Bacterial genome editing methods are used to engineer strains for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but traditional techniques using endogenous RecA-mediated pathways are inefficient and laborious. Phage encoded RecT proteins can improve HR over 1000-fold, but these proteins have limited portability between species. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus, and Caulobacter crescentus we investigated the host-limited functionality of RecTs. We find that these proteins specifically recognize the 7 C-terminal amino acids of the bacterial single-stranded DNA-binding protein (SSB), and are portable between species only if compatibility with this host domain is maintained. Furthermore, in some species, we find that co-expressing otherwise incompatible RecTs with a paired bacterial SSB is sufficient to establish functionality. Finally, we demonstrate that high-efficiency HR surpasses the mutational capacity of more widely used error-prone methods for genome diversification, and can be used to identify exceptional phenotypes inaccessible through sequential nucleotide conversions.### Competing Interest StatementG.M.C. is a founder of 64-x, Enevolv, and GRO Biosciences. G.T.F, T.M.W., and G.M.C. are named inventors on a patent application related to the technologies described in this article. Other potentially relevant financial interests are listed at http://arep.med.harvard.edu/gmc/tech.html.

Characterizing the portability of RecT-mediated oligonucleotide recombination