Organisms that permit hypermutation of target genes without off-target mutagenesis of the host genome enable the accelerated, continuous evolution of genes for new or enhanced functions. We develop and optimize an orthogonal DNA replication system in Escherichia coli that uses components from bacteriophage Φ29. The minimal system requires just two Φ29 genes to maintain the replicon and replicons can be efficiently engineered in vivo. We generate a highly mutagenic Φ29 DNA polymerase that introduces mutations at a frequency approaching 10-4 per base per generation (one mutation in a 1-kb gene every ten generations). Our system is stable for hundreds of generations and enables the continuous, accelerated evolution of new gene functions. We demonstrate the rapid evolution of a tetracycline resistance gene to confer resistance to tigecycline at higher levels than achieved with previously reported systems. We further evolve a 1,000-fold increase in β-lactamase activity for a third-generation cephalosporin in just 3 days.
© 2026. The Author(s).