Production of Clostridium thermocellum cohesion 3 fusion proteins for cell surface docking on recombinant Saccharomyces cerevisiae
Consolidated bioprocessing (CBP) for the production of bioethanol aims to harness engineered microbes capable of saccharifying and fermenting waste lignocellulosic biomass in one industrial step. With the goal of collaboratively producing a Saccharomyces cerevisiae strain designed for the attachment of cellulolytic mini-cellulosomes at a Clostridium thermocellum dockerin 3 displayed on its cell surface, I have produced two Cohesin-3 fusion proteins to validate the availability of the cell-surface dockerin, and verified the availability of the fused cohesion through the use of a fluorescence assay for cellulose binding, which relies on the cohesion/dockerin interaction. The novel attachment system of the yeast cell-surface dockerin 3 will allow high-throughput analysis of mini-cellulosomes. A wheat-germ based cell-free expression system was used to produce the two Cohesin 3 fusions of CFP and C. thermocellum beta-glucosidase. The first will be used to detect dockerin localization, and the second to demonstrate yeast growth on cellobiose, the beta-glA substrate.