INSERTION INTO ASPERGILLUS-NIDULANS OF FUNCTIONAL UDP-GLCNAC - ALPHA-3-D-MANNOSIDE BETA-1,2-N-ACETYLGLUCOSAMINYL-TRANSFERASE-I, THE ENZYME CATALYZING THE FIRST COMMITTED STEP FROM OLIGOMANNOSE TO HYBRID AND COMPLEX N-GLYCANS

Filamentous fungi are capable of secreting relatively large amounts of heterologous recombinant proteins. Recombinant human glycoproteins ex pressed in this system, however, carry only carbohydrates of the oligo mannose type limiting their potential use in humans. One approach to t he problem is genetic engineering of the fungal host to permit product ion of comp lex and hybrid N-glycans. UDP-GlcNAc: alpha 3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I (GnT I) is essential for th e conversion of oligomannose to hybrid and complex N-glycans in higher eukaryotic cells. Since GnT I is not produced by fungi, we have intro duced into the genome of Aspergillus nidulans the gene encoding full-l ength rabbit GnT I and demonstrated the expression of GnT I enzyme act ivity at levels appreciably higher than occurs in most mammalian tissu es. All the GnT I activity in the Aspergillus transformants remains in tracellular suggesting that the rabbit trans-membrane sequence may be capable of targeting GnT I to the fungal Golgi apparatus.