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

The N-glycosylation in Saccharomyces cerevisiae is of the high-mannose type, which affects the activity of the secreted heterologous glycoproteins. Cellobiohydrolase I (Tr-Cel7A) from Trichoderma reesei, is thus hyperglycosylated when expressed in S. cerevisiae. In the present work, three genes encoding the endogenous mannosyltransferases, Och1p, Mnn9p and Mnn1p, involved in glycoprotein processing in the S. cerevisiae Golgi apparatus, were individually or combinatorially disrupted to investigate the effect of the glycosylation extent on the activity of the secreted Tr-Cel7A. The glycosylation of the recombinant Tr-Cel7A was decreased and its extracellular activity was increased in all the deletion mutants. The simultaneous deletion of och1 and mnn1 has the most improvement on extracellular Tr-Cel7A activity. After expressed the α-1,2-mannosidase (Tr-Mds1p) from T. reesei in mnn1Δ/och1Δ strain, the Tr-Cel7A activity was further increased up to 320 ± 8% higher than that of the wild type strain. Such activity improvement was due not only to the higher secretion yield but also to the increased specific activity resulted from the changes in glycosylation. The results thus indicated that protein glycosylation engineering in S. cerevisiae was an effective approach to improve the extracellular activity of Tr-Cel7A