Abstract
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.
Highlights
The suitable glycosylation in secretory pathway is crucial for the correct folding, stability, bioactivity and extracellular activity of secreted heterologous proteins [1]
The deletion of the endogenous mannosyltransferases decreased the glycosylation of Tr-Cel7A: The secreted Tr-Cel7A from S. cerevisiae was proved to be Nhyperglycosylated [9,10,11]
To decrease the elongation of the outer chain of the N-linked oligosaccharides, the yeast α-1,6-mannosyltransferases, Och1p and Mnn9p, and the α-1,3mannosyltransferase, Mnn1p, which were involved in the Nglycosylation process, were disrupted singly or doubly
Summary
The suitable glycosylation in secretory pathway is crucial for the correct folding, stability, bioactivity and extracellular activity of secreted heterologous proteins [1]. The mannose residues can reach to about 200 to form a complex mannan [4], which has a potential to affect the activity of Nglycosylated glycoprotein including the heterologous proteins expressed in S. cerevisiae [5]. Wang et al [6] screened S. cerevisiae gene-knockout strains of glycosylation-related genes for improved extracellular activity of a heterologous exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. They found that the degree of N-glycosylation plays an important role in heterologous cellulase activity and both over- and under-glycosylation may alter the enzyme activity of cellulases in S. cerevisiae. The Tr-Cel7A was generally hyperglycosylated with lower secretion in S. cerevisiae compared with it in T. reesei [9,10,11]
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