Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes.
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 alpha-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-alpha-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley beta-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and beta-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing beta-glucan as the sole carbon source and could directly ferment 45 g of beta-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.
D002480:Cellulase / Q000378:metabolism; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004800:Enzymes, Immobilized; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D005813:Genes, Synthetic; D005818:Genetic Engineering; D005822:Genetic Vectors; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000431:Ethanol; D002482:Cellulose; D002480:Cellulase