Spatiotemporal manipulation of the mismatch repair system of Pseudomonas putida accelerates phenotype emergence

Developing complex phenotypes in industrially-relevant bacteria is a major goal of metabolic engineering, which encompasses the implementation of both rational and random approaches. In the latter case, several tools have been developed towards increasing mutation frequencies--yet the precise spatiotemporal control of mutagenesis processes continues to represent a significant technical challenge. Pseudomonas species are endowed with one of the most efficient DNA mismatch repair (MMR) systems found in bacteria. Here, we investigated if the endogenous MMR system could be manipulated as a general strategy to artificially alter mutation rates in Pseudomonas species. To bestow a conditional mutator phenotype in the platform bacterium Pseudomonas putida, we constructed inducible mutator devices to modulate the expression of the dominant-negative mutLE36K allele. Regulatable overexpression of mutLE36K in a broad-host-range, easy-to-cure plasmid format resulted in a transitory inhibition of the MMR machinery, leading to a significant increase (up to 438-fold) in mutation frequencies and a heritable fixation of genome mutations. Following such accelerated mutagenesis-followed-by selection approach, three phenotypes were successfully evolved: resistance to antibiotics streptomycin and rifampicin and reversion of a synthetic uracil auxotrophy. Thus, these mutator devices could be applied to accelerate evolution of metabolic pathways in long-term evolutionary experiments, alternating cycles of (inducible) mutagenesis coupled to selection schemes.