The ␣-1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 (ScOCH1) is responsible for the outer chain initiation of N-linked oligosaccharides. To identify the genes involved in the first step of outer chain biosynthesis in the methylotrophic yeast Hansenula polymorpha, we undertook the functional analysis of three H. polymorpha genes, HpHOC1, HpOCH1, and HpOCR1, that belong to the OCH1 family containing seven members with significant sequence identities to ScOCH1. The deletions of these H. polymorpha genes individually resulted in several phenotypes suggestive of cell wall defects. Whereas the deletion of HpHOC1 (Hphoc1⌬) did not generate any detectable changes in N-glycosylation, the null mutant strains of HpOCH1 (Hpoch1⌬) and HpOCR1 (Hpocr1⌬) displayed a remarkable reduction in hypermannosylation. Although the apparent phenotypes of Hpocr1⌬ were most similar to those of S. cerevisiae och1 mutants, the detailed structural analysis of N-glycans revealed that the major defect of Hpocr1⌬ is not in the initiation step but rather in the subsequent step of outer chain elongation by ␣-1,2-mannose addition. Most interestingly, Hpocr1⌬ showed a severe defect in the O-linked glycosylation of extracellular chitinase, representing HpOCR1 as a novel member of the OCH1 family implicated in both N-and O-linked glycosylation. In contrast, addition of the first ␣-1,6-mannose residue onto the core oligosaccharide Man 8 GlcNAc 2 was completely blocked in Hpoch1⌬ despite the comparable growth of its wild type under normal growth conditions. The complementation of the S. cerevisiae och1 null mutation by the expression of HpOCH1 and the lack of in vitro ␣-1,6-mannosyltransferase activity in Hpoch1⌬ provided supportive evidence that HpOCH1 is the functional orthologue of ScOCH1. The engineered Hpoch1⌬ strain with the targeted expression of Aspergillus saitoi ␣-1,2-mannosidase in the endoplasmic reticulum was shown to produce human-compatible high mannosetype Man 5 GlcNAc 2 oligosaccharide as a major N-glycan.

To optimize the secretory expression of recombinant human serum albumin (HSA) under the control of methanol oxidase (MOX) promoter in the methylotrophic yeast Hansenula polymorpha DL-1, we analyzed several parameters affecting the expression of HSA from the MOX promoter. Removal of the 5'-untranslated region derived from HSA cDNA in the expression cassette led to at least a fivefold improvement of HSA expression efficiency at the translational level. With the optimized expression cassette, the gene dosage effect on HSA expression was abolished and thus, a single copy of the expression vector integrated into the MOX locus became sufficient for the maximal expression of HSA. Northern blot analysis revealed that the levels of HSA transcript did not increase any further upon increasing copy number. The mox-disrupted (mox Delta) transformant was constructed, in which the genomic MOX gene was transplaced with the HSA expression cassette, to examine the effect of the methanol oxidase-deficient phenotype of the host on HSA expression. The mox Delta transformant showed higher levels of HSA production in shake-flask cultures than the MOX wild-type transformant, especially at low concentrations of methanol and a twofold higher specific HSA production rate in fed-batch fermentation with an abrupt induction mode. The native prepro signal sequence of HSA secreted in H. polymorpha was correctly processed and the mature recombinant protein had a pI value identical to that of the authentic HSA. Our results suggest that the H. polymorpha expression systems developed in this study are suitable for large-scale production of recombinant albumin.

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...