Ong, S.; Ghode, P.; Narenderan, A.; Willenborg, F.; Eden, T. V.; Lao, S.; Marsh, C. O.; Yew, W. S.; Fredens, J.

Directed evolution excels at optimizing individual proteins, but simultaneous evolution of multiple genes remains a significant challenge. Here, we establish a robust phage-based system for accelerated evolution of large gene clusters of up to 39 kilobases. Lytic Selection and Evolution (LySE) selectively replicates and mutagenizes the target gene cluster, carried on a phagemid through alternating cycles of lysis and transduction in Escherichia coli. We engineered a hypermutagenic T7 DNA polymerase (T7 DNAP) that increases mutation rates of the replicating phagemid 7,000-fold during the lytic cycle. We further optimized the mutational spectrum by fusing the T7 DNAP to a dual adenine-cytosine deaminase to install all possible transition mutations at similar frequencies. LySE-mediated transduction enables selection for desired metabolic functions by coupling gene cluster expression to host fitness, while eliminating genomic off-target mutations by refreshing the host in each cycle. Using LySE, we evolved a 25-fold increase in tigecycline resistance in 5 cycles, and a 50.9% increase in end-point biomass of a bacterial strain that utilizes the PET monomer, ethylene glycol, as its sole carbon source.