Document Type

PhD diss.

Date of Degree

2010

Degree Name

PhD (Doctor of Philosophy)

Department

Civil and Environmental Engineering

First Advisor

Timothy E. Mattes

Abstract

Polaromonas sp. strain JS666 is the only isolated bacterium capable of aerobic growth using the groundwater pollutant cis-1,2-dichloroethene (cDCE) as a sole carbon source. Its genome has a wealth of evidence of recent gene acquisition through horizontal gene transfer, and contains gene clusters predicted to encode enzymes allowing the metabolism of a wide variety of xenobiotic compounds. Culture growth using each of these hypothesized substrates was tested experimentally, and many were confirmed as sole carbon sources for strain JS666. In addition to pollutant degradation, many of these metabolic pathways have applicability in the field of biocatalysis, as does the genome-assisted pathway prediction approach to biocatalyst discovery.

During (or immediately following) growth on cDCE, cultures of Polaromonas sp. strain JS666 oxidize ethene to epoxyethane at an increased rate, and also cometabolically oxidize several other chlorinated ethenes. Given the involvement of a monooxygenase in other species' 1-chloroethene (vinyl chloride) oxidation, it was hypothesized that alkene oxidation in strain JS666 was due to the activity of a monooxygenase that also was responsible for the first step in cDCE oxidation. The alkene oxidation activity of strain JS666 was investigated using gene expression analysis, proteomics, and whole-cell kinetic assays. Results of these experiments pointed to the upregulation of a cyclohexanone monooxygenase (CHMO) during growth on cDCE and during oxidation of ethene.

To determine the activity of this cyclohexanone monooxygenase, its gene was cloned and heterologously expressed in an E. coli host. Our CHMO expression system exhibited activity on cyclohexanone, but not cDCE or ethene, disproving our hypothesis about its involvement in alkene oxidation. The heterologously expressed monooxygenase was also investigated for enantioselective oxidation of racemic cyclic ketones to chiral lactones, and was discovered to have very high enantioselectivity with the tested compounds. Chiral lactones and other single-enantiomer oxidation products are valuable for fine chemical synthesis, and their biocatalytic production is more environmentally sustainable and often less expensive than traditional techniques.

The research described in the following chapters illustrates the many opportunities that arise when the fields of bioremediation and biocatalysis converge. The shared research goals and methods of these two areas lend themselves to interdisciplinary research, and increased communication and crossover between them should provide benefits for both environmental remediation and sustainable chemical synthesis.

Pages

xi, 114

Bibliography

103-114

Copyright

Copyright 2010 Anne Kathryn Alexander