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147e

Proteome Changes after Metabolic Engineering to Enhance Aerobic Mineralization of Cis-1,2-Dichloroethylene

Jintae Lee1, Cao Li1, Saw Yen Ow2, Martin E. Barrios-Llerena2, Wilfred Chen3, Thomas K. Wood1, and Phillip C. Wright2. (1) Artie McFerrin Department of Chemical Engineering, Texas A&M University, 200 Jack E. Brown Building, MS 3122, College Station, TX 77843-3122, (2) Department of Chemical and Process Engineering, The University of Sheffield, Biological & Environmental Systems Group, Sheffield, S1 3JD, United Kingdom, (3) Chemical and Environmental Engineering, University of California, Riverside, Bourns Hall A242, University of California, Riverside, Riverside, CA 92521

We engineered E. coli to degrade cis-1,2-dichloroethylene (cis-DCE) by expressing the 6 genes of an evolved toluene ortho-monooxygenase (TOM-Green from B. cepacia G4, which formed a reactive epoxide) with either (1) g-glutamylcysteine synthetase (GSHI*, which forms glutathione) and the glutathione S-transferase IsoILR1 from Rhodococcus AD45 (which adds glutathione to the reactive cis-DCE epoxide) or (2) with an evolved epoxide hydrolase from A. radiobacter AD1 (EchA F108L/I219L/C248I, which converts the reactive cis-DCE epoxide to a diol). Here, the impact of this metabolic engineering was assessed through a quantitative shotgun proteomics technique (iTRAQ) by tracking the changes due to the sequential addition of TOM-Green, IsoILR1, and GSHI* and due to adding the evolved EchA vs. the wild-type enzyme to TOM-Green. For the TOM-Green/EchA system, 8 proteins out of 268 identified proteins were differentially expressed in the strain expressing EchA F108L/I219L/C248I relative to wild-type EchA. For the TOM-Green/IsoILR1/GSHI* system, the expression level of 49 proteins was changed out of 364 identified proteins. The induced proteins due to the addition of TOM-Green, IsoILR1, and GSHI* were involved in the oxidative defense mechanism, pyruvate metabolism, and glutathione synthesis. Enzymes involved in indole synthesis, fatty acid synthesis, gluconeogenesis, and the tricarboxylic acid cycle were repressed. Hence, the metabolic engineering that leads to enhanced aerobic degradation of 1 mM cis-DCE and reduced toxicity from cis-DCE epoxide results in enhanced synthesis of glutathione coupled with an induced stress response as well as repression of fatty acid synthesis, gluconeogenesis, and the tricarboxylic acid cycle.