Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae.
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 ethanol-fermenting 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 cell-surface 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 N-terminal domain, a central domain with repeated amino acid residues, and a C-terminal domain. The N-terminal 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 N-terminal coding sequence of Ina to the coding sequence of an N-terminal-truncated 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 N-terminal anchoring motif of Ina from P. syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z. palmae.
Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, 4-101 Koyamacho-Minami, Tottori, Tottori 680-8552, Japan.
Motoki
M
Kojima
Tomohiro
T
Akahoshi
Kenji
K
Okamoto
Hideshi
H
Yanase
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D040143:Cellulomonas / Q000201:enzymology* / Q000235:genetics; D000431:Ethanol / Q000378:metabolism*; D015870:Gene Expression; D044129:Halomonadaceae / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D021381:Protein Transport; D044224:Pseudomonas syringae / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D000431:Ethanol; D002480:Cellulase