Computer-Assisted Engineering of the Synthetic Pathway for Biodegradation of a Toxic Persistent Pollutant

Abstract

Anthropogenic halogenated compounds were unknown to nature until the industrial revolution, and microorganisms have not had sufficient time to evolve enzymes for their degradation. The lack of efficient enzymes and natural pathways can be addressed through a combination of protein and metabolic engineering. We have assembled a synthetic route for conversion of the highly toxic and recalcitrant 1,2,3-trichloropropane to glycerol in Escherichia coli, and used it for a systematic study of pathway bottlenecks. Optimal ratios of enzymes for the maximal production of glycerol, and minimal toxicity of metabolites were predicted using a mathematical model. The strains containing the expected optimal ratios of enzymes were constructed and characterized for their viability and degradation efficiency. Excellent agreement between predicted and experimental data was observed. The validated model was used to quantitatively describe the kinetic limitations of currently available enzyme variants and predict improvements required for further pathway optimization. This highlights the potential of forward engineering of microorganisms for the degradation of toxic anthropogenic compounds.

A Pseudomonas putida Strain Genetically Engineered for 1,2,3-Trichloropropane Bioremediation

“…Insertion of oxygen preferentially occurs on the terminal carbon, yielding chlorinated carbonyl compounds that undergo an elimination reaction to produce the very toxic compound 2-chloroacrolein (14). Genetic engineering may offer a strategy for producing a strain for the aerobic degradation of TCP (15)(16)(17). Theoretical calculations have indicated that a number of transformations, including reductive dechlorination, reductive ␤-elimination, dehydrochlorination, and nucleophilic substitution by OH Ϫ , are thermodynamically favorable (18).…”

Detection of Chloroalkanes by Surface-Enhanced Raman Spectroscopy in Microfluidic Chip

“…One of the proposed strategies for decontamination of water from TCP is bioremediation. Haloalkane dehalogenase was engineered for enhanced efficiency of the degradation of TCP [35] and coupled with another two enzymes haloalcohol dehalogenase and epoxide hydrolase to convert TCP via several intermediates to glycerol and chloride ions [24][25][26]36]. The bacteria containing these enzymes are tested to be used for bioremediation purposes [35,37].…”

“…With SK307, we developed a simple and versatile optofluidic SERS-based diagnostic and analytical system which provides fast and reliable quantitative measurement of organic chemicals, such as halogenated hydrocarbons. Specifically we were able to detect a toxic environmental pollutant 1,2,3-trichloropropane (TCP) [24][25][26] in water at submillimolar concentrations. We also tested our optofluidic SERS system with other analytes such 2,3-dichloropropan-1-ol (DCP) and trichloromethane (CHCl3).…”

Modular Microfluidic Paper‐Based Devices for Multi‐Modal Cascade Catalysis

“…Biocatalytic cascades are particularly successful because enzymes can be selected to have similar temperature and pH requirements in aqueous buffers. [5] Investigating complex systems can have enormous potential, but as molecular complexity increases, chemical transformations increase in parallel. [4] Kurumbang, et al synthetically derived a method for decreasing halogenated hydrocarbons that threaten human health and the environment using a five-step pathway and three enzymes.…”

“…[4] Kurumbang, et al synthetically derived a method for decreasing halogenated hydrocarbons that threaten human health and the environment using a five-step pathway and three enzymes. [5] Investigating complex systems can have enormous potential, but as molecular complexity increases, chemical transformations increase in parallel. Bimodal catalysts take advantage of two worlds of materials.…”