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Applied and Environmental Microbiology, October 2009, p. 6087-6093, Vol. 75, No. 19
0099-2240/09/$08.00+0     doi:10.1128/AEM.01538-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Functional Assembly of Minicellulosomes on the Saccharomyces cerevisiae Cell Surface for Cellulose Hydrolysis and Ethanol Production{triangledown}

Shen-Long Tsai,1 Jeongseok Oh,1 Shailendra Singh,1 Ruizhen Chen,2 and Wilfred Chen1*

Department of Chemical and Environmental Engineering, University of California at Riverside, Riverside, California 92521,1 School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Dr. NW, Atlanta, Georgia 30032-01002

Received 30 June 2009/ Accepted 7 August 2009

We demonstrated the functional display of a miniscaffoldin on the Saccharomyces cerevisiae cell surface consisting of three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f). Incubation with Escherichia coli lysates containing an endoglucanase (CelA) fused with a dockerin domain from C. thermocellum (At), an exoglucanase (CelE) from C. cellulolyticum fused with a dockerin domain from the same species (Ec), and an endoglucanase (CelG) from C. cellulolyticum fused with a dockerin domain from R. flavefaciens (Gf) resulted in the assembly of a functional minicellulosome on the yeast cell surface. The displayed minicellulosome retained the synergistic effect for cellulose hydrolysis. When a β-glucosidase (BglA) from C. thermocellum tagged with the dockerin from R. flavefaciens was used in place of Gf, cells displaying the new minicellulosome exhibited significantly enhanced glucose liberation and produced ethanol directly from phosphoric acid-swollen cellulose. The final ethanol concentration of 3.5 g/liter was 2.6-fold higher than that obtained by using the same amounts of added purified cellulases. The overall yield was 0.49 g of ethanol produced per g of carbohydrate consumed, which corresponds to 95% of the theoretical value. This result confirms that simultaneous and synergistic saccharification and fermentation of cellulose to ethanol can be efficiently accomplished with a yeast strain displaying a functional minicellulosome containing all three required cellulolytic enzymes.

* Corresponding author. Mailing address: Department of Chemical and Environmental Engineering, University of California at Riverside, Riverside, CA 92521. Phone: (951) 827-2473. Fax: (951) 827-5696. E-mail: wilfred{at}engr.ucr.edu

{triangledown} Published ahead of print on 14 August 2009.

Applied and Environmental Microbiology, October 2009, p. 6087-6093, Vol. 75, No. 19
0099-2240/09/$08.00+0     doi:10.1128/AEM.01538-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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