title: N-Glycosylation deficiency enhanced heterologous production of a Bacillus licheniformis thermostable α-amylase in Saccharomyces cerevisiae.
abstract: Expression of foreign enzymes in yeast is a traditional genetic engineering approach; however, useful secretory enzymes are not produced in every case. The hyperthermostable α-amylase encoded by the AmyL gene of Bacillus licheniformis was expressed in Saccharomyces cerevisiae; however, it was only weakly produced and was degraded by the proteasome. To determine the cause of low α-amylase production, AmyL was expressed in a panel of yeast mutants harboring knockouts in non-essential genes. Elevated AmyL production was observed in 44 mutants. The knockout genes were classified into six functional categories. Remarkably, all non-essential genes required for N-linked oligosaccharide synthesis and a gene encoding an oligosaccharyl transferase subunit were identified. Immunoblotting demonstrated that differently underglycosylated forms of AmyL were secreted from oligosaccharide synthesis-deficient mutants, while a fully glycosylated form was produced by wild-type yeast, suggesting that N-linked glycosylation of AmyL inhibited its secretion in yeast. Mutational analysis of six potential N-glycosylation sites in AmyL revealed that the N33Q and N309Q mutations remarkably affected AmyL production. To achieve higher AmyL production in yeast, all six N-glycosylation sites of AmyL were mutated. In wild-type yeast, production of the resulting non-glycosylated form of AmyL was threefold higher than that of the glycosylated form.
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, 2-16-1 Tokiwadai, Ube, Japan. hoshida@yamaguchi-u.ac.jp
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.forename: Tsuneyasu
authors.initials: T
authors.lastname: Fujita
authors.forename: Kamonchai
authors.initials: K
authors.lastname: Cha-aim
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D004252:DNA Mutational Analysis; D055786:Gene Knockout Techniques; D006031:Glycosylation; D060847:Metabolic Engineering; D020125:Mutation, Missense; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D000516:alpha-Amylases