Heterologous expression of a Clostridium minicellulosome in Saccharomyces cerevisiae.
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 beta-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.
Lilly
Mariska
M
Department of Food and Agricultural Sciences, Faculty of Applied Science, Cape Peninsula University of Technology, Cape Town, South Africa.
Fierobe
Henri-Pierre
HP
van Zyl
Willem H
WH
Volschenk
Heinrich
H
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D026481:Blotting, Far-Western; D002480:Cellulase / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D044603:Cellulosomes / Q000235:genetics / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014242:Trichoderma / Q000235:genetics*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002482:Cellulose; D002480:Cellulase