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N-glycosylation deficiency enhanced heterologous production of a Bacillus licheniformis thermostable α-amylase in Saccharomyces cerevisiae

  • Applied genetics and molecular biotechnology
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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.

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Acknowledgments

This study was supported in part by the Industrial Technology Research Grant Program in 2009 from New Energy and Industrial Technology Development Organization (NEDO) of Japan and by Asian Core University Program between Japan Society for the Promotion of Science (JSPS) and the National Research Council of Thailand (NRCT). We thank Dr. N. Decklerck, Genetique Moleculaire et Cellulaire, France; Dr. D. Wolf, Stuttgart University, Germany; and Dr. H. Shimoi, National Research Institute, Japan for the kind gifts of the AmyL gene, the proteasome-deficient yeast strain, and TAA expression plasmid, respectively. We are also grateful to the DNA Core Facility at the Center for Gene Research of Yamaguchi University.

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  1. Kamonchai Cha-aim

    Present address: Faculty of Agricultural Products Innovation and Technology, Srinakharinwirot University, 114 Sukhumvit, North-Khong Toey, Wattana District, Bangkok, Thailand

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  1. Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, 2-16-1 Tokiwadai, Ube, Japan

    Hisashi Hoshida, Tsuneyasu Fujita, Kamonchai Cha-aim & Rinji Akada

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  1. Hisashi Hoshida
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Hoshida, H., Fujita, T., Cha-aim, K. et al. N-glycosylation deficiency enhanced heterologous production of a Bacillus licheniformis thermostable α-amylase in Saccharomyces cerevisiae . Appl Microbiol Biotechnol 97, 5473–5482 (2013). https://doi.org/10.1007/s00253-012-4582-2

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