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

• DOI: 10.1128/aem.68.10.5136-5141.2002
• Publication Date: 2002-09-25T18:02:42Z
• Authors: Yasuya Fujita; Shouji Takahashi; Mitsuyoshi Ueda; Atsuo Tanaka; Hirofumi Okada; Yasushi Morikawa; Takashi Kawaguchi; Motoo Arai; Hideki Fukuda; Akihiko Kondo
• Keywords: 2. Zero hunger; 0106 biological sciences; 0301 basic medicine; Ethanol; Recombinant Fusion Proteins; Genetic Vectors; Saccharomyces cerevisiae; Enzymes, Immobilized; 01 natural sciences; Peptide Fragments; Culture Media; 03 medical and health sciences; Cellulase; Microscopy, Fluorescence; Fermentation; Genes, Synthetic; Cellulose; Genetic Engineering; Chromatography, High Pressure Liquid

ABSTRACT

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 α-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-His 6 ) 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-α-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley β-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and β-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing β-glucan as the sole carbon source and could directly ferment 45 g of β-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.