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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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Optimization of the cellulosome and ethanol production activity by controlling the combination ratio of different cell populations. (A) The optimal ratio of mini CipA (cohesin) to CelA (dockerin). CelA was regarded as a representative of total cells harboring dockerin-fused enzymes. EBY100 cells displaying mini CipA and secreting CelA were combined at the indicated ratios, and the released reducing sugars from PASC were detected using the harvested cells. (B) The optimal ratio of CelA to CBHII. The optimal CelA:CBHII ratio for PASC hydrolysis was determined while fixing the amount of cells displaying mini CipA as 1/4 of total cells. (C) The optimal ratio of BGLI to cellulosome for ethanol production. The optimal amount of BGL1 for ethanol production was determined while fixing the mini CipA:CelA:CBHII ratio of 2:3:3. The bar graph represents inoculation portion of cells containing each plasmid. The square line graph represents released reducing sugars from PASC in each ratio. The triangle line graph represents ethanol concentration after three-day fermentation in each ratio.
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Figure 3: Optimization of the cellulosome and ethanol production activity by controlling the combination ratio of different cell populations. (A) The optimal ratio of mini CipA (cohesin) to CelA (dockerin). CelA was regarded as a representative of total cells harboring dockerin-fused enzymes. EBY100 cells displaying mini CipA and secreting CelA were combined at the indicated ratios, and the released reducing sugars from PASC were detected using the harvested cells. (B) The optimal ratio of CelA to CBHII. The optimal CelA:CBHII ratio for PASC hydrolysis was determined while fixing the amount of cells displaying mini CipA as 1/4 of total cells. (C) The optimal ratio of BGLI to cellulosome for ethanol production. The optimal amount of BGL1 for ethanol production was determined while fixing the mini CipA:CelA:CBHII ratio of 2:3:3. The bar graph represents inoculation portion of cells containing each plasmid. The square line graph represents released reducing sugars from PASC in each ratio. The triangle line graph represents ethanol concentration after three-day fermentation in each ratio.

Mentions: First, in order to simplify possible combinations, we investigated the mini CipA portion of total population that makes the amount of displayed enzymes on the cell-surface maximum. EBY100 containing p424-s.s-CelA (EBY100/CelA) was regarded as a representative of cells secreting dockerin-fused enzymes. EBY100/CelA and EBY100 containing pCT-mini CipA (EBY100/mini CipA) were co-cultured for 24 h in synthetic galactose casamino acids (SG-CAA) medium in various ratios. The harvested cells were incubated with phosphoric acid swollen cellulose (PASC), and the released reducing sugars were measured by dinitrosalicylic acid (DNS) method. Detection of the CelA endoglucanase activity in the harvested cells indicates successful binding of the secreted CelA to the mini CipA displayed on the surface (Figure 3A). We also confirmed mini CipA-dependent display of CelA on the yeast surface by indirect immunofluorescence (data not shown). The amount of released reducing sugars reached a peak at mini CipA:CelA ratio of 1:3.


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)

Optimization of the cellulosome and ethanol production activity by controlling the combination ratio of different cell populations. (A) The optimal ratio of mini CipA (cohesin) to CelA (dockerin). CelA was regarded as a representative of total cells harboring dockerin-fused enzymes. EBY100 cells displaying mini CipA and secreting CelA were combined at the indicated ratios, and the released reducing sugars from PASC were detected using the harvested cells. (B) The optimal ratio of CelA to CBHII. The optimal CelA:CBHII ratio for PASC hydrolysis was determined while fixing the amount of cells displaying mini CipA as 1/4 of total cells. (C) The optimal ratio of BGLI to cellulosome for ethanol production. The optimal amount of BGL1 for ethanol production was determined while fixing the mini CipA:CelA:CBHII ratio of 2:3:3. The bar graph represents inoculation portion of cells containing each plasmid. The square line graph represents released reducing sugars from PASC in each ratio. The triangle line graph represents ethanol concentration after three-day fermentation in each ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Optimization of the cellulosome and ethanol production activity by controlling the combination ratio of different cell populations. (A) The optimal ratio of mini CipA (cohesin) to CelA (dockerin). CelA was regarded as a representative of total cells harboring dockerin-fused enzymes. EBY100 cells displaying mini CipA and secreting CelA were combined at the indicated ratios, and the released reducing sugars from PASC were detected using the harvested cells. (B) The optimal ratio of CelA to CBHII. The optimal CelA:CBHII ratio for PASC hydrolysis was determined while fixing the amount of cells displaying mini CipA as 1/4 of total cells. (C) The optimal ratio of BGLI to cellulosome for ethanol production. The optimal amount of BGL1 for ethanol production was determined while fixing the mini CipA:CelA:CBHII ratio of 2:3:3. The bar graph represents inoculation portion of cells containing each plasmid. The square line graph represents released reducing sugars from PASC in each ratio. The triangle line graph represents ethanol concentration after three-day fermentation in each ratio.
Mentions: First, in order to simplify possible combinations, we investigated the mini CipA portion of total population that makes the amount of displayed enzymes on the cell-surface maximum. EBY100 containing p424-s.s-CelA (EBY100/CelA) was regarded as a representative of cells secreting dockerin-fused enzymes. EBY100/CelA and EBY100 containing pCT-mini CipA (EBY100/mini CipA) were co-cultured for 24 h in synthetic galactose casamino acids (SG-CAA) medium in various ratios. The harvested cells were incubated with phosphoric acid swollen cellulose (PASC), and the released reducing sugars were measured by dinitrosalicylic acid (DNS) method. Detection of the CelA endoglucanase activity in the harvested cells indicates successful binding of the secreted CelA to the mini CipA displayed on the surface (Figure 3A). We also confirmed mini CipA-dependent display of CelA on the yeast surface by indirect immunofluorescence (data not shown). The amount of released reducing sugars reached a peak at mini CipA:CelA ratio of 1:3.

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