Currently available tools for multiplex bacterial genome engineering are optimized
for a few laboratory model strains, demand extensive prior modification of the host
strain, and lead to the accumulation of numerous off-target modifications. Building
on prior development of multiplex automated genome engineering (MAGE), our work addresses
these problems in a single framework. Using a dominant-negative mutant protein of
the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression
of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide
integration. By integrating all necessary components into a broad-host vector, we
developed a new workflow we term pORTMAGE. It allows efficient modification of multiple
loci, without any observable off-target mutagenesis and prior modification of the
host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE
simultaneously allows genome editing and mutant library generation in other biotechnologically
and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a
set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli.
Despite over 100 million y of divergence between the two species, mutational effects
remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid
allows bacterial species of interest to become an efficient host for genome engineering.
These advances pave the way toward biotechnological and therapeutic applications.
Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis
across a wide range of bacterial species.