Promotion of Extracellular Activity of Cellobiohydrolase I from Trichoderma reesei by Protein Glycosylation Engineering in Saccharomyces cerevisiae

Abstract

The N-glycosylation in Saccharomyces cerevisiae is of the high-mannose type, which affects the activity of thesecreted heterologous glycoproteins. Cellobiohydrolase I (Tr-Cel7A) from Trichoderma reesei, is thushyperglycosylated when expressed in S. cerevisiae. In the present work, three genes encoding the endogenousmannosyltransferases, Och1p, Mnn9p and Mnn1p, involved in glycoprotein processing in the S. cerevisiae Golgiapparatus, were individually or combinatorially disrupted to investigate the effect of the glycosylation extent on theactivity of the secreted Tr-Cel7A. The glycosylation of the recombinant Tr-Cel7A was decreased and its extracellularactivity was increased in all the deletion mutants. The simultaneous deletion of och1 and mnn1 has the mostimprovement on extracellular Tr-Cel7A activity. After expressed the �-1,2-mannosidase (Tr-Mds1p) from T. reesei inmnn1�/och1� strain, the Tr-Cel7A activity was further increased up to 320 � 8% higher than that of the wild typestrain. Such activity improvement was due not only to the higher secretion yield but also to the increased specificactivity resulted from the changes in glycosylation. The results thus indicated that protein glycosylation engineeringin S. cerevisiae was an effective approach to improve the extracellular activity of Tr-Cel7A.