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Published July 2012 | Supplemental Material
Journal Article Open

High-throughput enzyme evolution in Saccharomyces cerevisiae using a synthetic RNA switch


Metabolic engineering can produce a wide range of bulk and fine chemicals using renewable resources. These approaches frequently require high levels of activity from multiple heterologous enzymes. Directed evolution techniques have been used to improve the activity of a wide range of enzymes but can be difficult to apply when the enzyme is used in whole cells. To address this limitation, we developed generalizable in vivo biosensors using engineered RNA switches to link metabolite concentrations and GFP expression levels in living cells. Using such a sensor, we quantitatively screened large enzyme libraries in high throughput based on fluorescence, either in clonal cultures or in single cells by fluorescence activated cell sorting (FACS). By iteratively screening libraries of a caffeine demethylase, we identified beneficial mutations that ultimately increased the enzyme activity in vivo by 33 fold and the product selectivity by 22 fold. As aptamer selection strategies allow RNA switches to be readily adapted to recognize new small molecules, these RNA-based screening techniques are applicable to a broad range of enzymes and metabolic pathways.

Additional Information

© 2012 Elsevier Inc. Received 7 December 2011. Revised 9 April 2012. Accepted 17 April 2012. Available online 25 April 2012. The authors wish to thank M.M.Y. Chen for building and characterizing the original library from which the CDM1 demethylase was isolated and for assistance in troubleshooting the initial stages of the project; F.H. Arnold for serving as an invaluable resource on the directed evolution process; A.M. Sawayama for providing an introduction to directed evolution techniques; K.M. Hawkins for assisting with the design of the yeast expression system; J.C. Liang for aiding in the FACS screening; D.N. Macklin for cloning and characterizing several of the enzymes in new expression plasmids; and C.D. Snow for providing the homology model of CDM1. This work was supported by the National Science Foundation (CBET-0917638; fellowship to J.K.M.), the Institute for Collaborative Biotechnologies (U.S. Army Research Office DAAD19-03-D-0004), and the Alfred P. Sloan Foundation (fellowship to C.D.S.).

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