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The identification of yet-uncharacterized RDases is crucial for advancing our understanding of dehalogenation processes in both natural and engineered environments, particularly for bioremediation. In this study, we developed a heterologous expression system in\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      DH5\u03b1 to produce a chloroform-reducing RDase (TmrA) from\n                      <jats:italic toggle=\"yes\">Dehalobacter<\/jats:italic>\n                      sp. UNSWDHB, using a pTrcHisA vector without requiring an additional vector for vitamin B\n                      <jats:sub>12<\/jats:sub>\n                      transporters. By co-expressing the pRSFDuet-BtuCEDFB plasmid, we further enhanced active TmrA production without relying on the T7 promoter system. Using this system, we achieved the first heterologous expression of an RDase (the debrominase Ros_A3X954) from a marine heterotrophic\n                      <jats:italic toggle=\"yes\">Roseobacter<\/jats:italic>\n                      strain, without chemical cofactor reconstitution. Ros_A3X954 catalyzes conversion of 2,6-dibromophenol to 2-bromophenol with a specific activity of 21.8 \u00b1 1.0 nmol s\n                      <jats:sup>\u22121<\/jats:sup>\n                      mg\n                      <jats:sup>\u22121<\/jats:sup>\n                      . The enzyme exhibited optimal activity at pH 6.0 and demonstrated significant thermal stability by maintaining full catalytic capacity across a broad temperature range (20\u201350\u00b0C), a key advantage for applications in thermally fluctuating marine environments. These findings expand our understanding of RDase functional diversity in common marine microbiota and highlight the significant application potential of\n                      <jats:italic toggle=\"yes\">Roseobacter<\/jats:italic>\n                      for bioremediation of bromophenol pollution.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Organohalide-respiring bacteria (OHRB) play a crucial role in bioremediation by degrading halogenated pollutants using cobamide-dependent reductive dehalogenases (RDases). While these enzymes hold great promise for bioremediation, their practical application has been limited by difficulties in production and heterologous expression. This study successfully developed an\n                        <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                        DH5\u03b1 expression system for RDases without requiring vitamin B\n                        <jats:sub>12<\/jats:sub>\n                        transporters expression vector, significantly simplifying production process. A bromophenol-degrading RDase (Ros_A3X954) from\n                        <jats:italic toggle=\"yes\">Roseobacter<\/jats:italic>\n                        sp. was discovered, exhibiting high activity at pH 6.0 and thermal stability (20\u201350\u00b0C). This study not only provides a scalable platform for RDase exploration but also highlights\n                        <jats:italic toggle=\"yes\">Roseobacter<\/jats:italic>\n                        \u2019s ecological role in bromine cycling, advancing bioremediation strategies for persistent pollutants.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.01980-25","type":"journal-article","created":{"date-parts":[[2026,4,3]],"date-time":"2026-04-03T12:53:33Z","timestamp":1775220813000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Developing an\n                    <i>E. coli<\/i>\n                    heterologous expression system for characterizing a marine debrominase from\n                    <i>Roseobacter<\/i>\n                    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