Surface Display of a Functional Minicellulosome by Intracellular Complementation Using a Synthetic Yeast Consortium and Its Application to Cellulose Hydrolysis and Ethanol Production

ABSTRACT In this paper, we report the surface assembly of a functional minicellulosome by using a synthetic yeast consortium. The basic design of the consortium consisted of four different engineered yeast strains capable of either displaying a trifunctional scaffoldin, Scaf-ctf (SC), carrying three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f), or secreting one of the three corresponding dockerin-tagged cellulases (endoglucanase [AT], exoglucanase [EC/CB], or β-glucosidase [BF]). The secreted cellulases were docked onto the displayed Scaf-ctf in a highly organized manner based on the specific interaction of the three cohesin-dockerin pairs employed, resulting in the assembly of a functional minicellulosome on the yeast surface. By exploiting the modular nature of each population to provide a unique building block for the minicellulosome structure, the overall cellulosome assembly, cellulose hydrolysis, and ethanol production were easily fine-tuned by adjusting the ratio of different populations in the consortium. The optimized consortium consisted of a SC:AT:CB:BF ratio of 7:2:4:2 and produced almost twice the level of ethanol (1.87 g/liter) as a consortium with an equal ratio of the different populations. The final ethanol yield of 0.475 g of ethanol/g of cellulose consumed also corresponded to 93% of the theoretical value. This result confirms the use of a synthetic biology approach for the synergistic saccharification and fermentation of cellulose to ethanol by using a yeast consortium displaying a functional minicellulosome.

[1]  M. Ueda,et al.  Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes , 2002, Applied and Environmental Microbiology.

[2]  Bryce J. Stokes,et al.  Biomass as Feedstock for A Bioenergy and Bioproducts Industry: The Technical Feasibility of a Billion-Ton Annual Supply , 2005 .

[3]  L. Lynd,et al.  Utilization of cellobiose by recombinant β -glucosidase -expressing strains of Saccharomyces cerevisiae : characterization and evaluation of the sufficiency of expression , 2005 .

[4]  M. Ueda,et al.  Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme , 2004, Applied and Environmental Microbiology.

[5]  Jiong Hong,et al.  Cloning and functional expression of thermostable β-glucosidase gene from Thermoascus aurantiacus , 2007, Applied Microbiology and Biotechnology.

[6]  F. Smith,et al.  Colorimetric Method for Determination of Sugars and Related Substances , 1956 .

[7]  I. Pretorius,et al.  Domain engineering of Saccharomyces cerevisiae exoglucanases , 2005, Biotechnology Letters.

[8]  Lee R Lynd,et al.  Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae. , 2007, Metabolic engineering.

[9]  L. Lynd,et al.  How biotech can transform biofuels , 2008, Nature Biotechnology.

[10]  E. Nevoigt,et al.  Progress in Metabolic Engineering of Saccharomyces cerevisiae , 2008, Microbiology and Molecular Biology Reviews.

[11]  S. Champ,et al.  CelE, a Multidomain Cellulase fromClostridium cellulolyticum: a Key Enzyme in the Cellulosome? , 2000, Journal of bacteriology.

[12]  H. Kang,et al.  δ-Integration of endo/exo-glucanase and β-glucosidase genes into the yeast chromosomes for direct conversion of cellulose to ethanol , 1999 .

[13]  E. Bayer,et al.  Action of Designer Cellulosomes on Homogeneous Versus Complex Substrates , 2005, Journal of Biological Chemistry.

[14]  M. Penttilä,et al.  Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae. , 1988, Gene.

[15]  Huimin Zhao,et al.  Yeast Surface Display of Trifunctional Minicellulosomes for Simultaneous Saccharification and Fermentation of Cellulose to Ethanol , 2009, Applied and Environmental Microbiology.

[16]  L. Lynd,et al.  Consolidated bioprocessing of cellulosic biomass: an update. , 2005, Current opinion in biotechnology.

[17]  Shen-Long Tsai,et al.  Functional Assembly of Minicellulosomes on the Saccharomyces cerevisiae Cell Surface for Cellulose Hydrolysis and Ethanol Production , 2009, Applied and Environmental Microbiology.

[18]  G. O'neill,et al.  Expression and Secretion of a Cellulomonas fimi Exoglucanase in Saccharomyces cerevisiae , 1988, Applied and Environmental Microbiology.

[19]  David K. Johnson,et al.  Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production , 2007, Science.

[20]  Lee R Lynd,et al.  Cellulose utilization by Clostridium thermocellum: bioenergetics and hydrolysis product assimilation. , 2005, Proceedings of the National Academy of Sciences of the United States of America.