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Cellulosic ethanol production using a yeast consortium displaying a minicellulosome and β-glucosidase.

Kim S, Baek SH, Lee K, Hahn JS - Microb. Cell Fact. (2013)

Bottom Line: Consolidated bioprocessing (CBP), combining cellulase production, saccharification, and fermentation into one step, has been proposed as the most efficient way to reduce the production cost of cellulosic bioethanol.On the other hand, BGLI was independently assembled to the cell surface since we newly found that it already has a cell adhesion characteristic.A mixture of cells with the optimized mini CipA:CelA:CBHII:BGLI ratio of 2:3:3:0.53 produced 1.80 g/l ethanol after 94 h, indicating about 20% increase compared with a consortium composed of an equal amount of each cell type (1.48 g/l).

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic of Korea.

ABSTRACT

Background: Cellulosic biomass is considered as a promising alternative to fossil fuels, but its recalcitrant nature and high cost of cellulase are the major obstacles to utilize this material. Consolidated bioprocessing (CBP), combining cellulase production, saccharification, and fermentation into one step, has been proposed as the most efficient way to reduce the production cost of cellulosic bioethanol. In this study, we developed a cellulolytic yeast consortium for CBP, based on the surface display of cellulosome structure, mimicking the cellulolytic bacterium, Clostridium thermocellum.

Results: We designed a cellulolytic yeast consortium composed of four different yeast strains capable of either displaying a scaffoldin (mini CipA) containing three cohesin domains derived from C. thermocellum, or secreting one of the three types of cellulases, C. thermocellum CelA (endoglucanase) containing its own dockerin, Trichoderma reesei CBHII (exoglucanase) fused with an exogenous dockerin from C. thermocellum, or Aspergillus aculeatus BGLI (β-glucosidase). The secreted dockerin-containing enzymes, CelA and CBHI, were randomly assembled to the surface-displayed mini CipA via cohesin-dockerin interactions. On the other hand, BGLI was independently assembled to the cell surface since we newly found that it already has a cell adhesion characteristic. We optimized the cellulosome activity and ethanol production by controlling the combination ratio among the four yeast strains. A mixture of cells with the optimized mini CipA:CelA:CBHII:BGLI ratio of 2:3:3:0.53 produced 1.80 g/l ethanol after 94 h, indicating about 20% increase compared with a consortium composed of an equal amount of each cell type (1.48 g/l).

Conclusions: We produced cellulosic ethanol using a cellulolytic yeast consortium, which is composed of cells displaying mini cellulosomes generated via random assembly of CelA and CBHII to a mini CipA, and cells displaying BGLI independently. One of the advantages of this system is that ethanol production can be easily optimized by simply changing the combination ratio of the different populations. In addition, there is no limitation on the number of enzymes to be incorporated into this cellulosome structure. Not only cellulases used in this study, but also any other enzymes, including cellulases and hemicellulases, could be applied just by fusing dockerin domains to the enzymes.

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Design of a minicellulosome structure on the yeast surface. (A) Schematic diagram of the overall concept of this research. The basic design of this research is composed of four different yeast strains, one for displaying the mini CipA, and the others for secreting three types of cellulases. Mini CipA, a modified scaffoldin including a CBM and three cohesin domains, was expressed as an Aga2-fusion protein. The dockerin fused-enzymes, C. thermocellum CelA and T. reesei CBHII, were randomly assembled to mini CipA via cohesin-dockerin interactions, whereas the secreted A. aculeatus BGLI was independently bound to the cell surface through its own cell surface adhesion characteristic. Coh, cohesin; CBM, carbohydrate-binding module; DocA and DocS, native dockerin in CelA and exogenous dockerin from C. thermocellum CelS, respectively. (B) Construction of a surface display vector, pCT-mini CipA. Mini CipA, a modified scaffoldin containing a CBM and three cohesin domains, was expressed under the control of GAL1 promoter as an Aga2-fusion protein for displaying on the yeast surface. (C) Construction of cellulase secretion vectors. α-factor prepro-peptide (s.s) was used as a signal sequence.
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Figure 1: Design of a minicellulosome structure on the yeast surface. (A) Schematic diagram of the overall concept of this research. The basic design of this research is composed of four different yeast strains, one for displaying the mini CipA, and the others for secreting three types of cellulases. Mini CipA, a modified scaffoldin including a CBM and three cohesin domains, was expressed as an Aga2-fusion protein. The dockerin fused-enzymes, C. thermocellum CelA and T. reesei CBHII, were randomly assembled to mini CipA via cohesin-dockerin interactions, whereas the secreted A. aculeatus BGLI was independently bound to the cell surface through its own cell surface adhesion characteristic. Coh, cohesin; CBM, carbohydrate-binding module; DocA and DocS, native dockerin in CelA and exogenous dockerin from C. thermocellum CelS, respectively. (B) Construction of a surface display vector, pCT-mini CipA. Mini CipA, a modified scaffoldin containing a CBM and three cohesin domains, was expressed under the control of GAL1 promoter as an Aga2-fusion protein for displaying on the yeast surface. (C) Construction of cellulase secretion vectors. α-factor prepro-peptide (s.s) was used as a signal sequence.

Mentions: The basic design of this research is composed of four different yeast strains, one of which for displaying the mini CipA, and the others for secreting one of the three types of cellulases (Figure 1A). Mini CipA, a modified C. thermocellum scaffoldin containing a CBM and three cohesin domains, was expressed under the control of GAL1 promoter as an Aga2-fusion protein on the surface of yeast EBY100 strain (Figure 1B). In order to hydrolyze cellulose to glucose, endoglucanase CelA from C. thermocellum, exoglucanase CBHII from Trichoderma reesei, and β-glucosidase BGLI from Aspergillus aculeatus were secreted using α-factor prepro-peptide (Figure 1C). CelA and CBHII were expressed with the native dockerin (DocA) and exogenous dockerin from C. thermocellum CelS (DocS), respectively.


Cellulosic ethanol production using a yeast consortium displaying a minicellulosome and β-glucosidase.

Kim S, Baek SH, Lee K, Hahn JS - Microb. Cell Fact. (2013)

Design of a minicellulosome structure on the yeast surface. (A) Schematic diagram of the overall concept of this research. The basic design of this research is composed of four different yeast strains, one for displaying the mini CipA, and the others for secreting three types of cellulases. Mini CipA, a modified scaffoldin including a CBM and three cohesin domains, was expressed as an Aga2-fusion protein. The dockerin fused-enzymes, C. thermocellum CelA and T. reesei CBHII, were randomly assembled to mini CipA via cohesin-dockerin interactions, whereas the secreted A. aculeatus BGLI was independently bound to the cell surface through its own cell surface adhesion characteristic. Coh, cohesin; CBM, carbohydrate-binding module; DocA and DocS, native dockerin in CelA and exogenous dockerin from C. thermocellum CelS, respectively. (B) Construction of a surface display vector, pCT-mini CipA. Mini CipA, a modified scaffoldin containing a CBM and three cohesin domains, was expressed under the control of GAL1 promoter as an Aga2-fusion protein for displaying on the yeast surface. (C) Construction of cellulase secretion vectors. α-factor prepro-peptide (s.s) was used as a signal sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585817&req=5

Figure 1: Design of a minicellulosome structure on the yeast surface. (A) Schematic diagram of the overall concept of this research. The basic design of this research is composed of four different yeast strains, one for displaying the mini CipA, and the others for secreting three types of cellulases. Mini CipA, a modified scaffoldin including a CBM and three cohesin domains, was expressed as an Aga2-fusion protein. The dockerin fused-enzymes, C. thermocellum CelA and T. reesei CBHII, were randomly assembled to mini CipA via cohesin-dockerin interactions, whereas the secreted A. aculeatus BGLI was independently bound to the cell surface through its own cell surface adhesion characteristic. Coh, cohesin; CBM, carbohydrate-binding module; DocA and DocS, native dockerin in CelA and exogenous dockerin from C. thermocellum CelS, respectively. (B) Construction of a surface display vector, pCT-mini CipA. Mini CipA, a modified scaffoldin containing a CBM and three cohesin domains, was expressed under the control of GAL1 promoter as an Aga2-fusion protein for displaying on the yeast surface. (C) Construction of cellulase secretion vectors. α-factor prepro-peptide (s.s) was used as a signal sequence.
Mentions: The basic design of this research is composed of four different yeast strains, one of which for displaying the mini CipA, and the others for secreting one of the three types of cellulases (Figure 1A). Mini CipA, a modified C. thermocellum scaffoldin containing a CBM and three cohesin domains, was expressed under the control of GAL1 promoter as an Aga2-fusion protein on the surface of yeast EBY100 strain (Figure 1B). In order to hydrolyze cellulose to glucose, endoglucanase CelA from C. thermocellum, exoglucanase CBHII from Trichoderma reesei, and β-glucosidase BGLI from Aspergillus aculeatus were secreted using α-factor prepro-peptide (Figure 1C). CelA and CBHII were expressed with the native dockerin (DocA) and exogenous dockerin from C. thermocellum CelS (DocS), respectively.

Bottom Line: Consolidated bioprocessing (CBP), combining cellulase production, saccharification, and fermentation into one step, has been proposed as the most efficient way to reduce the production cost of cellulosic bioethanol.On the other hand, BGLI was independently assembled to the cell surface since we newly found that it already has a cell adhesion characteristic.A mixture of cells with the optimized mini CipA:CelA:CBHII:BGLI ratio of 2:3:3:0.53 produced 1.80 g/l ethanol after 94 h, indicating about 20% increase compared with a consortium composed of an equal amount of each cell type (1.48 g/l).

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic of Korea.

ABSTRACT

Background: Cellulosic biomass is considered as a promising alternative to fossil fuels, but its recalcitrant nature and high cost of cellulase are the major obstacles to utilize this material. Consolidated bioprocessing (CBP), combining cellulase production, saccharification, and fermentation into one step, has been proposed as the most efficient way to reduce the production cost of cellulosic bioethanol. In this study, we developed a cellulolytic yeast consortium for CBP, based on the surface display of cellulosome structure, mimicking the cellulolytic bacterium, Clostridium thermocellum.

Results: We designed a cellulolytic yeast consortium composed of four different yeast strains capable of either displaying a scaffoldin (mini CipA) containing three cohesin domains derived from C. thermocellum, or secreting one of the three types of cellulases, C. thermocellum CelA (endoglucanase) containing its own dockerin, Trichoderma reesei CBHII (exoglucanase) fused with an exogenous dockerin from C. thermocellum, or Aspergillus aculeatus BGLI (β-glucosidase). The secreted dockerin-containing enzymes, CelA and CBHI, were randomly assembled to the surface-displayed mini CipA via cohesin-dockerin interactions. On the other hand, BGLI was independently assembled to the cell surface since we newly found that it already has a cell adhesion characteristic. We optimized the cellulosome activity and ethanol production by controlling the combination ratio among the four yeast strains. A mixture of cells with the optimized mini CipA:CelA:CBHII:BGLI ratio of 2:3:3:0.53 produced 1.80 g/l ethanol after 94 h, indicating about 20% increase compared with a consortium composed of an equal amount of each cell type (1.48 g/l).

Conclusions: We produced cellulosic ethanol using a cellulolytic yeast consortium, which is composed of cells displaying mini cellulosomes generated via random assembly of CelA and CBHII to a mini CipA, and cells displaying BGLI independently. One of the advantages of this system is that ethanol production can be easily optimized by simply changing the combination ratio of the different populations. In addition, there is no limitation on the number of enzymes to be incorporated into this cellulosome structure. Not only cellulases used in this study, but also any other enzymes, including cellulases and hemicellulases, could be applied just by fusing dockerin domains to the enzymes.

Show MeSH
Related in: MedlinePlus