Organochlorine compounds are widely used in industry, and are persistent pollutants. 1,2-dichloroethane (DCA) in particular is a problem locally as a groundwater contaminant at the Botany Industrial Park in south Sydney. Although some naturally-occurring bacteria can biodegrade DCA, such isolates are difficult to genetically manipulate, and are not easily amenable to strain improvement (e.g. to expand their substrate range). Thus, we aimed to construct a synthetic DCA biodegradation pathway in E.coli, to allow us to better understand the evolutionary processes that lead to bacterial growth on xenobiotics, and to enable directed evolution approaches to be used to enhance the activity of the DCA-degrading bacteria.

We used three DCA biodegradation genes from Xanthobacter (dhlA, dhlB, aldA) and one human metabolic gene (adh1b1) to construct our pathway. The NAD-requiring human alcohol dehydrogenase gene was used here since the corresponding Xanthobacter gene requires an unusual cofactor not found in E.coli (PQQ). The four catabolic genes were cloned into a single plasmid under the control of the tetracycline promoter.

Inducible co-expression of  active AldA, DhlB and DhlA were all successfully achieved in response to tetracycline induction, as revealed by enzyme assays and SDS-PAGE. Tetracycline-induced cells effectively degraded both DCA and chloroacetate (a key pathway intermediate). However, no expression of Adh1B1 protein was observed, and the recombinant E.coli could not grow on DCA. Sequencing revealed a point mutation in the adh1b1 gene introduced during cloning, which may have caused the metabolic pathway to be incomplete.

This study provides an excellent foundation for the construction of a complete heterologous DCA pathway, with 3 out of the 4 genes cloned together and proven functional.