N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae.

Hongting Tang,Mengying Zhang,Yu Shen,Xiaoming Bao,Meihui Song,Mengyang Xu,Jiajing Wang,Jin Hou,Shenghuan Wang

doi:10.1038/srep25654

Hongting Tang, Mengying Zhang + Show 7 more

Open Access

https://doi.org/10.1038/srep25654

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Journal: Scientific Reports	Publication Date: May 9, 2016
Citations: 53	License type: CC BY 4.0

Affiliation: Shandong University

Abstract

Saccharomyces cerevisiae is a robust host for heterologous protein expression. The efficient expression of cellulases in S. cerevisiae is important for the consolidated bioprocess that directly converts lignocellulose into valuable products. However, heterologous proteins are often N-hyperglycosylated in S. cerevisiae, which may affect protein activity. In this study, the expression of three heterologous proteins, β-glucosidase, endoglucanase and cellobiohydrolase, was found to be N-hyperglycosylated in S. cerevisiae. To block the formation of hypermannose glycan, these proteins were expressed in strains with deletions in key Golgi mannosyltransferases (Och1p, Mnn9p and Mnn1p), respectively. Their extracellular activities improved markedly in the OCH1 and MNN9 deletion strains. Interestingly, truncation of the N-hypermannose glycan did not increase the specific activity of these proteins, but improved the secretion yield. Further analysis showed OCH1 and MNN9 deletion up-regulated genes in the secretory pathway, such as protein folding and vesicular trafficking, but did not induce the unfolded protein response. The cell wall integrity was also affected by OCH1 and MNN9 deletion, which contributed to the release of secretory protein extracellularly. This study demonstrated that mannosyltransferases disruption improved protein secretion through up-regulating secretory pathway and affecting cell wall integrity and provided new insights into glycosylation engineering for protein secretion.

Highlights

Α -1,6-mannosyltransferase Och1p increased the production of the active form of human tissue-type plasminogen activator[12]
OCH1 initiates the α -1,6-hypermannose glycan elongation by the first α -1,6-mannose addition, MNN9 determines whether the Man9GlcNAc2 oligosaccharide is elongated with α -1,6-mannose backbone or stopped by the addition of α -1, 2-mannose and MNN1 is responsible for the terminal α -1,3-mannose addition
The results showed that protein folding-related genes, such as KAR2 and SSA1, protein trafficking-related genes, including BOS1, ERV25, SNC2 and SSO1, and genes involved in ER-associated degradation (ERAD), such as DER1 and HRD3 were up-regulated by the OCH1 and MNN9 deletions

Summary

Introduction

Α -1,6-mannosyltransferase Och1p increased the production of the active form of human tissue-type plasminogen activator[12]. Deletion of the enzyme complex M-Pol II component Mnn10p improved secretion of multiple recombinant proteins and endogenous invertase[13,14]. These previous studies showed that N-glycosylation modification can increase protein secretion. It was found that three recombinant cellulases, Cel3A, CelA and Cel7A, were N-hyperglycosylated when expressed in S. cerevisiae These three heterologous proteins were expressed in strains with deletions in key Golgi mannosyltransferase gene (OCH1, MNN1 and MNN9) to block the formation of hypermannose glycan, and the extracellular activities of the three recombinant proteins were increased significantly. It was observed that the transcription of key components in the secretory pathway was up-regulated and the cell wall integrity was damaged, which may be responsible for the improvement of protein production

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Results

Conclusion