Metabolic engineering of a galactose assimilation pathway in the glycoengineered yeast pichia pastoris

摘要

METABOLIC ENGINEERING OF A GALACTOSE ASSIMILATION PATHWAY IN THE GLYCOENGINEERED YEAST PICHIA PASTORIS Abstract qodoctoseo'd I galctonse permaae (Gol2p) galoctoseh Leoir galactokinose (Gallp) goxlctose-1-p osphate UDP-galactose C4 ur tr(nsferose 7p) epnerose (Gall0p) U1P-golactose glucose-1 -phosphote Pho ucomtse (Galsp) glucose-6-phosphate Lower eukaryotic cells such as Pichiapastoris 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-l-phosphate uridyl-transferase, 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-I-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. (6731103_l):KZA