A Pseudomonas Putida Strain Genetically Engineered for 1,2,3-Trichloropropane Bioremediation
Ghufrana, S., Pavlova, M., Arif, M.I., Postema, C.P., Damborsky, J., Janssen, D.B.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY 80: 5467-5476 (2014)
1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment and attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3 dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium P. putida MC4-5222 on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4 5222 degraded >95% of influent TCP (0.33 mM) under continuous flow conditions, with stoichiometric release of inorganic chloride. The results show the use of a laboratory-evolved dehalogenase and genetic engineering for obtaining an effective plasmid-free and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon.