Publication: N-glycosylation deficiency enhanced heterologous production of a Bacillus licheniformis thermostable α-amylase in Saccharomyces cerevisiae
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Issued Date
2013
Resource Type
File Type
application/pdf
ISSN
1757598
Other identifier(s)
2-s2.0-84878680062
Rights Holder(s)
Scopus
Bibliographic Citation
Applied Microbiology and Biotechnology. Vol 97, No.12 (2013), p.5473-5482
Suggested Citation
Hoshida H., Fujita T., Cha-Aim K., Akada R. N-glycosylation deficiency enhanced heterologous production of a Bacillus licheniformis thermostable α-amylase in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. Vol 97, No.12 (2013), p.5473-5482. doi:10.1007/s00253-012-4582-2 Retrieved from: https://hdl.handle.net/20.500.14740/6652
Author(s)
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. © 2013 Springer-Verlag Berlin Heidelberg.
Subject(s)
Bacillus licheniformis
Engineering approaches
Heterologous production
N-Glycosylation
N-glycosylation sites
N-linked glycosylation
Oligosaccharide synthesis
Oligosaccharyl transferase
Amylases
Bacteriology
Esterification
Gene encoding
Genetic engineering
Glycosylation
Oligosaccharides
Yeast
Amylase
Asparagine linked oligosaccharide
Bacterium
Enzyme
Enzyme activity
Genetic engineering
Mutation
Secretion
Yeast
Article
Bacillus licheniformis
Carbohydrate synthesis
Controlled study
Enzyme stability
Glycosylation
Heterologous expression
Immunoblotting
Knockout gene
Nonhuman
Saccharomyces cerevisiae
Wild type
Yeast
Alpha-Amylases
Bacillus
DNA Mutational Analysis
Gene Knockout Techniques
Glycosylation
Metabolic Engineering
Mutation, Missense
Protein Processing, Post-Translational
Recombinant Proteins
Saccharomyces cerevisiae
Bacillus licheniformis
Saccharomyces cerevisiae
Engineering approaches
Heterologous production
N-Glycosylation
N-glycosylation sites
N-linked glycosylation
Oligosaccharide synthesis
Oligosaccharyl transferase
Amylases
Bacteriology
Esterification
Gene encoding
Genetic engineering
Glycosylation
Oligosaccharides
Yeast
Amylase
Asparagine linked oligosaccharide
Bacterium
Enzyme
Enzyme activity
Genetic engineering
Mutation
Secretion
Yeast
Article
Bacillus licheniformis
Carbohydrate synthesis
Controlled study
Enzyme stability
Glycosylation
Heterologous expression
Immunoblotting
Knockout gene
Nonhuman
Saccharomyces cerevisiae
Wild type
Yeast
Alpha-Amylases
Bacillus
DNA Mutational Analysis
Gene Knockout Techniques
Glycosylation
Metabolic Engineering
Mutation, Missense
Protein Processing, Post-Translational
Recombinant Proteins
Saccharomyces cerevisiae
Bacillus licheniformis
Saccharomyces cerevisiae