Ou, Jingshen
(School of Bioscience and Bioengineering, South China University of Technology)
,
Cao, Yicheng
(School of Bioscience and Bioengineering, South China University of Technology)
In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the ...
In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications.
In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications.
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제안 방법
To compare the degradation capabilities of the free cellulases with those of the cellulases that were organized into minicellulosomes, hydrolysis efficiencies in parallel hydrolysis reaction conditions were measured. These reactions were set up using three types of cellulose substrates; namely, carboxymethyl cellulose (CMC), microcrystalline cellulose (MC), and filter paper (FP).
To further detect the localization and function of the CipA protein on the cell surface of P. pastoris, a microscopic inspection of its cellulose-binding capacity was performed. This binding was mediated by the CBM of the CipA protein anchor on the cell surface.
성능/효과
The incorporation of cellulases from different species into cellulosomes by the appending of dockerin tags to their termini may decrease hydrolytic activities for soluble cellulose but increase hydrolytic activities for microcrystalline cellulose [5, 7, 59]. In this study, the CipA of C. acetobutylicum had similarly enhanced hydrolytic activities for GH9 cellulases (Fig. 5) in both the cell-free state and CSD minicellulosomes, suggesting that the C. acetobutylicum CipA exerted superior synergistic effects for cellulases, such as the scaffoldin of other Clostridia.
In this study, the truncated scaffoldin of C. acetobutylicum was able to be expressed on the yeast cell surfaces and assemble with other species’ cellulases by cohesin-dockerin interactions, similar to other clostridial cellulosomes.
The results that are presented here indicate that the truncated scaffoldin from C. acetobutylicum may be successfully expressed on yeast cell surfaces and assembled with other species’ cellulases by cohesin-dockerin interactions.
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