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Applied and Environmental Microbiology, December 1998, p. 4857-4861, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Assimilation of Cellooligosaccharides by a Cell Surface-Engineered Yeast Expressing beta -Glucosidase and Carboxymethylcellulase from Aspergillus aculeatus

Toshiyuki Murai,1 Mitsuyoshi Ueda,1 Takashi Kawaguchi,2 Motoo Arai,2 and Atsuo Tanaka1,*

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501,1 and Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 599-8531,2 Japan

Received 8 June 1998/Accepted 30 September 1998

Since Saccharomyces cerevisiae lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies. The genes encoding FI-carboxymethylcellulase (CMCase) and beta -glucosidase from the fungus Aspergillus aculeatus were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha -agglutinin and introduced into S. cerevisiae. The delivery of CMCase and beta -glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from Rhizopus oryzae for beta -glucosidase, respectively. The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase and beta -glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant. The display of CMCase and beta -glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy. The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source. The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography. This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides. This is the first step in the assimilation of cellulosic materials by S. cerevisiae expressing heterologous cellulase genes.

* Corresponding author. Mailing address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-5524. Fax: 81-75-753-5534. E-mail: atsuo{at}sbchem.kyoto-u.ac.jp.

Applied and Environmental Microbiology, December 1998, p. 4857-4861, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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