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OVERVIEW:
Enzymes are biological polymers of amino acids that function on the nanoscale
by assembling products individually in their active site. The goal of this
REU project is to engineer enzymes for environmental biotechnology: both
for the degradation of priority pollutants and for green chemistry (the
synthesis of chemicals while minimizing wastes). The most-frequently-listed
pollutants in our soil and groundwater are the suspected human carcinogens
perchloroethylene (PCE) and trichloroethylene (TCE). Current remediation
technologies are inadequate for the cost-effective cleanup of sites contaminated
by these chemicals. For green chemistry, the pharmaceutical precursor (R)-1-phenylethane-1,2-diol
will be emphasized as it is a valuable chiral building block for optically-pure
isoproterenol analogues. The goals of this project are to create a new
paradigm in bioremediation of chlorinated solvents by metabolically engineering
microorganisms for the rapid, long-term, aerobic biodegradation of PCE
and TCE as well as to produce enantiomerically-pure (R)-1-phenylethane-1,2-diol
from racemic styrene oxide using bacterial cells in water. We will achieve
these goals by pursuing four specific objectives: (1) use DNA shuffling
to create an optimized monooxygenase that initiates the degradation of
PCE and TCE (Fig. 1), (2) evolve an epoxide hydrolase to reduce the toxicity
of the chlorinated aliphatic degradation products, (3) evaluate the fate
of the modified cells and introduced genes in soil and bioreactor settings,
and (4) evolve the epoxide hydrolase for production of (R)-1-phenylethane-1,2-diol.
The undergraduate students that assist the overall project goals will concentrate
on screening mutants for an improved monooxygenase and an enhanced epoxide
hydrolase using spectrophotometric screens that we have developed. For
example, they will use the 96-well plate format and a spectrophotometer
to screen these enzymes for additional chloride release from mixtures of
PCE and TCE. These students will also learn the DNA shuffling technique
and other molecular biology techniques thereby gaining the ability to create
new enzymes for environmental restoration and responsible chemical synthesis.
Figure 1: Important hydroxylase residues of toluene monooxygenases found
by the Wood laboratory. The wild-type ToMO hydroxylase (Protein Data Bank
accession
code
1t0q (Sazinsky et al. 2004)) was visualized using Swiss-Pdb Viewer program
(DeepView) (Guex and Peitsch 1997; Peitsch 1995; Schwede et al. 2003) and
Pymol. The diiron center is shown in orange. Many of the beneficial positions
such as I100 (blue), A101 (yellow), E103 (orange), A107 (red), and A110 (green)
are located on the TouA B-helix (highlighted in red). Other beneficial positions
such as T201 (purple), F205 (light blue), and E214 (pink) are located in
the TouA E-helix (highlighted in yellow). Position Q141 and M180 are shown
in light pink and dark pink, respectively.
