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 YlOCH1 gene encoding a putative alpha1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro alpha1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that that YlOCH1 is a functional orthologue of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained single predominant species, the core oligosaccharide Man8GlcNAc2 (M8), whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man8-12GlcNAc2 (M8-M12). Digestion with alpha1,2- and alpha1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first alpha1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 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.
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