发明申请
- 专利标题: METABOLIC ENGINEERING OF A GALACTOSE ASSIMILATION PATHWAY IN THE GLYCOENGINEERED YEAST PICHIA PASTORIS
- 专利标题(中): 葡萄糖生物化学烟草中糖基化途径的代谢工程
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申请号: US13202002申请日: 2010-02-24
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公开(公告)号: US20120003695A1公开(公告)日: 2012-01-05
- 发明人: Robert C. Davidson , Piotr Bobrowicz , Dongxing Zha
- 申请人: Robert C. Davidson , Piotr Bobrowicz , Dongxing Zha
- 国际申请: PCT/US2010/025163 WO 20100224
- 主分类号: C12P21/00
- IPC分类号: C12P21/00 ; C12N9/64 ; C12N15/81 ; C12N9/16 ; C12N9/24 ; C12N1/19 ; C12N9/72
摘要:
Lower eukaryotic cells such as Pichia pastoris that normally cannot use galactose as a carbon source but which have been genetically engineered according to the methods herein to use galactose as a sole source of carbon are described. The cells are genetically engineered to express several of the enzymes comprising the Leloir pathway. In particular, the cells are genetically engineered to express a galactokinase, a UDP-galactose-C4-epimerase, and a galactose-1-phosphate uridyltransferase, and optionally a galactose permease. In addition, a method is provided for improving the yield of glycoproteins that have galactose-terminated or -containing N-glycans in cells that have been genetically engineered to produce glycoproteins with N-glycans having galactose residues but which normally lack the enzymes comprising the Leloir pathway comprising transforming the cells with one or more nucleic acid molecules encoding a galactokinase, a UDP-galactose-C4-epimerase, and a galactose-1-phosphate uridyltransferase. The methods and host cells described enable the presence or lack of the ability to assimilate galactose as a selection method for making recombinant cells. The methods and host cells are shown herein to be particularly useful for making immunoglobulins and the like that have galactose-terminated or containing N-glycans.
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