Synthetic RNA switches for yeast metabolic engineering: screening recombinant enzyme libraries

Directed evolution is a powerful technique for increasing the activity of poorly active enzymes, for example when an enzyme is engineered to accept a new substrate or function in a new environment. Since enzyme activity greatly depends on the enzyme environment, screening should be performed under the same conditions as the ultimate application of the enzyme. When an enzyme will be used in live cells, RNA biosensors offer a powerful and flexible method of linking the desired phenotype, production of a small molecule of interest, to an easily measured phenotype, such as fluorescence. Here, we describe methods for screening enzyme libraries using an RNA biosensor, showing examples from the evolution of a P450 monooxygenase.