LySE: A tunable phage-bacteria platform for rapid, on-target evolution of large gene clusters
LySE is a T7 phage–based system that merges discrete, checkpointed mutagenesis with continuous-like selection to accelerate evolution of large gene clusters (up to ~39 kb) while preventing off-target host mutations. It uses a biocontained phagemid, an accessory plasmid expressing a hypermutagenic T7 DNA polymerase, and cycles of lysis and transduction to rapidly mutate and select GOIs. Variants v8 and v9 achieve mutation rates up to 3.82×10−5 substitutions per base, with balanced transition mutations aided by TadA-8e and TadDE deaminases. Demonstrations include evolving tetA for tigecycline resistance and a five-gene ethylene glycol assimilation pathway, outperforming ALE in on-target evolution and enabling rapid optimization of complex metabolic pathways with potential applications in plastics recycling and beyond.