Mimicking nature's synergy: engineering FN3-based protein scaffolds for multi-enzyme assembly
| Author(s) | Chen, Long | |
| Date Accessioned | 2015-12-02T15:00:08Z | |
| Date Available | 2015-12-02T15:00:08Z | |
| Publication Date | 2015 | |
| Abstract | Multi-enzyme cascade reactions are common in nature. Compared to free-floating enzyme catalysis, enzyme co-localization, existing in some natural systems, can improve the efficiency of the reactions thanks to kinetic benefits. In engineering multi-enzyme reactions, multi-functional enzyme complexes can be constructed to mimic nature's synergy and "substrate-channeling" behavior. This thesis focuses on engineering the spatial organization of enzymes using an antibody mimic, the 10th human fibronectin type III (FN3) domain, as the building block. Due to the small size, good solubility and stability of FN3, along with the diversity of FN3-binders, a complex protein scaffold platform can be built. By employing the specific and tight binding interactions between FN3-binders and their corresponding monobodies (mutated FN3), FN3-binder fused enzymes can be docked onto the protein scaffold and result in the formation of synthetic "substrate-channels". Two particular pathways have been studied and executed. First, the scaffold was designed to be displayed on the cell surface of S. cerevisiae for designer cellulosome construction which aims at efficient hydrolysis of biomass. Experimental results showed that three engineered FN3 domains have orthogonal and high-affinity binding with their targets. Second, the scaffold was designed to be applied in E.coli cells to assemble the metabolic enzymes involved in the conversion of methanol for the production of n-butanol in order to achieve a metabolic flux enhancement. | en_US |
| Advisor | Chen, Wilfred | |
| Degree | M.Ch.E. | |
| Department | University of Delaware, Department of Chemical and Biomolecular Engineering | |
| Unique Identifier | 930786148 | |
| URL | http://udspace.udel.edu/handle/19716/17255 | |
| Publisher | University of Delaware | en_US |
| URI | http://search.proquest.com/docview/1728728262?accountid=10457 | |
| dc.subject.lcsh | Fibronectins. | |
| dc.subject.lcsh | Enzymes. | |
| dc.subject.lcsh | Saccharomyces cerevisiae -- Biotechnology. | |
| dc.subject.lcsh | Escherichia coli -- Biotechnology. | |
| dc.subject.lcsh | Protein binding. | |
| Title | Mimicking nature's synergy: engineering FN3-based protein scaffolds for multi-enzyme assembly | en_US |
| Type | Thesis | en_US |