Heterologous Production of Functional Chloroform Reductive Dehalogenase

Abstract

BackgroundReductive dehalogenases (RDase) are key enzymes involved in the respiratory process of anaerobic organohalide respiring bacteria (ORB). RDases are membrane‐associated proteins whose activity is dependent on protein‐associated cobalamin and iron‐sulfur clusters. The heterologous expression of respiratory RDases is reported to be extremely challenging. Dehalobacter strain UNSWDHB is an ORB capable of respiring chloroform (CF, or trichloromethane) as an electron acceptor for the generation of cellular energy and as a consequence, dechlorinates CF to dichloromethane. TmrA enzyme is responsible for this reaction (i.e. CF‐RDase) and has been purified from Dehalobacter strain UNSWDHB and partly characterized. This study explores heterologous expression of the same enzyme.MethodsWe cloned a codon optimized tmrA with an N‐terminal hexa‐His‐tag replacing the TAT signal sequence and potential transmembrane helix region into the pPT7 plasmid. The pPT7‐tmrA plasmid was transformed into Bacillus megaterium MS941 containing the pT7‐RNAP plasmid. Xylose‐inducible expression of tmrA was performed aerobically in TB media at 17°C. The cells were disrupted anaerobically using a French press. CF dechlorination activity of the soluble protein fraction was measured by gas chromatography using Ti(III)citrate and methyl viologen as electron donors.ResultsWe expressed the N‐His tagged TmrA protein in B. megaterium cells. Soluble protein fractions exhibited CF dechlorination activity in vitro. Further biochemical characterizations will be undertaken on the purified enzyme.ConclusionThis is the first report of heterologous expression of soluble and active respiratory RDase in B. megaterium. The CF reducing TmrA enzyme was expressed in cobalamin‐producing B. megaterium cells, and dichloromethane production could be demonstrated in the soluble protein fractions.Support or Funding InformationThe authors wish to acknowledge the University of New SouthWales (UNSW) for providing scholarship support for BJ.