Augmenting weak enzyme activity

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. 

In principle, directed evolution procedures could be repeated indefinitely until any desired activity has been attained. At some point, however, the catalyst will be sufficiently active that the host cell grows like the wild-type strain, making selection for further improvement difficult. This is true for the modified hexamer even though it is still an order of magnitude less efficient than the homodimeric MjCM [37]. Since total activity depends on the catalyst concentration as well as specific activity, reducing the available catalyst concentration can further increase selection pressure. In practice, intracellular protein concentrations can be lowered in a variety of ways, including the use of low copy plasmids [101], weak promoters [102] and inefficient ribosome binding sites [103]. 

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