Heterologous expression of a Clostridium minicellulosome in Saccharomyces cerevisiae
Date
2009
Authors
Lilly M.
Fierobe H.-P.
Van Zyl W.H.
Volschenk H.
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Abstract
The yeast Saccharomyces cerevisiae was genetically modified to assemble a minicellulosome on its cell surface by heterologous expression of a chimeric scaffoldin protein from Clostridium cellulolyticum under the regulation of the phosphoglycerate kinase 1 (PGK1) promoter and terminator regulatory elements, together with the β-xylanase 2 secretion signal of Trichoderma reesei and cell wall protein 2 (Cwp2) of S. cerevisiae. Fluorescent microscopy and Far Western blot analysis confirmed that the Scaf3p is targeted to the yeast cell surface and that the Clostridium thermocellum cohesin domain is functional in yeast. Similarly, functionality of the C. thermocellum dockerin domain in yeast is shown by binding to the Scaf3 protein in Far Western blot analysis. Phenotypic evidence for cohesin-dockerin interaction was also established with the detection of a twofold increase in tethered endoglucanase enzyme activity in S. cerevisiae cells expressing the Scaf3 protein compared with the parent strain. This study highlights the feasibility to future design of enhanced cellulolytic strains of S. cerevisiae through emulation of the cellulosome concept. Potentially, Scaf3p-armed yeast could also be developed into an alternative cell surface display strategy with various tailor-made applications. © 2009 Federation of European Microbiological Societies.
Description
Keywords
cell membrane protein, cell membrane protein 2, cellulosome, cohesin, endo 1,4 beta xylanase, fungal protein, glucan synthase, phosphoglycerate kinase, phosphoglycerate kinase 1, protein dockerin, protein scaf3, unclassified drug, article, Clostridium, Clostridium cellulolyticum, Clostridium thermocellum, controlled study, enzyme activity, enzyme regulation, heterologous expression, nonhuman, protein assembly, protein binding, protein domain, protein function, protein interaction, protein targeting, Saccharomyces cerevisiae, signal transduction, Bacterial Proteins, Blotting, Far-Western, Cellulase, Cellulose, Cellulosomes, Clostridium thermocellum, Membrane Proteins, Microscopy, Fluorescence, Promoter Regions, Genetic, Protein Binding, Protein Sorting Signals, Recombinant Proteins, Saccharomyces cerevisiae, Trichoderma, Clostridium, Clostridium cellulolyticum, Clostridium thermocellum, Hypocrea jecorina, Saccharomyces cerevisiae
Citation
FEMS Yeast Research
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