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Applied and Environmental Microbiology, October 2002, p. 5136-5141, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.5136-5141.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes

Yasuya Fujita,¹ Shouji Takahashi,² Mitsuyoshi Ueda,² Atsuo Tanaka,² Hirofumi Okada,³ Yasushi Morikawa,³ Takashi Kawaguchi,⁴ Motoo Arai,⁴ Hideki Fukuda,¹ and Akihiko Kondo^5*

Division of Molecular Science, Graduate School of Science and Technology of Kobe University,¹ Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501,⁵ Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501,² Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188,³ Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 599-8531, Japan⁴

Received 15 April 2002/ Accepted 9 July 2002

For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on -agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His₆) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII--agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley ß-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and ß-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing ß-glucan as the sole carbon source and could directly ferment 45 g of ß-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.

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* Corresponding author. Mailing address: Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan. Phone: 81-78-803-6196. Fax: 81-78-803-6206. E-mail: kondo{at}cx.kobe-u.ac.jp.

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Applied and Environmental Microbiology, October 2002, p. 5136-5141, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.5136-5141.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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