Biology Reference
In-Depth Information
in synthetic biology applications. Furthermore, the ability to engineer these proteins
allows synthetic biologists to develop customized genetic circuits. Regulatory proteins
engineered to respond to novel small molecule inputs are also finding use as
molecular reporters for high-throughput screening of enzyme and metabolic pathway
libraries (production of the effector/input molecule activates expression of an easily
detectable reporter gene/protein such as GFP). Examples in which regulatory proteins
were engineered are provided below.
THE XYLR REGULATOR
The transcriptional activator XylR from the soil bacterium
Pseudomonas putida
is natively
induced by benzene derivatives such as xylene. de Lorenzo and coworkers sought to develop
biosensors that detect toxic benzene derivatives in soil.
74,75
A mutant library of XylR was
constructed by randomly mutating (via error-prone PCR) the XylR effector-binding domain
and the
to the DNA-binding domain (DBD). A selection/counterselection
system was developed in a
P. putida
uracil auxotroph, analogous to the yeast URA3 selection
system. Here, XylR variants that respond to the presence of 2,4-dinitrotoluene (DNT),
a xenobiotic used in the polymer industry, activate expression of
pyrF
encoding orotidine-
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-phosphate decarboxylase, thereby complementing uracil auxotrophy. Meanwhile,
in the presence of uracil and fluoroorotic acid (FOA), leaky XylR variants or variants that
respond to the decoy compound xylene express
pyrF
in the absence of DNT, resulting in
production of a toxic compound and eliminating nonspecific clones. Using this system,
a XylR variant with four amino acid substitutions showed a strong response to 1 mM DNT.
A DNT-sensing
P. putida
strain was engineered with luciferase as the reporter, and thus emits
light in the presence of DNT within a soil sample.
75,76
Additional work has focused on
developing stable and environmentally safe strains and formulations for delivery of
aromatic biosensors to soil using water-soluble gelatin capsules.
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THE ARAC REGULATOR
The well-characterized AraC regulatory protein represses transcription from promoter PBAD
in the absence of effector, and activates transcription in the presence of its native effector
L-arabinose.
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Cirino and coworkers sought to alter the effector specificity of AraC,
to create novel molecular reporters. They initially engineered AraC to specifically respond to
D-arabinose, the rarer isomer of the native inducer.
79
Based on analysis of the AraC L-arabinose
binding pocket, four amino acid positions were chosen for simultaneous saturation
mutagenesis. Dual screening using FACS isolated AraC variants that induce expression of
GFP (at PBAD) in the presence of D-arabinose. Variants that cause leaky GFP expression or
show responsiveness to L-arabinose (in the absence of D-arabinose) were selected against.
AraC variants were recovered that showed strong induced gene expression in the presence of
0.1 mM D-arabinose, with essentially no response to L-arabinose. It is noteworthy that similar
attempts to isolate AraC variants from a randommutagenesis library were unsuccessful.
Isoprenoids receive increasing attention as a valuable class of secondary metabolites,
and have been the target in numerous synthetic biology studies. Mevalonate is the product
of HMG-CoA reductase and a key intermediate in isoprenoid biosynthesis.
80
To facilitate
combinatorial library screening of pathways involving HMG-CoA reductase, Tang and
Cirino next sought a mevalonate-responsive AraC variant.
81
A similar strategy as described
for D-arabinose was used, only this time a five-site saturation mutagenesis library was
created, and dual screening via FACS was performed in the presence and absence of 30 mM
mevalonate. After several rounds of screening, a mevalonate-responsive variant was selected
that showed a relatively linear increase in expression of reporter protein (GFP or LacZ)
from PBAD as the mevalonate concentration was increased from 10 mM to 100 mM. The
authors then demonstrated the utility of this mevalonate reporter system by using it to
screen a library of mutations in the ribosomal binding site (RBS) region upstream of
