Abstract
BackgroundCellulosic 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.ResultsWe 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).ConclusionsWe 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.
Highlights
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
Construction of a minicellulosome structure on the yeast surface 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)
CelA and CBHII were expressed with the native dockerin (DocA) and exogenous dockerin from C. thermocellum CelS (DocS), respectively
Summary
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. Cellulosic biomass is the most abundant materials in nature and relatively apart from the ethical concerns raised against the use of food-based materials such as corn and sugarcane [4,5]. Despite these advantages, the recalcitrant nature of cellulosic biomass and the high cost of cellulase are the major obstacles to utilize cellulosic biomass [6,7]. Exoglucanase further hydrolyzes the cellulose chains from their reducing or non-reducing ends, releasing cellodextrins, mainly cellobiose, which are converted to glucose by β-glucosidase [8,9]
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