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

School of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea.
Applied and Environmental Microbiology (Impact Factor: 3.67). 08/2007; 73(14):4446-54. DOI: 10.1128/AEM.02058-06
Source: PubMed

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 alpha-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 alpha-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 Man8GlcNAc2, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man8GlcNAc2 to Man12GlcNAc2. Digestion with alpha-1,2- and alpha-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 alpha-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.

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    • "Integrated vectors developed that help genetic stability of the recombinant elements, even in continuous and large-scale fermentation processes Well-established commercial vector systems and host strains (Invitrogen) A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal a-1,3-linked mannose residues Bretthauer (2003) Genome sequencing: Reference strain GS115; 9216 Kb (5040 ORFs); Accession number PRJNA39439, PRJEA37871 De Schutter et al. (2009) Hansenula polymorpha GRAS status Gellissen et al. (2005) Stringently regulated strong promoters (MOX, FMDH, etc.) A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes Stable, multicopy integration of foreign DNA into chromosomal locations Thermotolerant (growth up to 45 °C), resistant to heavy metals and oxidative stress Can assimilate nitrates A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal a-1,3-linked mannose residues Kang et al. (1998) and Kim et al. (2004) Genome sequencing: Reference strain DL1; 9056 Kb (5325 ORFs); Accession number PRJNA60503 Ravin et al. (2013) Yarrowia lipolytica An oleaginous yeast, based on its ability to accumulate large amounts of lipids Madzak et al. (2004) GRAS status Can grow in hydrophobic environments, that is able to metabolize triglycerides, fatty acids, n-alkanes, and n-paraffins as carbon sources for the bioremediation of environments contaminated with oil spills Can secrete a variety of proteins via cotranslational translocation and efficient secretion signal recognition similar to higher eukaryotes Availability of a commercial expression kit (YEASTERN BIOTECH CO., LTD.) Salt tolerance A lesser extent of hypermannosylation compared to S. cerevisiae; a lack of the immunogenic terminal a-1,3-mannose linkages Song et al. (2007) Genome sequencing: Reference strain CLIB122; 20 503 Kb (7042 ORFs); Accession number PRJNA12414 Dujon et al. (2004) Schizosaccharomyces pombe A fission yeast, reflecting proliferation of higher eukaryotic cells Takegawa et al. (2009) Many cellular processes similar to those of higher eukaryotes, such as mRNA splicing, posttranslational modification (including protein galactosylation), cell cycle control, etc. Transcription start site similar to that in higher eukaryotes Expression vectors for high-level expression developed Presence of galactose in both O-and N-linked glycans Ballou et al. (1994) Genome sequencing: Reference strain "
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    ABSTRACT: The production of recombinant therapeutic proteins is one of the fast growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic posttranslational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several non-conventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins.This article is protected by copyright. All rights reserved.
    FEMS Yeast Research 08/2014; DOI:10.1111/1567-1364.12195 · 2.82 Impact Factor
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    • "In the Golgi apparatus of yeasts, the Man8GlcNAc2 N-glycans are further extended by the addition of mannose and phospho-mannose residues. This elongation is initiated by the α-1,6-mannosyltransferase Och1p [3], [4]. In contrast, higher eukaryotes first trim the glycans to Man5GlcNAc2 by Golgi mannosidases I and then further modify them to complex type glycans [5]–[7]. "
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    ABSTRACT: 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.
    PLoS ONE 06/2012; 7(6):e39976. DOI:10.1371/journal.pone.0039976 · 3.23 Impact Factor
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    • "Analysis of the N-glycan profile of cell wall mannoproteins showed that only the YlOCH1 knock-out strain accumulated the Man8GlcNAc2 precursor glycan. These results are in agreement with a previous report [11]. Thus, though the function of Och1p in Y. lipolytica had been questioned [10], we conclude that the Y. lipolytica Och1p, like the S. cerevisiae α-1,6-mannosyltransferase Och1p [7,13,20,21], is critical for the extension of N-linked oligosaccharide chains, and that it is at least responsible for the addition of the first α-1,6-linked mannose to the core Man8GlcNAc2 oligosaccharide. "
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    ABSTRACT: Background Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation. Results We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases YlOch1p and YlMnn9p; the former inactivation yielded a strain producing homogeneous Man8GlcNAc2 glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that YlOch1p and YlMnn9p, despite some initial uncertainty about their function, are most likely the α-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained α-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man5GlcNAc2. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme. Conclusions We generated a Y. lipolytica expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man8GlcNAc2 or Man5GlcNAc2 N-glycans. This platform expands the utility of Y. lipolytica as a heterologous expression host and makes it possible to produce glycoproteins with homogeneously glycosylated N-glycans of the human high-mannose-type, which greatly broadens the application scope of these glycoproteins.
    Microbial Cell Factories 05/2012; 11(1):53. DOI:10.1186/1475-2859-11-53 · 4.22 Impact Factor
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