Bridging continuous and discrete evolution through a controllable, hypermutagenic phage-bacteria system

Directed evolution methods face trade-offs between the control of discrete approaches and the throughput of modern continuous systems. Here, we engineered a method called lytic selection and evolution (LySE) for near-continuous evolution of bacterial gene clusters while maintaining discrete checkpoints. We developed a hypermutagenic T7 DNA polymerase variant fused to a dual adenine-cytosine deaminase to install all possible transition mutations at similar frequencies. By relieving pressure from maintaining genome fidelity, we obtained mutation rates of 3.82 × 10−5 substitutions per base. For biocontainment, the T7 DNA polymerase was encoded on an accessory plasmid, while the target gene cluster was encoded on a T7 DNA polymerase-lacking T7 phagemid. Alternating cycles of lysis and transduction enable selective replication and mutagenesis of target genes, while off-target genomic mutations are discarded. LySE evolved a 25-fold increase in tetA-encoded tigecycline resistance in 5 cycles, and a 50.9% increase in endpoint biomass of a bacterial strain that uses the polyethylene terephthalate monomer, ethylene glycol, as its sole carbon source. Our method balances speed and control for directed bacterial evolution. The lytic selection and evolution (LySE) system uses a plasmid-encoded, hypermutagenic T7 DNA polymerase and T7 phagemid-encoded target genes to enable controllable, near-continuous directed evolution of large gene clusters.