activity in the Yl och1 null mutant indicated that Yl OCH1 is a functional ortholog of S. cerevisiae OCH1 . The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yl och1 null mutant contained a single predominant species, the core oligosaccharide Man 8 GlcNAc 2 , whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man 8 GlcNAc 2 to Man 12 GlcNAc 2 . Digestion with α-1,2- and α-1,6-mannosidase of the oligosaccharides from the wild-type and Yl och1 mutant strains strongly supported the possibility that the Yl och1 mutant strain has a defect in adding the first α-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that Yl OCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica . Therefore, the Yl och1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.