Shuang-Yan Tang, Hossein Fazelinia, Patrick C. Cirino
Postdoctoral Scholar, Chemical Engineering
The Pennsylvania State University
Engineered regulatory proteins have applications in metabolic engineering and biosensing. Use of regulatory proteins for reporting small molecules or controlling metabolic pathways in response to specific metabolites requires a highly selective protein-effector interaction that is not influenced by similar compounds. We have developed FACS-based screens to engineer the effector specificity of the AraC dual regulatory protein, which naturally regulates the ara operon in response to L-arabinose in E. coli. Heterologous expression of L-Arabinose-producing enzymes was used to validate the use of this system for reporting in vivo metabolite production. We are designing AraC variants which activate transcription in response to D-arabinose and not the native isomer L-arabinose, while still tightly repressing transcription in the absence of inducer. Structure analysis of the AraC binding pocket with docked D-arabinose was used to suggest mutant library design strategies. Saturation mutagenesis of four binding pocket residues followed by dual screening yielded mutants inducible by D-arabinose and not by L-arabinose. Sequence analysis of mutants yields insights into the contribution of various residue positions to the resulting induction and repression phenotypes.