Targeted mutagenesis in E. coli: A powerful tool for the generation of random mutant libraries

Extract: Mutagenesis is widely used in the fields of genetics, enzyme catalysis, ligand-binding recognition, metabolic regulation, control of gene expression, and to study mechanisms of DNA repair. Mutations are also introduced to optimize enzyme performance. This area has attracted renewed interest with the increased use of biocatalysts in chemical and pharmaceutical synthesis, in bioremediation, and in biotechnology. Individual mutations are introduced, based on detailed structural and functional information, to generate enzymes or other proteins with novel properties and to change the properties of regulatory sequences such as promoters and origins of replication (site-directed mutagenesis). Alternatively, variants of interest can be identified in large libraries harboring random substitutions (random mutagenesis). Unlike site-directed mutagenesis, random mutagenesis requires little or no previous information of the targeted genes. This approach, however, typically allows only a small fraction of all possible mutants to be analyzed. This is due to a number of factors including the inordinate numbers of possible mutations, the need to identify individual mutants of interest from a large pool, the generation of non-functional mutants, and the nature of the genetic code.