title: A comparison of directed evolution approaches using the beta-glucuronidase model system.
abstract: Protein engineers can alter the properties of enzymes by directing their evolution in vitro. Many methods to generate molecular diversity and to identify improved clones have been developed, but experimental evolution remains as much an art as a science. We previously used DNA shuffling (sexual recombination) and a histochemical screen to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with improved beta-galactosidase (BGAL) activity. Here, we employ the same model evolutionary system to test the efficiencies of several other techniques: recursive random mutagenesis (asexual), combinatorial cassette mutagenesis (high-frequency recombination) and a versatile high-throughput microplate screen. GUS variants with altered specificity evolved in each trial, but different combinations of mutagenesis and screening techniques effected the fixation of different beneficial mutations. The new microplate screen identified a broader set of mutations than the previously employed X-gal colony screen. Recursive random mutagenesis produced essentially asexual populations, within which beneficial mutations drove each other into extinction (clonal interference); DNA shuffling and combinatorial cassette mutagenesis led instead to the accumulation of beneficial mutations within a single allele. These results explain why recombinational approaches generally increase the efficiency of laboratory evolution.
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Rowe
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Geddie
authors.forename: Omar B
authors.initials: OB
authors.lastname: Alexander
authors.forename: Ichiro
authors.initials: I
authors.lastname: Matsumura
mesh_terms: D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005787:Gene Frequency; D005838:Genotype; D005966:Glucuronidase / Q000235:genetics*; D008958:Models, Molecular; D010641:Phenotype; D015202:Protein Engineering; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001616:beta-Galactosidase; D005966:Glucuronidase