Proteome changes after metabolic engineering to enhance aerobic mineralization of cis-1,2-dichloroethylene.
Metabolically engineered Escherichia coli has previously been used to degrade cis-1,2-dichloroethylene (cis-DCE). The strains express the six genes of an evolved toluene ortho-monooxygenase from Burkholderia cepacia G4 (TOM-Green, which formed a reactive epoxide) with either (1) gamma-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 Agrobacterium radiobacter AD1 (EchA F108L/I219L/C248I which converts the reactive cis-DCE epoxide to a diol). Here, the impact of this metabolic engineering for bioremediation was assessed by investigating the changes in the proteome 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 versus 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 (e.g., EchA, protein chain elongation factor EF-Ts, 50S ribosomal subunits L7/L12/L32/L29, cysteine synthase A, glycerophosphodiester phosphodiesterase, iron superoxide dismutase). 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 (e.g., 30S ribosomal subunit proteins S3 and S16, 50S ribosomal subunit protein L20, alkyl hydroperoxide reductase, lactate dehydrogenase, acetate kinase, cysteine synthase A). Enzymes involved in indole synthesis, fatty acid synthesis, gluconeogenesis, and the tricarboxylic acid cycle were repressed (e.g., tryptophanase, acetyl-CoA carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase). Hence, the metabolic engineering that leads to enhanced aerobic degradation of 1 mM cis-DCE (2.4-4-fold more chloride ions released) 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.
Lee
Jintae
J
Artie McFerrin Department of Chemical Engineering and Department of Biology, 220 Jack E. Brown Building, Texas A & M University, College Station, Texas 77843-3122, USA.
Cao
Li
L
Ow
Saw Yen
SY
Barrios-Llerena
Martin E
ME
Chen
Wilfred
W
Wood
Thomas K
TK
Wright
Phillip C
PC
D000332:Aerobiosis; D016960:Agrobacterium tumefaciens / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016956:Burkholderia cepacia / Q000201:enzymology; D004000:Dichloroethylenes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005721:Glutamate-Cysteine Ligase / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000235:genetics; D015202:Protein Engineering; D020543:Proteome / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology
D001426:Bacterial Proteins; D004000:Dichloroethylenes; D020543:Proteome; D006899:Mixed Function Oxygenases; C093480:toluene ortho-monooxygenase; D005982:Glutathione Transferase; D005721:Glutamate-Cysteine Ligase