Authors: Motoki Kojima Tomohiro Akahoshi Kenji Okamoto Hideshi Yanase
Publish Date: 2012/10/03
Volume: 96, Issue: 4, Pages: 1093-1104
Abstract
In order to reduce the cost of bioethanol production from lignocellulosic biomass we developed a tool for cell surface display of cellulolytic enzymes on the ethanologenic bacterium Zymobacter palmae Z palmae is a novel ethanolfermenting bacterium capable of utilizing a broad range of sugar substrates but not cellulose Therefore to express and display heterologous cellulolytic enzymes on the Z palmae cell surface we utilized the cellsurface display motif of the Pseudomonas ice nucleation protein Ina The gene encoding Ina from Pseudomonas syringae IFO3310 was cloned and its product was comprised of three functional domains an Nterminal domain a central domain with repeated amino acid residues and a Cterminal domain The Nterminal domain of Ina was shown to function as the anchoring motif for a green fluorescence protein fusion protein in Escherichia coli To express a heterologous cellulolytic enzyme extracellularly in Z palmae we fused the Nterminal coding sequence of Ina to the coding sequence of an Nterminaltruncated Cellulomonas endoglucanase Z palmae cells carrying the fusion endoglucanase gene were shown to degrade carboxymethyl cellulose Although a portion of the expressed fusion endoglucanase was released from Z palmae cells into the culture broth we confirmed the display of the protein on the cell surface by immunofluorescence microscopy The results indicate that the Nterminal anchoring motif of Ina from P syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z palmae
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