Overexpression and Secretion of Cellulolytic Enzymes by δ-Sequence-Mediated Multicopy Integration of Heterologous DNA Sequences into the Chromosomes of Saccharomyces cerevisiae
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概要
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Saccharomyces cerevisiae transformants which secrete high levels of cellulolytic enzymes, with chromosome-integrated multicopies of heterologous DNA sequences encoding the celluloytic enzymes were constructed. An expression construct of β-glucosidase and carboxymethyl cellulase directed by the GAP promoter was integrated into the chromosomes of the haploid S. cerevisiae using the δ sequence-mediated integration system. Southern blot analysis of the chromosomes prepared from various integrants and separated by pulse-field gel electrophoresis demonstrated that the integration occurred mainly in a particular chromosome and the copy number of the integration was variable. The amount of enzymes secreted by the transformants correlated with the copy number of integration. For each enzyme, the highest activity was about 1.4-fold that produced by the transformant harboring the same expression cassette on a YEp-type plasmid. The δ-integrated exogenous DNA was mitotically stable in rich medium. A haploid double transformant which coexpresses and secretes β-glucosidase and carboxymethyl cellulase was further constructed by genetic crossing of the haploid transformant that produces a high level of the enzyme, followed by meiotic segregation of the resulting diploid strain. The haploid double transformant, but neither of the single transformant, could grow on a plate containing carboxymethyl cellulose as a sole carbon source. It is suggested that the δ-sequence-mediated integration system is a very useful means for the genetic engineering of yeast, especially when overproduction and secretion of multiple heterologous enzymes are desired.
- 社団法人日本生物工学会の論文
- 1994-05-25
著者
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Miyahara Kohji
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima Uni
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HIRATA Dai
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima Uni
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HATANO TAKUSHI
Department of Bioengineering, Faculty of Engineering, Fukuyama University
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FUKUI SAKUZO
Department of Bioengineering, Faculty of Engineering, Fukuyama University
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Hirata D
Niigata Prefectural Institute Of Brewing
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Hirata Dai
From The Department Of Fermentation Technology Faculty Of Engineering Hiroshima University
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Hirata Dai
Center For Gene Science Hiroshima University
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Hirata Dai
Department Of Fermentation Technology Hiroshima University
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MIYAKAWA Tokichi
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima Uni
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MATSUZAKI HIROAKI
Department of Bioengineering, Faculty of Engineering, Fukuyama University
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Mochizuki Daisuke
Department Of Fermentation Technology Faculty Of Engineering Hiroshima University
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Matsuzaki Hiroaki
Department Of Bioengineering Faculty Of Engineering Fukuyama University
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Fukui Sakuzo
Department Of Bioengineering Faculty Of Engineering Fukuyama University
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Hatano Takushi
Department Of Bioengineering Faculty Of Engineering Fukuyama University
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Hirata D
Hiroshima Univ. Higashi‐hiroshima Jpn
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Miyakawa T
Department Of Molecular Biotechnology Graduate School Of Advanced Sciences Of Matter Hiroshima Unive
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Miyakawa Tokichi
Department Of Fermentation Technology Faculty Of Engineering Hiroshima University
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Miyahara Kohji
Department Of Clinical Physiology Tokyo Metropolitan Institute Of Gerontology
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Miyakawa Tokichi
Department Of Fermentation Technology
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Miyahara Kohji
Department Of Applied Life Science Sojo University
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