Title

Enzyme and metabolic engineering for bioremediating chlorinated ethenes and producing (R)-1-phenylethane-1,2-diol, 1-naphthol, and indigoids

Date of Completion

January 2004

Abstract

The goal of this study was to engineer bacteria for the biodegradation of chlorinated ethenes by optimizing enzymes through saturation mutagenesis and site-directed mutagenesis and by cloning genes to form new metabolic pathways. As a continuation of previous efforts to optimize toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 using DNA shuffling for the biodegradation of chlorinated ethenes, saturation mutagenesis was performed at position V106 of the TOM alpha subunit, yielding variant V106F with three times better chloroform degradation activity and variant V106E with two times greater 1-naphthol formation from naphthalene. A statistical program was also developed to predict the number of saturation mutagenesis colonies that must be screened. Further, recombinant strains were created with enhanced degradation of chlorinated ethenes due to reaction with the epoxide intermediates. First, a recombinant Escherichia coli strain with enhanced degradation of cis-1,2-dichloroethylene was created by engineering a novel pathway consisting of eight new genes including a DNA-shuffled TOM (to initiate oxidation of cis-1,2-dichloroethylene), a newly-discovered glutathione S-transferase from Rhodococcus AD45 (to react with cis-1,2-dichloroethylene epoxide), and an overexpressed E. coli mutant γ-glutamylcysteine synthetase. This metabolic pathway engineering led to 3.5-fold enhanced cis-dichloroethylene degradation and better trichloroethylene and trans-dichloroethylene degradation. Second, the epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA) was engineered to accept cis-1,2-dichloroepoxyethane as a substrate by accumulating beneficial mutations from three round of saturation mutagenesis at three selected active site residues: F108, 1219, and C248. The EchA F 108L/1219L/C248I variant co-expressed with a DNA-shuffled TOM enhanced the degradation of cis-dichloroethylene an infinite extent compared to wild-type EchA at low concentrations (6.8 μM) and up to 10 fold at high concentrations (540 μM). ^ Efforts have also been contributed to optimize enzymes as biocatalysts for chemical synthesis. First, the sites of TOM alpha subunit (V106 and A113) that are responsible for the development of different cell colors were identified and saturation mutagenesis was performed at these sites to engineer the enzyme for regiospecific hydroxylation of indole. (Abstract shortened by UMI.)^