252320 Endogenous Molecular Biosensors From Engineered Regulatory Proteins AraC and TetR

Tuesday, October 30, 2012: 1:42 PM
Westmoreland West (Westin )
Christopher S. Frei1, Joseph A. Gredell1, Shuang-Yan Tang2 and Patrick C. Cirino1, (1)Chemical & Biomolecular Engineering, University of Houston, Houston, TX, (2)Penn State University, University Park, PA

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.

Extended Abstract: File Not Uploaded