Functional assembly of minicellulosomes on the Saccharomyces cerevisiae cell surface for cellulose hydrolysis and ethanol production

Abstract

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 beta-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.

FIG. 1.

Functional assembly of minicellulosomes on the yeast cell surface. A trifunctional scaffoldin (Scaf-ctf) consisting of an internal CBD flanked by three divergent cohesin (C) domains from C. thermocellum (t), C. cellulolyticum (c), and R. flavefaciens (f) was displayed on the yeast cell surface. Three different cellulases (E1, E2, and E3) fused with the corresponding dockerin domain (either Dt, Dc, or Df) were expressed in E. coli. Cell lysates containing these cellulases were mixed with yeast cells displaying Scaf-ctf for the functional assembly of the minicellulosome.

FIG. 2.

Phase-contrast and immunofluorescence micrographs of yeast cells displaying minicellulosomes. (A) Cells displaying either scaffoldin Scaf-c, Sacf-ct, or Sacf-ctf. Functional assembly of three dockerin-tagged cellulases (CelE-Dc [Ec], CelA-Dt [At], or CelG-Df [Gf]) on cells displaying (B) Sacf-ctf, (C) Sacf-ct, or (D) Scaf-c. Cells were probed with either anti-c-Myc or anti-c-His₆ serum and fluorescently stained with a goat anti-mouse IgG conjugated with Alexa Fluor 488. Cells displaying only the scaffoldins were used as controls.

FIG. 3.

Fluorescence intensity of cells either displaying scaffoldin Sacf-ctf or with different combinations of dockerin-tagged cellulases (At [A], Ec [E], and Gf [G]) docked on the displayed Sacf-ctf. Cells were probed with either anti-c-Myc or anti-c-His₆ serum and fluorescently stained with goat anti-mouse IgG conjugated with Alexa Fluor 488. Whole-cell fluorescence was determined with a fluorescence microplate reader. Cells displaying only Scaf-ctf were used as controls. RFU, relative fluorescence units.

FIG. 4.

Whole-cell hydrolysis of CMC by different cellulase pairs (CelE-Dc [Ec], CelA-Dt [At], or CelG-Df [Gf]) docked on the displayed Scaf-ctf protein. Cells displaying only Scaf-ctf were used as controls.

FIG. 5.

Production of glucose (A) and reducing sugars (B) from the hydrolysis of PASC by free enzymes and by surface-displayed cellulosomes. Reactions were conducted either with different cellulase pairs (CelE-Dc [Ec], CelA-Dt [At], or β-glucosidase-Df [BglA]) docked on the displayed Scaf-ctf protein or with the corresponding purified cellulases. Cells displaying only Scaf-ctf were used as controls.

FIG. 6.

Time profiles of ethanol production (A) and cellulose hydrolysis (B) from PASC by control strain EBY100 plus free enzymes and yeast cells displaying functional cellulosomes. Fermentations were conducted either with different cellulase pairs (CelE-Dc [Ec], CelA-Dt [At], or β-glucosidase-Df [BglA]) docked on cells displaying Scaf-ctf or with control strain EBY100 plus the corresponding purified cellulases. Cells displaying only Scaf-ctf were used as controls. The individual enzyme amounts were the same in all cases.