Advances in Phage-Assisted Continuous Evolution and Application to Overcoming Bioinsecticide Resistance
| Autor: | Badran, Ahmed |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: | |
| Druh dokumentu: | Text |
| Popis: | The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits, constituting a substantial and increasingly large portion of the total global production of various crops, including cotton, corn, maize and soybeans. Bt toxins are exquisitely selective for targeted pests, typically do not affect off-target insects, and are completely orthogonal to human biology. However, the development of insect resistance to Bt toxins endangers their long-term effectiveness. In this thesis, I describe the development of methodology for the systematic directed evolution of novel Bt toxins to selectively affect resistant insects. Using Phage-Assisted Continuous Evolution (PACE), a previously developed general platform for the continuous directed evolution of biomolecules, I developed a highly sensitized selection for novel protein-protein interactions. This system robustly reported on interactions spanning affinities from low micromolar to picomolar. However, attempts at using this system for the directed evolution of novel protein-protein interactions were largely unsuccessful, presumably as a consequence of low mutagenesis efficiency. To increase the utility of the platform, I sought to enhance the mutagenesis levels afforded by PACE, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency, and narrow mutational spectra. I used a systematic, mechanism-driven approach to create a potent, inducible, broad-spectrum, and vector-based mutagenesis system in E. coli that enhances mutation rates by 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro mutagenesis methods. This system enabled the high-frequency, broad-spectrum mutagenesis of chromosomal, episomal, and viral nucleic acids in vivo, and dramatically enhanced the success of PACE experiments, highlighting the importance of mutagenesis efficiency on selection outcome. Using this enhanced mutagenesis approach and the previously described sensitized selection platform, I was able to evolve variants of the commonly used Bt toxin Cry1Ac that bind toxin binding region of a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not targeted by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome Bt toxin resistance in insects and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. In conclusion, these finding offer a novel mechanism of overcoming what is quickly becoming among the largest issue overshadowing the continued success of Bt toxins for pest control and management, and establish a platform for the detection and evolution of a wide array of protein-protein interactions. Chemical Biology |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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