Microbial Expression of Recombinant TmrA; a Chloroform Reducing Reductive Dehalogenase

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Speaker: Dr. Christopher Marquis, University of New South Wales

Abstract:

Organohalides are recalcitrant, toxic environmental pollutants. The presence of these compounds at elevated levels in soil and aquifers has led to a focus on methods to remediate these sites. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise organohalides as electron acceptors for cellular energy and growth, producing lesser-halogenated compounds that may be more biodegradable and less toxic. Consequently, microbial reductive dehalogenation via organohalide respiration represents a promising solution for clean-up of organohalide pollutants and has been successfully applied in bioremediation of contaminated sites. Therefore, an understanding of the structure–function relationship of RDases is of considerable interest. Dehalobacter sp. UNSWDHB is an OHRB capable of respiring highly toxic chloroform (CF) and converting it to dichloromethane (DCM). TmrA has been identified as the key RDase responsible for this conversion and different strategies for functional expression of recombinant TmrA have been evaluated in my lab. Today, I will describe efforts to express functional recombinant TmrA in different host organisms, including Escherichia coli, Shimwella blattae and Bacillus megaterium. We were successful in obtaining RDAse activity from refolded inclusion bodies expressed in E. coli that were refolded in the presence of FeCl3, Na2S and cobalamin under anaerobic conditions. We were also successful in generating in a soluble and functional form active TmrA in the corrinoid-producing B. megaterium however, the specific activity estimated for the recombinant TmrA was 11-fold lower than the activity of the native TmrA. TmrA was also expressed in a soluble and active form in S. blattae. Co-expression with two different chaperone proteins from the original host did not increase the level of soluble expression however activity assays showed that removing the TAT signal from TmrA increases the dechlorination activity compared to when the TAT signal is present. Finally, we explored the potential of utilising Cupravidius necator as an expression host for TmrA, with a view to assessing the potential for using this host as a chloroform remediating organism.