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

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 ␣-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro ␣-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 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 Yloch1 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 ␣-1,2-and ␣-1,6-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 ␣-1,6-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.Yeast can secrete a variety of proteins in much the same way that mammalian cells do. The presence of yeast-specific outerchain mannosylation, however, has been a primary hindrance to the exploitation of yeast for therapeutic glycoprotein production, because glycoproteins decorated with yeast-specific glycans are immunogenic and show poor pharmacokinetic properties in humans (1, 24). In the budding yeast Saccharomyces cerevisiae, the N-linked oligosaccharides assembled on glycoproteins include hypermannose structures with outer chains that may contain up to 200 mannose units (6). Elongation of the outer chain is initiated by the Och1 protein, which adds the first ␣-1,6-linked mannose to the core N-linked oligosaccharides upon their arrival in the Golgi apparatus in S. cerevisiae (17). Following the addition of the first ␣-1,6-mannose by Och1p, the core oligosaccharide is elongated by additional ␣-1,6-mannosyltransferases, Mnn9p and Van1p, which extend the ␣-1,6-linked polymannose backbone, and the core oligosaccharide is further branched by the addition of ␣-1,2-and ␣-1,3-linked mannoses (5, 8). Other yeast species, including Pichia pastoris, Hansenula polymorpha, and Schizosaccharomyces pombe, also use the Och1 protein to extend the mannose outer chain of N-glycans (14, 24, 26, 28). Therefore, the elimination of the Och1 protein was performed to block the yeast-specific outer-chain mannosylation, followed by further engineering of yeast N-glycosylation pathways for the production of glycoproteins with hum...

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“…This process is initiated in Golgi α-1,6-mannosyltransferase (Och1p), adding a first α-1,6-linked mannose to the core oligosaccharide Man8GlcNAc2 [23]. After the addition of α-1,6-linked mannose by Och1p, the linear backbone of the outer oligosaccharide chain is extended by additional α-1,6-mannosyl transferases encoded by gene MNN9 and further branched by the addition of α-1,3-linked mannoses encoded by gene MNN1 [36]. The terminal α-1,3-linked mannose units that are added to the outer oligosaccharide chain are particularly immunogenic, and the serum collected from rabbits injected with whole yeast cells contains a large amount of antibodies that recognize this α-1,3-linked mannose epitope [9].…”

Essential Role of Yl MPO1 , a Novel Yarrowia lipolytica Homologue of Saccharomyces cerevisiae MNN4 , in Mannosylphosphorylation of N- and O-Linked Glycans

“…The oligonucleotides used for this study are listed in Table S1 in the supplemental material. The Y. lipolytica target genes were disrupted via the PCR-based gene disruption method described by Song et al (38). To disrupt the YlMPO1 gene, primer set YlMPO1-NF and YlMPO1-NR and primer set YlMPO1-CF and YlMPO1-CR were designed to amplify the 5Ј and 3Ј flanking regions (571 bp for the 5Ј region and 477 bp for the 3Ј region) of the YlMPO1 gene, respectively.…”

“…The PCR fragments amplified by Taq polymerase (Bioneer, Daejeon, South Korea) were fused via a linker sequence (AGATCTACGGATCCATGG) using the YlMPO1-NF and YlMPO1-CR primers, and the product (1,066 bp) was subcloned into the pDrive vector (Qiagen, Hilden, Germany). The BamHI/BglIItreated TcR-YlURA3-TcR cassette from pYIUB (38) was inserted into the BglII site of the linker sequence of the fused PCR product. This disruption cassette was linearized via digestion with BamHI/HindIII and used to transform the Y. lipolytica SMS397A strain.…”

“…Phenotypic analysis was conducted on YPD solid medium containing 10 g/ml calcofluor white (CFW; Sigma), 50 g/ml Congo red (CR; Sigma), 7.5 mM sodium orthovanadate (Van; Sigma), or 40 g/ml hygromycin B (Hyg B; Sigma). YPDm medium (38) was used for the expression of Trichoderma reesei endoglucanase I (EGI).…”

“…Recently, we reported that the N-linked glycans of Y. lipolytica are composed of neutral and acidic sugars lacking a terminal ␣1,3-linked mannose (38). The neutral sugars of Nlinked glycans are high-mannose oligosaccharides, principally Man 7-12 GlcNAc 2 , and the acidic sugars of N-linked glycans are composed of monomannosylphosphorylated Man 7-9 GlcNAc 2 sugars.…”