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: 3.95). 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 · 2.44 Impact Factor
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    • "Whereas S. cerevisiae, P. pastoris, and H. polymorpha are the most frequently used yeasts for recombinant protein production, there is a growing interest in the dimorphic yeast Y. lipolytica. Although it was suggested that the Y. lipolytica Och1p α-1,6-mannosyltransferase might play a minor role in the outer-chain elongation of N-glycosylation (Barnay-Verdier et al. 2004), it was shown that knocking out the Y. lipolytica OCH1 gene results in glycoproteins predominantly modified with Man 8 GlcNAc 2 glycans (Song et al. 2007; De Pourcq et al., unpublished results). Subsequent introduction of the T. reesei α-1,2-mannosi- dase–HDEL construct used also for P. pastoris engineering generated a strain producing Man 5 GlcNAc 2 glycans on its glycoproteins (De Pourcq et al., unpublished results). "
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    ABSTRACT: With the increasing demand for recombinant proteins and glycoproteins, research on hosts for producing these proteins is focusing increasingly on more cost-effective expression systems. Yeasts and other fungi are promising alternatives because they provide easy and cheap systems that can perform eukaryotic post-translational modifications. Unfortunately, yeasts and other fungi modify their glycoproteins with heterogeneous high-mannose glycan structures, which is often detrimental to a therapeutic protein's pharmacokinetic behavior and can reduce the efficiency of downstream processing. This problem can be solved by engineering the glycosylation pathways to produce homogeneous and, if so desired, human-like glycan structures. In this review, we provide an overview of the most significant recently reported approaches for engineering the glycosylation pathways in yeasts and fungi.
    Applied Microbiology and Biotechnology 08/2010; 87(5):1617-31. DOI:10.1007/s00253-010-2721-1 · 3.81 Impact Factor
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    • "After addition of this first ␣1,6-Man by Och1p, additional ␣1,6-mannosyltransferases will extend the ␣1,6-chain, which then becomes the substrate for trans-or medial-Golgiresiding mannosyltransferases and phosphomannosyltransferases that add yet more Man sugars to the growing N-glycan structure (Dean, 1999; Gemmill and Trimble, 1999). The Och1p protein has been proven to be an initiating ␣1,6-mannosyltransferase that plays a key role in the addition of the first mannose to the core oligosaccharide in several yeast species, including Pichia pastoris (Choi et al., 2003), Yarrowia lipolytica (Barnay-Verdier et al., 2004; Song et al., 2007), Schizosaccharomyces pombe (Yoko-o et al., 2001), and Hansenula. Polymorpha (Kim et al., 2006). "
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    ABSTRACT: Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding alpha1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative alpha1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The N-linked oligosaccharides assembled on a secretory glycoprotein, HSA/GM-CSF in Kloch1 mutant, contained oligosaccharide Man(13-14)GlcNAc(2), and in Kloch1 mnn1 mutant, contained oligosaccharide Man(9-11)GlcNAc(2), whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man(>30)GlcNAc(2). Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in K. lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation, most possibly encodes alpha1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production.
    Journal of Biotechnology 07/2009; 143(2):95-102. DOI:10.1016/j.jbiotec.2009.06.016 · 2.88 Impact Factor
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