An Orthogonal DNA Replication System in Yeast

An extranuclear replication system, consisting of an orthogonal DNA plasmid–DNA polymerase pair, was developed in Saccharomyces cerevisiae. Engineered error-prone DNA polymerases showed complete mutational targeting in vivo: per-base mutation rates on the plasmid were increased substantially and remained stable with no increase in genomic rates. Orthogonal replication serves as a platform for in vivo continuous evolution and as a system whose replicative properties can be manipulated independently of the host’s. Specifically, its application towards (1) the study of the reversibility of enzyme evolution, and (2) the evolution of global transcription factors for improved yeast tolerance to environmental stresses (e.g. high levels glucose and ethanol, low pH, and high heat), are discussed.