Hypermutation of specific genomic loci of Pseudomonas putida for continuous evolution of target genes

The ability of T7 RNA polymerase (RNAP^T7 ) fusions to cytosine deaminases (CdA) for entering C➔T changes in any DNA segment downstream of a T7 promoter was exploited for hyperdiversification of defined genomic portions of Pseudomonas putida KT2440. To this end, test strains were constructed in which the chromosomally encoded pyrF gene (the prokaryotic homologue of yeast URA3) was flanked by T7 transcription initiation and termination signals and also carried plasmids expressing constitutively either high-activity (lamprey's) or low-activity (rat's) CdA-RNAP^T7 fusions. The DNA segment-specific mutagenic action of these fusions was then tested in strains lacking or not uracil-DNA glycosylase (UDG), that is ∆ung/ung⁺ variants. The resulting diversification was measured by counting single nucleotide changes in clones resistant to 5-fluoroorotic acid (5FOA), which otherwise is transformed by wild-type PyrF into a toxic compound. Although the absence of UDG dramatically increased mutagenic rates with both CdA-RNAP^T7 fusions, the most active variant - pmCDA1 - caused extensive appearance of 5FOA-resistant colonies in the wild-type strain not limited to C➔T but including also a range of other changes. Furthermore, the presence/absence of UDG activity swapped cytosine deamination preference between DNA strands. These qualities provided the basis of a robust system for continuous evolution of preset genomic portions of P. putida and beyond.