ETfinder: harnessing conserved C-terminal tails of single-stranded DNA-binding proteins for mining and engineering...

Nonmodel microorganisms offer substantial potential as next-generation microbial chassis (NGMCs), yet most lack efficient and broadly transferable genome-editing systems. Here we present ETfinder, a framework that uses the conserved C-terminal tail of host single-stranded DNA-binding proteins (SSB-Ct) as a biochemical constraint to guide the discovery of RecET recombineering systems. Applied to Rhodobacter sphaeroides, ETfinder identified 91 candidates from 18 841 α-proteobacterial genomes, and all five experimentally tested RecT homologs supported measurable double-stranded DNA (dsDNA) recombineering, with the Paracoccaceae SJ630 system reaching 8.9 × 10² colony-forming units (CFU) per μg of dsDNA and 100% editing accuracy. Testing in Halomonas further showed that RecT proteins from evolutionarily distant taxa remain functional within the same halophilic chassis, indicating that SSB-Ct-guided selection enriches for portable recombination modules beyond phylogenetic proximity. To facilitate broad adoption, we compiled 25 529 RecT-SSB pairs into a curated database and implemented ETfinder as a standalone, locally deployable toolkit for mining, ranking, and phylogenetic visualization. This framework prioritizes high-compatibility homologs, reduces experimental screening burden, and expands the accessible genome-editing toolbox for NGMCs. ETfinder is freely available at https://github.com/lvdongyuan/ETfinder.