Biodegradation of 1,2,3-Trichloropropane Through Directed Evolution and Heterologous Expression of a Haloalkane Dehalogenase Gene
Bosma, T., Damborsky, J., Stucki, G., Janssen, D.B.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY 68: 3582-3587 (2002)
Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we obtained an organism that is capable of growth on 1,2,3-trichloropropane (TCP). The highly toxic and recalcitrant chemical TCP is a waste product generated from the manufacture of the industrial commodity chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP have been unsuccessful, prohibiting the development of a biological process for groundwater treatment. The key step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol by a haloalkane dehalogenase. We used random mutagenesis and screening on eosine-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp. m15-3 (DhaA). A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild type dehalogenase. The 2,3-dichloro-1-propanol utilizing bacterium Agrobacterium radiobacter AD1 expressing the evolved haloalkane dehalogenase under control of a constitutive promoter was able to utilize TCP as sole carbon- and energy source. These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of toxic and environmentally recalcitrant chemicals.