Rational engineering of synthetic pathway for biodegradation of anthropogenic compound

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Nagendra Kurumbang, Pavel Dvorak, Jaroslav Bendl, Jan Brezovsky, Zbynek Prokop, Jiri Damborsky

Loschmidt Laboratories, Czech Republic

Anthropogenic compounds introduced into the biosphere by humans since the industrial revolution are often recalcitrant and persist in the environment. 1,2,3-trichloropropane (TCP) is such an anthropogenic compound which is produced in chemical industries as a solvent and also as a by-product during manufacture of epichlorohydrin. Due to improper disposal, this chemical spreads in the environment and retains over decades in ground water, causing a serious health threat. There is no single microorganism in nature capable of degrading TCP completely to the harmless compound. To address this problem, we assembled a synthetic biochemical pathway using a haloalkane dehalogenase (DhaA) and two associated enzymes, haloalcohol dehalogenase (HheC) and epoxide hydrolase (EchA), for complete conversion of TCP to glycerol (GLY)1. We used a wild type and two engineered variants of DhaA, one with improved activity2 and another with increased selectivity3. The synthetic pathway was introduced into Escherichia coli. Toxicity level of TCP and its intermediate to the E. coli was determined. Then the TCP degradation pathway was rationally engineered by tuning the parameters of individual enzyme. The optimal enzyme ratios for maximum GLY production and minimum toxicity of metabolites were predicted by a mathematical modelling. A number of E. coli strains were constructed based on these predictions and characterized for their potential to survive, degrade and utilize TCP as a source of carbon and energy for their growth. We achieved a good agreement between predicted and experimental data in the designed constructs in terms of enzyme ratios and their degradation profile. The results demonstrate the rational engineering of TCP biodegradation pathway in vivo by using protein and metabolic pathway engineering.
References 1. Bosma et al., Appl. Env. Microbiol. 68: 3582-3587, 2002 2. Pavlova et al., Nat. Chem. Biol. 5: 727-733, 2009
3. van Leeuwen et al., ChemBioChem. 13: 137-148, 2012