title: N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae.
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.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China.
authors.forename: Hongting
authors.initials: H
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China.
authors.forename: Shenghuan
authors.initials: S
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China.
authors.forename: Jiajing
authors.initials: J
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China.
authors.forename: Meihui
authors.initials: M
authors.lastname: Song
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China.
authors.forename: Mengyang
authors.initials: M
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China.
authors.forename: Mengying
authors.initials: M
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Shen
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Hou
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Bao
mesh_terms: D002473:Cell Wall / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D017353:Gene Deletion; D006031:Glycosylation; D008364:Mannosyltransferases / Q000235:genetics; D008970:Molecular Weight; D016062:Porosity; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D055571:Secretory Pathway; D014158:Transcription, Genetic; D056811:Unfolded Protein Response; D043324:beta-Fructofuranosidase / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D008364:Mannosyltransferases; D043324:beta-Fructofuranosidase