(593z) Novel Endogenous Molecular Biosensors From Engineered Regulatory Proteins AraC and TetR

NOVEL ENDOGENOUS MOLECULAR BIOSENSORS FROM ENGINEERED REGULATORY
PROTEINS ARAC AND TETR

Christopher Frei, Joseph Gredell, Shuang-Yan Tang, Patrick C. Cirino

University of Houston, S294
Engineering Bldg 1, Houston, TX, 77204-4004, USA

T: 832-842-6306, cfrei@uh.edu

Endogenous molecular biosensors for specific, sensitive, and
high-throughput detection/monitoring of small molecules are emerging as
invaluable tools in metabolic engineering and synthetic biology.  Regulatory proteins controlled by ?effector?
molecules naturally couple molecular recognition to changes in gene expression,
providing a platform for endogenous molecular reporters by linking in vivo molecular
synthesis to a readily detectable phenotype (e.g. GFP expression).  Numerous advances have been made in the detection
of endogenous small molecules through whole-cell regulatory protein-based
biosensors [1], but
limitations arise due to the narrow range of natural effectors recognized by
regulatory proteins.  We are developing
customized molecular biosensors by engineering regulatory protein effector
recognition through directed evolution techniques.  In general, these customized molecular
biosensors are applicable to high-throughput screening of combinatorial enzyme
libraries to improve enzymatic or microbial production of metabolites.

For these purposes, our initial focus was the well-studied L-arabinose-responsive
AraC dual regulatory protein from E. coli.
 Saturation mutagenesis libraries,
targeting up to 5 residues (~10^7 mutants), were generated based on analysis of
the AraC ligand binding pocket. Using a pBAD-GFP
construct expressed in E. coli and
FACS, AraC variants responsive to D-arabinose, mevalonate,
and triacetic acid lactone (TAL) were isolated [2-4]. 
The mevalonate and TAL reporter systems were
subsequently used to screen for improved production of these compounds from E. coli expressed pathway-specific mutant
libraries.  Following the success of the
previously engineered AraC-based biosensors, further AraC libraries were screened
for a response to p-coumaric acid, trans-cinnamic acid, and vanillin.  Continued engineering of AraC effector
specificity is now aimed at exploring the range of ligands accessible with this
motif as well as improving the understanding of the molecular determinants of
ligand binding and regulatory switching.  

We next sought to engineer reporter systems for considerably
larger and more complex natural products such as polyketides.  The regulatory protein TetR from the Tn10
transposon was selected due to its well-characterized functions and ability to naturally
recognize large polycyclic molecules (i.e. tetracycline). Combinatorial mutant
libraries of TetR were created through restricted saturation mutagenesis.  Due to the sophisticated hydrogen bonding
network found within the ligand binding domain of TetR and the limitations of
FACS, our selection of specific target residues was critical in our library
design.  Restricted saturation
mutagenesis allowed us to scan more residues per library while maintaining potential
cooperative interactions and a workable library size (~10^7).  The TetR mutants were expressed in E. coli cells containing a TetR-regulated
GFP reporter construct.   The libraries
are being screened for specificity toward various polyketide compounds.  Improved understanding of effector
recognition will help optimize the mutational composition of various libraries
generated, resulting in more rapid screening and isolation of mutants
responsive to compounds of interest. 
These customized reporters will prove valuable in engineering
heterologous polyketide biosynthesis pathways. 

1.            Gredell,
J.A., C.S. Frei, and P.C. Cirino, Protein
and RNA engineering to customize microbial molecular reporting.
Biotechnology Journal, 2012. 7(4):
p. 477-499.

2.            Tang,
S.Y., H. Fazelinia, and P.C. Cirino, AraC
regulatory protein mutants with altered effector specificity. Journal of
the American Chemical Society, 2008. 130(15):
p. 5267-5271.

3.            Tang,
S.-Y., O. Akinterinwa, and P.C. Cirino, A
novel endogenous reporter of triacetic acid lactone (TAL) enables screening for
improved TAL production by E. coli. submitted.

4.            Tang,
S.-Y. and P.C. Cirino, Design and
Application of a Mevalonate-Responsive Regulatory Protein. Angewandte
Chemie International Edition, 2011. 50(5):
p. 1084-1086.

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