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Applied and Environmental Microbiology, July 2007, p. 4446-4454, Vol. 73, No. 14
0099-2240/07/$08.00+0     doi:10.1128/AEM.02058-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Yunkyoung Song,1 Min Hee Choi,1 Jeong-Nam Park,1,2 Moo Woong Kim,1,2 Eun Jung Kim,1,2 Hyun Ah Kang,2* and Jeong-Yoon Kim1*

School of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764,1 Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea2

Received 31 August 2006/ Accepted 8 May 2007

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.

* Corresponding author. Mailing address for Jeong-Yoon Kim: School of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea. Phone: 82-42-821-6419. Fax: 82-42-822-7367. E-mail: jykim{at}cnu.ac.kr. Mailing address for Hyun Ah Kang: Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea. Phone: 82-42-860-4378. Fax: 82-42-860-4594. E-mail: hyunkang{at}kribb.re.kr

{triangledown} Published ahead of print on 18 May 2007.

Applied and Environmental Microbiology, July 2007, p. 4446-4454, Vol. 73, No. 14
0099-2240/07/$08.00+0     doi:10.1128/AEM.02058-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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