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

ABSTRACT: 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

SUBMITTER: Velazquez E 

PROVIDER: S-EPMC9437889 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature