Engineering of the yeast Yarrowia lipolytica for the production of glycoproteins lacking the outer-chain mannose residues of N-glycans.
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.
Song
Yunkyoung
Y
School of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea.
Choi
Min Hee
MH
Park
Jeong-Nam
JN
Kim
Moo Woong
MW
Kim
Eun Jung
EJ
Kang
Hyun Ah
HA
Kim
Jeong-Yoon
JY
D002480:Cellulase / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D008049:Lipase / Q000737:chemistry / Q000235:genetics; D008361:Mannosidases / Q000378:metabolism; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D025062:Yarrowia / Q000235:genetics* / Q000378:metabolism*
D006023:Glycoproteins; D008562:Membrane Glycoproteins; C075636:OCH1 protein, S cerevisiae; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D008364:Mannosyltransferases; C058478:alpha 1,6-mannosyltransferase; D008049:Lipase; D008361:Mannosidases; C078166:alpha(1-6)mannosidase; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D002480:Cellulase