Developing an E. coli heterologous expression system for characterizing a marine debrominase from Roseobacter sp

Reductive dehalogenases (RDases) are a family of enzymes that employ a cobamide cofactor and two iron-sulfur clusters to selectively cleave carbon-halogen bonds. 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 Escherichia coli DH5α to produce a chloroform-reducing RDase (TmrA) from Dehalobacter sp. UNSWDHB, using a pTrcHisA vector without requiring an additional vector for vitamin B₁₂ 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 Roseobacter strain, without chemical cofactor reconstitution. Ros_A3X954 catalyzes conversion of 2,6-dibromophenol to 2-bromophenol with a specific activity of 21.8 ± 1.0 nmol s^-1 mg^-1. The enzyme exhibited optimal activity at pH 6.0 and demonstrated significant thermal stability by maintaining full catalytic capacity across a broad temperature range (20-50°C), 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 Roseobacter for bioremediation of bromophenol pollution.IMPORTANCEOrganohalide-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 Escherichia coli DH5α expression system for RDases without requiring vitamin B₁₂ transporters expression vector, significantly simplifying production process. A bromophenol-degrading RDase (Ros_A3X954) from Roseobacter sp. was discovered, exhibiting high activity at pH 6.0 and thermal stability (20-50°C). This study not only provides a scalable platform for RDase exploration but also highlights Roseobacter's ecological role in bromine cycling, advancing bioremediation strategies for persistent pollutants.