Directed evolution of beta-galactosidase from Escherichia coli by mutator strains defective in the 3'-->5' exonuclease activity of DNA polymerase III.
Directed evolution of Escherichia coli beta-galactosidase into variants featuring beta-glucosidase activity was challenged. To this end, mutagenesis of lacZ was performed by replication in E. coli CC954, a mutator strain containing a DNA polymerase III defective in 3'-->5' exonuclease activity. beta-Galactosidase variants can be isolated upon mutagenesis of lacZ hosted into the self-transmissible episome F'128. Optimal evolution of lacZ can be achieved by propagation of E. coli CC954/F'128 cultures for 15 generations; further growth of mutator cultures for 37 or 55 generations imposes a high mutational load on lacZ and hinders the selection of efficiently evolved clones.
Stefan
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Department of Industrial Chemistry, University of Bologna, Viale Risorgimento 4, 40136, Bologna, Italy.
Radeghieri
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Gonzalez Vara y Rodriguez
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Hochkoeppler
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D001483:Base Sequence; D004258:DNA Polymerase III / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics*; D005798:Genes, Bacterial; D007763:Lac Operon; D016296:Mutagenesis; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004258:DNA Polymerase III; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D001617:beta-Glucosidase; D001616:beta-Galactosidase