Evolution chorismate mutases

Genetic selection is perhaps the most powerful technique currently available for analyzing and directing the evolution of large populations of biomacromolecules. In this chapter, we focus on its use as a tool in studying the structure and function of chorismate mutases. The reader is also referred to other more general reviews [7 - 17]. 

It is in the realm of very large combinatorial libraries that selection rather than screening gains crucial importance. As the focus shifts from randomizing an eight-residue peptide to a 100 amino acid protein (the typical size of a small functional domain, for example a chorismate mutase domain), the number of sequence permutations rises to an astronomical 20100. The ability to assay even a tiny fraction of this sequence space in directed molecular evolution experiments demands selection, even though initial development of an appropriate system may be considerably more involved than the setup of a screening procedure. 

True Darwinian evolution involves multiple cycles of mutation and selection. This process can be mimicked in a laboratory setting to optimize the properties of an inefficient enzyme. The hexameric but weakly active chorismate mutase [95] described in Section 3.3.4.1 has been improved in this way [99]. Mutations were introduced into the gene encoding the hexamer subunit by DNA shuffling (Fig. 3.16) [5, 100], which mimics sexual recombination in vitro. Improved variants were selected, as before, by their ability to complement the chorismate mutase deficiency in bacteria. Plasmid DNA was isolated from the fastest growing cells and the entire procedure was repeated. 

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