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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\u2009841 \u03b1-proteobacterial genomes, and all five experimentally tested RecT homologs supported measurable double-stranded DNA (dsDNA) recombineering, with the Paracoccaceae SJ630 system reaching 8.9 \u00d7 10\u00b2 colony-forming units (CFU) per \u03bcg 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\u2009529 RecT\u2013SSB 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.<\/jats:p>","DOI":"10.1093\/nar\/gkag818","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T11:13:14Z","timestamp":1786101194000},"source":"Crossref","is-referenced-by-count":0,"title":["ETfinder: harnessing conserved C-terminal tails of single-stranded DNA-binding proteins for mining and engineering RecET systems in non-model microbial chassis"],"prefix":"10.1093","volume":"54","author":[{"given":"Dongyuan","family":"Lv","sequence":"first","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering (SKLBE) and School of Biotechnology, East China University of Science and Technology (ECUST) , Shanghai 200237 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mindong","family":"Liang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering (SKLBE) and School of Biotechnology, East China University of Science and Technology (ECUST) , Shanghai 200237 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiyang","family":"Gu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering (SKLBE) and School of Biotechnology, East China University of Science and Technology (ECUST) , Shanghai 200237 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangying","family":"Zhu","sequence":"additional","affiliation":[{"name":"Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology , Shanghai 200237 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9874-5892","authenticated-orcid":false,"given":"Hailong","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, School of Life Science, Shandong University , Jinan 250100 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-3279-2972","authenticated-orcid":false,"given":"Menglei","family":"Xia","sequence":"additional","affiliation":[{"name":"Metabolism and Fermentation Process Control, College of Biotechnology, Ministry of Education, Tianjin University of Science and Technology , Tianjin 300457 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinkang","family":"Hao","sequence":"additional","affiliation":[{"name":"Metabolism and Fermentation Process Control, College of Biotechnology, Ministry of Education, Tianjin University of Science and Technology , Tianjin 300457 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Youyuan","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering (SKLBE) and School of Biotechnology, East China University of Science and Technology (ECUST) , Shanghai 200237 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7827-2696","authenticated-orcid":false,"given":"Weishan","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linquan","family":"Bai","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University , Shanghai 200240 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiagao","family":"Cheng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering (SKLBE) and School of Biotechnology, East China University of Science and Technology (ECUST) , Shanghai 200237 ,","place":["China"]},{"name":"Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology , Shanghai 200237 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lixin","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering (SKLBE) and School of Biotechnology, East China University of Science and Technology (ECUST) , Shanghai 200237 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2272-0159","authenticated-orcid":false,"given":"Gao-Yi","family":"Tan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering (SKLBE) and School of Biotechnology, East China University of Science and Technology (ECUST) , Shanghai 200237 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