Engineering of biochemical pathway for degradation of Anthropogenic environmental pollutant 1,2,3-trichloropropane
Pavel Dvořák1, Zbyněk Prokop1, Jan Brezovský1, David Bednář1 and Jiří Damborský1
1Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Masaryk University, Kamenice 5/A13, 625 00 Brno
Toxic anthropogenic compound 1,2,3-trichloropropane (TCP) is typically released into the environment as a result of its manufacture and use as a solvent, extractive agent and an intermediate in the production of some other industrially relevant chemicals. It has been detected in low concentrations in surface, drinking and ground water, and is anticipated human carcinogen . TCP is likely to persist for long period of time in the groundwater environment, because no living organism posses the complete biochemical pathway for its utilization . The persistence problem observed with various man-made environmental pollutants, including TCP, could be possibly solved by assembling missing biochemical pathways using protein and metabolic engineering.
The aim of this project is to assemble the biochemical pathway for complete degradation of TCP in vitro, using immobilized enzymes, and in vivo, employing suitable microbial host. We proposed pathway consisting of haloalkane dehalogenase DhaA from Rhodococcus sp., haloalcohol dehalogenase HheC and epoxide hydrolase EchA from Agrobacterium radiobacter AD1. These enzymes are expected to catalyze five subsequent steps of TCP degradation to the harmless product glycerol, which can be further utilized in nature through the microbial glycolysis. The genes dhaA, hheC and echA were synthesized using the sequences available in public databases. Purified enzymes were used for determination of kinetic parameters with the target substrates of proposed pathway. Degradation of TCP and its intermediates by respective enzymes was followed using gas chromatography methods. Possible pathway bottlenecks were identified according to the obtained data. Detected poor catalytic efficiency of wild-type DhaA with TCP was improved 26-times by the application of computer-assisted design and focused directed evolution . Resulting mutant DhaA31 has four amino acid substitutions within the main tunnel and the slot tunnel, which connects the buried active site of enzyme with bulk solvent. Bulky hydrophobic residues introduced into the tunnels limit the access of water molecules into the active site, otherwise preventing formation of the reactive complex.
Once the problem of poor DhaA activity was solved, we have studied degradation of TCP in a multi-enzyme reaction with DhaA31, HheC and EchA in buffer solution. Almost complete conversion of 5 mM TCP to the final product glycerol was observed at 37°C, 30°C and 19°C, thus confirming the concept of TCP degradation through assembled biochemical pathway in vitro. In the future, we will study this metabolic pathway with immobilized enzymes and enzymes expressed in the suitable host microorganism or bacterial chassis. Comparison of in vitro and in vivo systems engineered for degradation of TCP will provide unique information on conversion of non-natural compounds by assembled biochemical pathway.
This work was financially supported by the grants LC06010, MSM0021622412 and CZ.1.05/2.1.00/01.0001 from the Czech Ministry of Education and by the biotechnology company LentiKats. The authors express their thanks to Eva Hrdličková for excellent laboratory support.
1. Toxicological review of 1,2,3-trichloropropane, U.S. Environmental Protection Agency (2009)
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3. M. Pavlová et al., Nat. Chem. Biol. 5 (2009) 727-733