Rapid evolution of novel traits in microorganisms

The use of natural microorganisms in biotransformations is frequently const rained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selectio n. This process of directed evolution can be accelerated in mutator strains , which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mu tation rate of the selected organisms immediately after selection to preven t the accumulation of undesirable spontaneous mutations. To enable this pro cess, we constructed temperature-sensitive plasmids that temporarily increa se the mutation frequency of their hosts by 20- to 4,000-fold. Under approp riate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approa ch, Nye were able to increase the tolerance of three bacterial strains to d imethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutatio n rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo m utagenesis.