Biodegradation of 1,2,3-Trichloropropane Through Directed Evolution and Heterologous Expression of a Haloalkane Dehalogenase Gene

Authors

Bosma, T., Damborsky, J., Stucki, G., Janssen, D.B.

Source

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 68: 3582-3587 (2002)

Abstract

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.

Full text

Citation

Bosma, T., Damborsky, J., Stucki, G., Janssen, D.B., 2002: Biodegradation of 1,2,3-Trichloropropane Through Directed Evolution and Heterologous Expression of a Haloalkane Dehalogenase Gene. Applied and Environmental Microbiology 68: 3582-3587.

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