Generating enzymes with enhanced catalytic activity via cell-mediated directed evolutionTechnology #m06-088
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Directed evolution holds promise for creating designer enzymes for a variety of applications, including biomedical research and renewable energy, and can be used to select molecules with improved binding and catalytic properties from large libraries of molecules. Directed evolution, however, has not yet been successfully applied to enzyme catalysis, as there are no suitable high-throughput selections for the large number of protein variants for most of these reactions. The technology described herein overcomes this issue by combining directed evolution with chemical complementation, a high-throughput in vivo selection for enzyme catalysis, and in vivo mutagenesis, to create large, high-quality libraries of DNA mutants. These libraries could potentially be utilized for the development of assays to select for efficient enzyme mutants or to quickly and inexpensively create an enzyme with any desired activity de novo.
Cell-mediated directed evolution improves the size and quality of mutant libraries
This technology combines directed evolution with in vivo chemical complementation and mutagenesis, to allow for the generation and selection of mutant molecules simultaneously. Directed evolution generates more efficient DNA mutants with enhanced catalytic activity, while chemical complementation serves as a high-throughput assay for enzyme catalysis based on the yeast three-hybrid assay. By carrying out both DNA shuffling and chemical complementation in vivo, this process eliminates the limitation of transformation efficiency on library size. This strategy allows for the generation of larger libraries of >1024 mutants, for the development of proteins with more significant changes in function than existing methods.
As a proof of concept, this technology has been tested for directed evolution for protein function for a number of enzyme classes, including cellulases, glycosidases, and aldolases.
- Biological catalyst design for use in research, medical diagnostics, and therapeutics
- Peptide-based drug discovery
- Development of assays for the selection of efficient enzyme mutants
- Generation of DNA libraries for directed evolution of novel or improved proteins
- Creation of an enzyme with any desired effect de novo
- Directed evolution of hydrolases in the energy sector
- More efficient production of ethanol for use as a renewable energy source
- Increased catalytic ability of cellulase for commercial food processing (drying beans, coffee) and textile manufacturing (denim)
- Cell-mediated directed evolution can generate large and high-quality libraries of mutants
- This selection process can cause more significant changes to enzyme function than existing methods, having a greater impact on catalytic activity
- Allows for selection of optimized enzymes with increased efficiency and activity, which could be more cost effective
- DNA shuffling occurs in vivo, allowing simultaneous mutation and selection
- In vivo DNA shuffling removes the limitation on library size due to DNA transformation efficiency
Patent Issued (US 9,169,481)
Tech Ventures Reference: IR M06-088, IR M06-108, IR M07-023