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 P-galactosidase into variants featur ing P-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 LII defective in 3 ' -->5 ' exonuclease activity. beta -G alactosidase variants can be isolated upon mutagenesis of lacZ hosted into the self-transmissible episome F ' 128, Optimal evolution of lacZ can be ac hieved 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 c lones. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.