A simple selection strategy for evolving highly efficient enzymes.
Combining tunable transcription with an enzyme-degradation tag affords an effective means to reduce intracellular enzyme concentrations from high to very low levels. Such fine-tuned control allows selection pressure to be systematically increased in directed-evolution experiments. This facilitates identification of mutants with wild-type activity, as shown here for an engineered chorismate mutase. Numerous selection formats and cell-based screening methodologies may benefit from the large dynamic range afforded by this easily implemented strategy.
Neuenschwander
Martin
M
Laboratory of Organic Chemistry, ETH Zurich, Hönggerberg HCI F 339, CH-8093 Zurich, Switzerland.
Butz
Maren
M
Heintz
Caroline
C
Kast
Peter
P
Hilvert
Donald
D
D002826:Chorismate Mutase / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D004925:Escherichia / Q000201:enzymology; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D016296:Mutagenesis; D012641:Selection, Genetic; D013752:Tetracycline; D014158:Transcription, Genetic
D002826:Chorismate Mutase; D013752:Tetracycline