Format

Send to

Choose Destination
Metab Eng. 2012 Jul;14(4):306-16. doi: 10.1016/j.ymben.2012.04.004. Epub 2012 Apr 25.

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

Author information

1
Department of Bioengineering, 1200 E. California Blvd., MC 210-41, California Institute of Technology, Pasadena, CA 91125, USA.

Abstract

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.

PMID:
22554528
DOI:
10.1016/j.ymben.2012.04.004
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center