Strains random mutagenesis

As an alternative to random mutagenesis by error-prone PCR, expression vectors containing the gene of interest may be propagated in mutator E. coli strains like XL 1-Red (Stratagene). This strains contains mutations in three DNA repair pathways and exhibits a more than 5000-fold increased spontaneous mutation rate (3.5 x 10-6), compared to wildtype E. coli (7 x 10-10) [22]. Provided that all mu-... 

A. Greener, M. Callahan, and B. Jerpseth, An efficient random mutagenesis technique using an E. colt mutator strain, Methods Mol. Biol. 1996, 57, 375-385. 

Classical strain improvement can be used, for example, to address the regulation of an amino acid by selecting mutants that are able to grow in the presence of a mimic that has similar regulation characteristics. The classical approach involves random mutagenesis and addresses the whole genome. Hence, the phenotype is primarily targeted and the actual mechanism is not easily subjected to rational control. 

In the cases described below we will generally focus on metabolic pathway engineering rather than on classical strain improvement, for obvious reasons. One should be aware, however, that the classical approach has strengths that can make it a powerful partner of the rational approach. Thus, regulation problems have been addressed by the development of a feedback-resistant enzyme, using selective pressure and random mutagenesis as described above, in a research species, followed by introduction of the altered gene in the production species via recombinant techniques. 

Henke, E., Bomscheuer, U.T. (1999), Directed evolution of an esterase from Pseudomonas fluorescens. Random mutagenesis by error-prone PCR or a mutator strain and identification of mutants showing enhanced enantioselectivity... 

Greener A, Callahan M, Jerpseth B (1996) An efficient random mutagenesis technique using an E. coli mutator strain. In Trower MK (ed) In vitro mutagenesis protocols. Humana Press, New Jersey... 

While still indispensable for successful industrial strain development programs, the drawback of brute-force random mutagenesis is the accumulation of irrelevant or even counterproductive mutations that often result in crippled production hosts.Low-cost whole genome resequencing techniques developed recently by... 

Rowlands RT (1984). Industrial strain improvement mutagenesis and random screening procedures. Enzyme Microb. Technol. 6 3-10. 

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