Directed evolution is a versatile method for the creation of proteins with novel properties. The evolution of enzymes has already provided variants with higher stability, increased efficiency, and altered specificity or enantioselectivity. As the trend of using enzymes in industrial processes, the synthesis of pharmaceuticals, and consumer health and cleaning products continues to grow, newer and better directed evolution techniques will be required to solve more complex problems. One major limitation of current directed evolution approaches is the random library used to search for improved proteins. Current technology enables average libraries on the order of 10'^8-10'^9 variants, while research has suggested that much larger libraries (>10'^13) will be necessary to discover enzymes with significantly altered specificities or entirely new functions. This proposal describes a new system in yeast that aims to overcome current limitations on library size.
Aim 1 describes a method for performing in vivo mutagenesis of a target gene within the budding yeast S. cerevisiae. This method relies on the highly efficient homologous recombination machinery in yeast. Mutagenic libraries, introduced on plasmids, will be liberated using a highly specific endonuclease. The resulting linear DNA strands will target and mutagenize a gene of interest through homologous recombination.
Aim 2 outlines a method for sharing mutagenic libraries between yeast to increase potential library size. By allowing two collections of yeast with complementary random libraries to mate, those two libraries can be shared among the entire population. A selective pressure will be applied, thus propagating only the best variants while essentially searching all possible mutants. Such a protocol has the potential to search very large virtual libraries well beyond the ability of existing technology.
Aim 3 proposes a directed evolution experiment that uses the methods developed in Aims 1 and 2. A new variant of Cre, a site-specific recombinase widely used in mouse genetics, will be evolved using the in vivo mutagenesis and mating procedures. This experiment should convincingly demonstrate the utility of these new methods.
This proposal describes a new technique for directed evolution of enzymes. New enzymes generated using this technique could be used in the discovery and synthesis of new drugs or as drugs themselves. The proposed methodology could provide access to proteins with uses as varied as cleaning products, environmental remediation, and artificial organs.
| Romanini, Dante W; Peralta-Yahya, Pamela; Mondol, Vanessa et al. (2012) A Heritable Recombination system for synthetic Darwinian evolution in yeast. ACS Synth Biol 1:602-9 |
