Nozaki, S.; Miwa, Y.

Escherichia coli is a well-established model organism in molecular biology and biotechnology. Despite its long history as a laboratory workhorse, the efficient single-step chromosomal integration of large DNA fragments remains a challenge. Currently known methods are either simple but have limitations on insert size, or flexible but laborious requiring plasmid construction or multi-step procedures. Here, we present PhAGE (Phage-Assisted Genome Engineering), which enables the integration of ~20 kb DNA fragments into E. coli genome within a single day. PhAGE method uses in vitro packaging of recombinant DNA into bacteriophage capsids, followed by general transduction to introduce pre-assembled DNA with flanking homology arms into recipient cells. This approach allows efficient and landing pad-free integration of large constructs into the target loci. We demonstrate its usefulness through rapid integration of multi-gene operons. PhAGE resolves the long-standing trade-off between simplicity and insert size in E. coli genome engineering, accelerating strain construction across a wide range of applications, from biosynthetic pathway engineering to genome-scale design.