Incorporation of Nasutitermes takasagoensis endoglucanase into cell surface-displayed minicellulosomes in Pichia pastoris X33

• Published in: Journal of microbiology and biotechnology Vol. 24; no. 9; pp. 1178 - 1188
• Main Authors: Ou, Jingshen, Cao, Yicheng
• Format: Journal Article
• Language: English
• Published: Korea (South) 한국미생물·생명공학회 01.09.2014
• Subjects: Animals; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cell Surface Display Techniques - methods; Cellulase - chemistry; Cellulase - genetics; Cellulase - metabolism; Cellulosomes - chemistry; Cellulosomes - genetics; Cellulosomes - metabolism; Insect Proteins - chemistry; Insect Proteins - genetics; Insect Proteins - metabolism; Isoptera - enzymology; Isoptera - genetics; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Pichia - cytology; Pichia - genetics; Pichia - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; 생물학
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.1402.02034

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.