High-Throughput Screening Of Combinatorial Regulatory Protein Libraries for Isolation of Novel Endogenous Molecular Biosensors

HIGH-THROUGHPUT SCREENING OF COMBINATORIAL REGULATORY PROTEIN LIBRARIES FOR ISOLATION OF NOVEL ENDOGENOUS MOLECULAR BIOSENSORS

Christopher Frei, Joseph Gredell,Shuai Qian, Patrick C. Cirino

University of Houston, S294 Engineering Bldg 1, Houston, TX, 77204-4004, USA
T: 832-842-6306, csfrei@uh.edu

Endogenous molecular biosensors based on effector-dependent transcriptional regulatory proteins are emerging and powerful biomolecular tools for specific, sensitive, and high-throughput detection/monitoring of intracellular molecules [1].  The naturally occurring proteins couple molecular recognition with changes in gene expression.  To direct the evolution of molecular recognition toward compounds of interest, we construct large libraries of regulatory protein variants and screen for ligand responsiveness using a GFP reporter system[2].  These customized molecular biosensors enable high-throughput screening of combinatorial biosynthesis libraries to improve enzymatic or microbial production of metabolites [3, 4]. 

Our group has shown success engineering AraC, a well-studied native E. coli dual-regulatory protein.  By generating multiple AraC saturation mutagenesis libraries and using positive and negative FACS-based screening, we have isolated variants with altered specificity toward D-arabinose (D-ara), mevalonate (mev), and triacetic acid lactone (TAL)[2-4].  We then explored the limits of engineering AraC for altered molecule recognition.  We sought variants that respond to many compounds including p-coumaric acid (pCA), theophylline, propionic acid, vanillin, malonic acid, levulinic acid, and several others. In the majority of cases AraC variants remaining after sorting showed minimal ligand responsiveness, although a notable exception was pCA-responsive variants.  Improvements in biosensor properties of variants isolated from the initial saturation mutagenesis library screening (e.g. reduced leaky expression and enhanced sensitivity) were also sought by further directed evolution.  Properties of select variants and mutation patterns that correlate with these properties will be described.

The isolation of responsive clones is both tedious and not guaranteed, and to streamline the process we aimed to optimize multiple parameters in the high-throughput screen.  First, schemes with different combinations of positive and negative sorting were tested for a subset of target ligands, and the responses of the populations and individual variants present at the sorting endpoints were compared.  The influence of reporter gene copy number and copy number variability on sorting results was also studied.  As expected, stable chromosomal integration of a single reporter gene reduced expression variability.  Also, replacing GFP with a variant having attenuated stability reduces the signal from leaky AraC variants, amplifies the on/off expression ratio, and could improve the accuracy of the positive sorting step.  

Finally, high-throughput sequencing of individual gene populations surviving different stages of sorting was also performed to develop a more comprehensive understanding of the sequence evolution of specific clones.  This sequencing study and the resulting insights into future screening and AraC protein library design will be discussed.

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.